register interest

Associate Professor Ben Davies

Research Area: Cell and Molecular Biology
Technology Exchange: ES cell / homologous recombination and Transgenesis
Scientific Themes: Physiology, Cellular & Molecular Biology
Keywords: Transgenic, Knock-out, Knock-down, Genetically modified, Embryonic Stem cell and Animal Model
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Fluorescent 1 and 2 cell embryos

Fluorescent 1 and 2 cell embryos

A single ES cell colony expressing Green Fluorescent Protein growing on a fibroblast feeder layer

A single ES cell colony expressing Green Fluorescent Protein growing on a fibroblast feeder layer

Microinjection of embryonic stem cells into a blastocyst

Microinjection of embryonic stem cells into a blastocyst

Genetically modified models represent one of the most powerful methods of functional gene analysis in vivo. Furthermore, the ability to introduce specific mutations into the genome enables models of human disease to be generated, facilitating insights into the pathophysiology of disease and providing a model with which therapeutic strategies and diagnostic tools can be optimized.

Our group provides groups within Oxford University access to transgenic technologies both on a fee-for-service type arrangement and on a collaborative basis. Technologies offered include embryo microinjection, embryonic stem cell transfection, Knock-out/-in construct design and in vivo shRNA mediated gene Knock-down. In addition, embryo rederivation and cryoconservation services are offered to facilitate the management, transfer and security of genetically modified strains.

The research activity of the group is focused on the development of novel methodologies for the generation of genetically modified models. The aims being to improve the reliability of the technology and to reduce the animal cost of research involving genetically modified models.

Name Department Institution Country
Professor Peter Donnelly FRS Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Simon Myers Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Prof Doug Higgs FRS (RDM) Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Mark McCarthy (RDM) OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Anna L Gloyn (RDM) OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Professor Robert Kleta UCL United Kingdom
Professor William Brown University of Nottingham United Kingdom
Hinch AG, Zhang G, Becker PW, Moralli D, Hinch R, Davies B, Bowden R, Donnelly P. 2019. Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm. Science, 363 (6433), pp. 1300-+. | Show Abstract | Read more

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

Hugar LA, Lopa SH, Yabes JG, Yu JA, Turner RM, Fam MM, MacLeod LC, Davies BJ, Smith AB, Jacobs BL. 2019. Palliative care use amongst patients with bladder cancer BJU International, | Show Abstract | Read more

© 2019 The Authors BJU International © 2019 BJU International Published by John Wiley & Sons Ltd Objectives: To describe the rate and determinants of palliative care use amongst Medicare beneficiaries with bladder cancer and encourage a national dialogue on improving coordinated urological, oncological, and palliative care in patients with genitourinary malignancies. Patients and methods: Using Surveillance, Epidemiology, and End Results-Medicare data, we identified patients diagnosed with muscle-invasive bladder cancer (MIBC) between 2008 and 2013. Our primary outcome was receipt of palliative care, defined as the presence of a claim submitted by a Hospice and Palliative Medicine subspecialist. We examined determinants of palliative care use using logistic regression analysis. Results: Over the study period, 7303 patients were diagnosed with MIBC and 262 (3.6%) received palliative care. Of 2185 patients with advanced bladder cancer, defined as either T4, N + , or M + disease, 90 (4.1%) received palliative care. Most patients that received palliative care (>80%, >210/262) did so within 24 months of diagnosis. On multivariable analysis, patients receiving palliative care were more likely to be younger, female, have greater comorbidity, live in the central USA, and have undergone radical cystectomy as opposed to a bladder-sparing approach. The adjusted probability of receiving palliative care did not significantly change over time. Conclusion(s): Palliative care provides a host of benefits for patients with cancer, including improved spirituality, decrease in disease-specific symptoms, and better functional status. However, despite strong evidence for incorporating palliative care into standard oncological care, use in patients with bladder cancer is low at 4%. This study provides a conservative baseline estimate of current palliative care use and should serve as a foundation to further investigate physician-, patient-, and system-level barriers to this care.

Anzilotti C, Swan DJ, Boisson B, Deobagkar-Lele M, Oliveira C, Chabosseau P, Engelhardt KR, Xu X, Chen R, Alvarez L et al. 2019. An essential role for the Zn2+ transporter ZIP7 in B cell development. Nat Immunol, 20 (3), pp. 350-361. | Show Abstract | Read more

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Dwivedi OP, Lehtovirta M, Hastoy B, Chandra V, Kleiner S, Jain D, Richard A-M, Beer N, Krentz N, Prasad R et al. 2018. Loss of ZnT8 function protects against diabetes by enhanced insulin secretion | Show Abstract | Read more

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) enriched in Western Finland protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived β-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human β-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

Gordon D, Dafinca R, Scaber J, Alegre-Abarrategui J, Farrimond L, Scott C, Biggs D, Kent L, Oliver PL, Davies B et al. 2019. Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction. Neurobiol Dis, 121 pp. 148-162. | Show Abstract | Read more

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Leonavicius K, Royer C, Preece C, Davies B, Biggins JS, Srinivas S. 2018. Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay. Proc Natl Acad Sci U S A, 115 (41), pp. 10375-10380. | Show Abstract | Read more

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.

Li R, Bitoun E, Altemose N, Davies R, Davies B, Myers S. 2018. A high-resolution map of non-crossover events reveals impacts of genetic diversity on mammalian meiotic recombination | Show Abstract | Read more

During meiotic recombination in most mammals, hundreds of programmed DNA Double-Strand Breaks (DSBs) occur across all chromosomes in each cell at sites bound by the protein PRDM9. Faithful DSB repair using the homologous chromosome is essential for fertility, yielding either non-crossovers, which are frequent but difficult to detect, or crossovers. In certain hybrid mice, high sequence divergence causes PRDM9 to bind each homologue at different sites, 'asymmetrically', and these mice exhibit meiotic failure and infertility, by unknown mechanisms. To investigate the impact of local sequence divergence on recombination, we intercrossed two mouse subspecies over five generations and deep-sequenced 119 offspring, whose high heterozygosity allowed detection of thousands of crossover and non-crossover events with unprecedented power and spatial resolution. Both crossovers and non-crossovers are strongly depleted at individual asymmetric sites, revealing that PRDM9 not only positions DSBs but also promotes their homologous repair by binding to the unbroken homologue at each site. Unexpectedly, we found that non-crossovers containing multiple mismatches repair by a different mechanism than single-mismatch sites, which undergo GC-biased gene conversion. These results demonstrate that local genetic diversity profoundly alters meiotic repair pathway decisions via at least two distinct mechanisms, impacting genome evolution and Prdm9-related hybrid infertility.

Ostrowski KA, Holt SK, Haynes B, Davies BJ, Fuchs EF, Walsh TJ. 2018. Evaluation of Vasectomy Trends in the United States. Urology, 118 pp. 76-79. | Show Abstract | Read more

OBJECTIVE: To use the Truven Health MarketScan database to better approximate the annual rate of vasectomies performed in the US population, to determine changes over time, regional differences, providers performing this, and to know if there is any monthly variation in vasectomy rates. MATERIALS AND METHODS: Claims data were evaluated from 2007 to 2015 to determine the annual prevalence of vasectomy by patient age and region in the United States. The cohort included men aged 18-64 years with at least 1 claim in any given year in Truven Health MarketScan. Provider type and place of service were evaluated in 2014 and 2015. Monthly evaluation of vasectomy prevalence compared with total claims was performed. RESULTS: The prevalence of vasectomies decreased from 2007 to 2015, across all age groups and in all locations of the country (P <.001). Using these data and the most recent US census data, an estimated 527,476 vasectomies were performed in the United States in 2015. The North Central and West regions (0.64% and 0.60%, respectively) had the highest annual prevalence of vasectomies. The month of March and the end of the year had the highest proportion of vasectomies performed. In both 2014 and 2015, a urologist in the office setting performed 82% of vasectomies. CONCLUSION: An estimated 527,476 vasectomies were performed in the United States in 2015. From 2007 to 2015 there was a decrease in the proportion of vasectomies performed in all age groups and in all locations of the country. The end of the year and the month of March are when the most vasectomies are performed.

Macleod LC, Turner RM, Lopa S, Hugar LA, Davies BJ, Ben-David B, Chelly JE, Jacobs BL, Nelson JB. 2018. Effect of multimodal analgesia with paravertebral blocks on biochemical recurrence in men undergoing open radical prostatectomy. Urol Oncol, 36 (8), pp. 364.e9-364.e14. | Show Abstract | Read more

BACKGROUND: Recent studies suggest that anesthetic technique during radical prostatectomy for prostate cancer may affect recurrence or progression. This association has previously been investigated in series that employ epidural analgesia. The objective of this study is to determine the association between the use of a multimodal analgesic approach incorporating paravertebral blocks and risk of biochemical recurrence following open radical prostatectomy. PATIENTS AND METHODS: Using a prospective database of 3,029 men undergoing open radical prostatectomy by a single surgeon, we identified 2,909 men who received no neoadjuvant androgen deprivation and had at least 1 year of follow up. We retrospectively compared patients who received general analgesia with opioid analgesia (1999-2003, n = 662) to those who received general analgesia with multimodal analgesia incorporating paravertebral blocks (2003-2014, n = 2,247). The primary outcome was time to biochemical recurrence. Biochemical recurrence-free interval was assessed using the Kaplan-Meier technique and compared using a multivariate Cox-proportional hazards regression model. RESULTS: In total, 395 patients (14%) experienced biochemical recurrence following radical prostatectomy, including 265 (12%) who received multimodal analgesia and 130 (20%) who did not (adjusted P = 0.27). After adjusting for age, race, body mass index, preoperative prostate specific antigen, grade, stage, perineural invasion, margin status, percent of tumor in the gland, and diameter of the dominant nodule, there was no difference in recurrence-free interval between groups (HR = 0.92, 95% CI: 0.73-1.17). CONCLUSION: Use of a multimodal analgesic approach incorporating paravertebral blocks is not associated with a reduced risk of biochemical recurrence following radical prostatectomy.

Ehsan M, Kelly M, Hooper C, Yavari A, Beglov J, Bellahcene M, Ghataorhe K, Poloni G, Goel A, Kyriakou T et al. 2018. Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion. J Mol Cell Cardiol, 121 pp. 287-296. | Show Abstract | Read more

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

Bandari J, Maganty A, MacLeod LC, Davies BJ. 2018. Manufacturing and the Market: Rationalizing the Shortage of Bacillus Calmette-Guérin. Eur Urol Focus, 4 (4), pp. 481-484. | Show Abstract | Read more

Bacillus Calmette-Guérin (BCG) is used as first-line intravesical therapy following tumor resection of non-muscle-invasive bladder cancer. Primary producers of BCG announced shortages within the last decade, leading to a worldwide shortage. We review the literature examining the BCG shortage and propose solutions to cope with this problem.

Reichold M, Klootwijk ED, Reinders J, Otto EA, Milani M, Broeker C, Laing C, Wiesner J, Devi S, Zhou W et al. 2018. Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure. J Am Soc Nephrol, 29 (7), pp. 1849-1858. | Show Abstract | Read more

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Morgan TN, Bandari J, Hale N, Davies B. 2017. Inguinal Herniation of Perinephric Tissue: Case Report and Review of the Literature. J Am Osteopath Assoc, 117 (12), pp. 786-788. | Show Abstract | Read more

Inguinal hernias containing a kidney or perinephric tissue are extremely rare and usually related to cases involving a kidney positioned in the pelvis. We report the case of a 79-year-old man who presented with abdominal pain and scrotal swelling. He was found on imaging to have an inferiorly displaced kidney with an inguinal herniation of Gerota fascia, as well as an obstructing ureteral stone with an associated forniceal rupture. The unusual renal anatomy, as well as the management of a forniceal rupture, is discussed.

Bandari J, Ayyash OM, Turner RM, Jacobs BL, Davies BJ. 2017. The lack of a relationship between physician payments from drug manufacturers and Medicare claims for abiraterone and enzalutamide. Cancer, 123 (22), pp. 4356-4362. | Show Abstract | Read more

BACKGROUND: Interactions between industry and prescribers have raised concerns regarding conflicts of interest. To the best of the authors' knowledge, quantitative data measuring these interactions have been limited until recently. In the current study, the authors sought to determine whether an association exists between industry payments and prescriber behavior with regard to abiraterone and enzalutamide. METHODS: Two Centers for Medicare and Medicaid Services databases were combined to analyze oncologists and urologists who received industry payments and/or prescribed abiraterone and enzalutamide. Correlation analysis was constructed on prescription count and industry payments. Multivariable median regression examined predictors of change in prescription count per dollar of industry payment. Stratifying prescribers by quantile evaluated threshold effects on prescribers. RESULTS: The number of prescriptions was similar between prescribers who did and those who did not receive industry payment for both drugs. The median industry payment amount to prescribers differed between prescribers and nonprescribers for abiraterone ($72 vs $56) and enzalutamide ($59 vs $31). Although no statistical association was found to exist between industry payment amount and prescription count for abiraterone prescribers, an association was found to exist for enzalutamide prescribers (rho = 0.31). A small change was found with regard to prescription count per dollar of industry payment for abiraterone (0.0007 prescriptions) and enzalutamide (0.0006 prescriptions). The amount of industry payment needed to predict one additional prescription was found to be lower in the fourth and fifth quantiles compared with the first through third quantiles. CONCLUSIONS: No difference in prescription count was found to exist between prescribers who received industry payments and those who did not. A positive correlation was noted between industry payments and prescription count for enzalutamide. Ease of adoption may affect differences between the 2 drugs. Cancer 2017;123:4356-62. © 2017 American Cancer Society.

Henry MA, Howard DH, Davies BJ, Filson CP. 2017. Variation in Use of Prostate Biopsy Following Changes in Prostate Cancer Screening Guidelines Journal of Urology, 198 (5), pp. 1046-1053. | Show Abstract | Read more

© 2017 American Urological Association Education and Research, Inc. Purpose Prostate biopsy rates have paralleled decreasing prostate specific antigen screening rates since 2012. We hypothesized that biopsy rates and the change in rates since 2012 would vary considerably across hospital referral regions. Materials and Methods Using Medicare data from 2012 through 2014 we identified prostate biopsies performed by physicians who performed 11 or more biopsies annually. We calculated annual biopsy rates and changes in rates from 2012 to 2014 across 306 hospital referral regions. We performed multivariable regression adjusting for factors associated with annual biopsy rates (eg percent of patients older than 75 who were screened with prostate specific antigen and percent of the population that was African American). We also estimated adjusted prostate biopsy rates and changes with time across regions. Results We identified 395,993 biopsies. The overall rates decreased from 11.68 biopsies per 1,000 men in 2012 to 10.23 per 1,000 in 2014 (−12.4%, p = 0.11). Biopsy rates were higher in regions in which a greater percentage of the population was African American (β = 0.810, 95% CI 0.235–1.384, p = 0.006), ambulatory surgical centers were available where biopsy could be performed (β = 0.892, 95% CI 0.108–1.676, p = 0.026) and prostate specific antigen testing occurred more frequently (β = 2.462, 95% CI 1.153–3.771, p <0.001). There was marked geographic variation in the adjusted average biopsy rate (median adjusted rate 9.08 biopsies per 1,000 men, IQR 7.65–10.76) and in the change in biopsy rates with time (median adjusted rate change −1.49 biopsies per 1,000 men, IQR −1.94–−1.22 per 1,000). Conclusions Since 2012 there has been considerable geographic variation in the performance of prostate biopsies as well as changes with time after prostate specific antigen recommendations changed. Characterizing the role of unmeasured patient and physician level factors is crucial to optimize the use and minimize the harms of prostate biopsy.

Bayne CE, Davies BJ. 2017. Chipping away at the body politic one study at a time: the case for more 'unprofessional' online content. BJU Int, 120 (5), pp. 609-610. | Read more

Davies B, Brown LA, Cais O, Watson J, Clayton AJ, Chang VT, Biggs D, Preece C, Hernandez-Pliego P, Krohn J et al. 2017. A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability. Hum Mol Genet, 26 (20), pp. 3869-3882. | Show Abstract | Read more

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Devoy A, Kalmar B, Stewart M, Park H, Burke B, Noy SJ, Redhead Y, Humphrey J, Lo K, Jaeger J et al. 2017. Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice. Brain, 140 (11), pp. 2797-2805. | Show Abstract | Read more

Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease-with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.

Bandari J, Ayyash OM, Emery SL, Wessel CB, Davies BJ. 2017. Marketing and Testosterone Treatment in the USA: A Systematic Review. Eur Urol Focus, 3 (4-5), pp. 395-402. | Show Abstract | Read more

CONTEXT: Testosterone replacement therapy (TRT) is currently approved by the Food and Drug Administration only for classic hypogonadism, although off-label indications have resulted in a dramatic expansion in prescriptions in the USA. Marketing may significantly affect prescriber behavior. OBJECTIVE: To systematically review all available evidence on marketing and TRT in the USA. EVIDENCE ACQUISITION: PubMed, Embase, and Scopus were searched up to July 2017 for all relevant publications reporting on assessments of the TRT market size, economic costs associated with hypogonadism, trends in TRT prescriptions, drug discontinuation rates, and advertising and sales efforts in the USA. EVIDENCE SYNTHESIS: Twenty retrospective studies were included in the final analysis. The market size for hypogonadism constitutes 5.6-76.8% of men in the USA, with the lower end of the range representing the strictest criteria for diagnosis. Men with a diagnosis of hypogonadism consume $14 118 in direct and indirect costs to the payer. Over the last 2 decades, TRT prescriptions have increased between 1.8- and 4-fold. After 1 yr, 80-85% of men discontinue TRT. There is an association between direct-to-consumer advertising and testosterone testing, TRT prescriptions, and TRT without testosterone testing. There is a high prevalence of misinformation on Internet advertising. CONCLUSIONS: Off-label indications have driven the dramatic expansion of TRT prescriptions over the last 2 decades. Direct-to-consumer advertising poses a unique challenge in the USA. Overtreatment can be avoided by applying strict diagnostic criteria for hypogonadism, which limits the addressable market for TRT. PATIENT SUMMARY: In this report, we reviewed the relationship between marketing and testosterone therapy in the USA. We found that many patients are prescribed testosterone without an appropriate diagnosis of hypogonadism, which may be related to the marketing efforts for off-label prescribing.

Cebrian-Serrano A, Davies B. 2017. CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools. Mamm Genome, 28 (7-8), pp. 247-261. | Show Abstract | Read more

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases.

Hanssen LLP, Kassouf MT, Oudelaar AM, Biggs D, Preece C, Downes DJ, Gosden M, Sharpe JA, Sloane-Stanley JA, Hughes JR et al. 2017. Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo. Nat Cell Biol, 19 (8), pp. 952-961. | Show Abstract | Read more

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

Lyon TD, Turner I I RM, McBride D, Wang L, Gingrich JR, Hrebinko RL, Jacobs BL, Davies BJ, Tarin TV. 2017. Preoperative immunonutrition prior to radical cystectomy: a pilot study. Can J Urol, 24 (4), pp. 8895-8901. | Show Abstract

INTRODUCTION: To investigate the use of a high-arginine immunonutrient supplement prior to radical cystectomy for bladder cancer. MATERIALS AND METHODS: We recruited 40 patients to consume a total of four high-arginine immunonutrient shakes per day for 5 days prior to radical cystectomy. The primary outcome measures were safety, tolerability and adherence to the supplementation regimen. Ninety-day postoperative outcomes were also compared between supplemented patients and a cohort of 104 prospectively identified non-supplemented radical cystectomy patients. Multivariable logistic regression models were used to compare overall complications, infectious complications, and readmission rates between groups. RESULTS: There were no serious adverse events during supplementation. Four patients (10%) stopped supplementation due to nausea (n = 2) and bloating (n = 2). Thirty-three patients (83%) consumed all prescribed shakes. Immunonutrient supplementation was not significantly associated with overall complications (adjusted odds ratio [OR] 1.08; 95% confidence interval [CI] 0.50-2.33), infectious complications (OR 1.23; 95% CI 0.49-3.07), or readmissions (OR 1.48; 95% CI 0.62-3.51) on multivariable analyses. CONCLUSIONS: Preoperative supplementation with a high-arginine immunonutrient shake was safe and well tolerated prior to radical cystectomy. Contrary to prior reports, immunonutrient supplementation was not associated with lower postoperative infectious complications in this cohort, perhaps owing to the 5 day supplementation period. Further study is needed to identify the optimal immunonutrient supplement regimen for radical cystectomy patients.

Vieira JM, Howard S, Villa Del Campo C, Bollini S, Dubé KN, Masters M, Barnette DN, Rohling M, Sun X, Hankins LE et al. 2017. BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease. Nat Commun, 8 pp. 16034. | Show Abstract | Read more

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin β4 (Tβ4) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with Tβ4 and is recruited by CCAAT/enhancer-binding protein β (C/EBPβ) to discrete regulatory elements in the Wt1 locus. BRG1-Tβ4 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair.

Cebrian-Serrano A, Zha S, Hanssen L, Biggs D, Preece C, Davies B. 2017. Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models. PLoS One, 12 (1), pp. e0169887. | Show Abstract | Read more

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply.

Lakhal-Littleton S, Wolna M, Chung YJ, Christian HC, Heather LC, Brescia M, Ball V, Diaz R, Santos A, Biggs D et al. 2016. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife, 5 (NOVEMBER2016), | Show Abstract | Read more

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain.

Lyon TD, Turner RM, Yabes JG, Woldemichael E, Davies BJ, Jacobs BL, Nelson JB. 2016. Preoperative Statin Use at the Time of Radical Prostatectomy Is Not Associated With Biochemical Recurrence or Pathologic Upgrading. Urology, 97 pp. 153-159. | Show Abstract | Read more

OBJECTIVE: To determine the association of statin use with oncological outcomes and risk of pathologic upgrading following radical prostatectomy. MATERIALS AND METHODS: Using a prospectively populated database of 3042 men who underwent open radical prostatectomy, patients were grouped according to reported statin use at the time of surgery. The primary outcome was time to biochemical recurrence. The secondary outcome was risk of pathologic upgrading among a subset of 1256 patients with Gleason pattern 3 + 3 = 6 on biopsy. A multivariable Cox model was used to assess risk of biochemical recurrence, and multivariable logistic regression was used to assess risk of pathologic upgrading. RESULTS: Eight hundred twenty-four men (27%) reported statin use at the time of radical prostatectomy. Statin users were older and had higher body mass index, higher Charlson Comorbidity Index, and lower pretreatment prostate-specific antigen values than statin nonusers. Over a median follow-up of 70 months (interquartile range: 36-107), a total of 455 men (15%) experienced biochemical recurrence. Statin use was not associated with biochemical recurrence (adjusted hazard ratio: 1.06, 95% confidence interval: 0.86-1.31). Of those men with biopsy Gleason 3 + 3 = 6 disease, 647 (52%) were upgraded to higher grade disease following radical prostatectomy; however, statin use was not associated with pathologic upgrading (adjusted odds ratio: 0.78, 95% confidence interval: 0.58-1.04). CONCLUSION: Preoperative statin use at the time of radical prostatectomy was not associated with biochemical recurrence or risk of pathologic upgrading in this cohort. These data add to the existing body of literature suggesting that statin use is not associated with more favorable clinical outcomes following radical prostatectomy.

Sacilotto N, Chouliaras KM, Nikitenko LL, Lu YW, Fritzsche M, Wallace MD, Nornes S, García-Moreno F, Payne S, Bridges E et al. 2016. MEF2 transcription factors are key regulators of sprouting angiogenesis. Genes Dev, 30 (20), pp. 2297-2309. | Show Abstract | Read more

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

Bandari J, Turner RM, Jacobs BL, Davies BJ. 2016. Urology Payments from Industry in the Sunshine Act Urology Practice, 3 (5), pp. 332-337. | Show Abstract | Read more

© 2016 American Urological Association Education and Research, Inc. Introduction Payments to practitioners from drug and device manufacturers or group purchasing organizations are reported in the Centers for Medicare and Medicaid Services (CMS) databases as a part of the Sunshine Act. Characterizing these payments is a necessary step in identifying conflicts of interest and the influence of payments on practice patterns, if any. Payments have never been analyzed in detail among urologists. Methods We reviewed the most recent CMS Open Payments database for the full year 2014, released on June 30, 2015. Urology practitioners were extracted and the database was analyzed for number of total payments, total dollar value of payments, mean, median and number of physicians, number of manufacturers, and number of drugs/biologicals. Data were further categorized according to provider specialty, form of payment, nature of payment, practitioner ownership and dispute status. Results Payments totaled $32,450,382. Practitioner payments were unevenly distributed, with a median payment of $15. The majority of payments were in the form of food and beverage. Female pelvic medicine practitioners received the highest payments out of the provider specialties. The largest categorical difference from the median was in the form of stock, options and other ownership interests ($24,050). Ownership status and disputed payments were associated with payment values above median values ($400 and $61, respectively). Conclusions There are major disparities in industry payments to urology practitioners. Whether this influences practice patterns remains to be seen, although identifying categorical differences in payments is an important first step in the process.

Armitage AE, Lim PJ, Frost JN, Pasricha S-R, Soilleux EJ, Evans E, Morovat A, Santos A, Diaz R, Biggs D et al. 2016. Induced Disruption of the Iron-Regulatory Hormone Hepcidin Inhibits Acute Inflammatory Hypoferraemia. J Innate Immun, 8 (5), pp. 517-528. | Show Abstract | Read more

Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation.

Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Crawford G, Anzilotti C, Steeples V, Ghaffari S et al. 2016. Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity. Proc Natl Acad Sci U S A, 113 (26), pp. E3706-E3715. | Show Abstract | Read more

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

Becker PW, Sacilotto N, Nornes S, Neal A, Thomas MO, Liu K, Preece C, Ratnayaka I, Davies B, Bou-Gharios G, De Val S. 2016. An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression. Arterioscler Thromb Vasc Biol, 36 (6), pp. 1209-1219. | Show Abstract | Read more

OBJECTIVE: The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells. APPROACH AND RESULTS: A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor-binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj. CONCLUSIONS: This study demonstrates a novel mechanism of arterial-venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.

Davies B, Hatton E, Altemose N, Hussin JG, Pratto F, Zhang G, Hinch AG, Moralli D, Biggs D, Diaz R et al. 2016. Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature, 530 (7589), pp. 171-176. | Show Abstract | Read more

The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

Dolatshad H, Biggs D, Diaz R, Hortin N, Preece C, Davies B. 2015. A versatile transgenic allele for mouse overexpression studies Mammalian Genome, 26 (11-12), pp. 598-608. | Show Abstract | Read more

© 2015, The Author(s). For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.

Dolatshad H, Biggs D, Diaz R, Hortin N, Preece C, Davies B. 2015. A versatile transgenic allele for mouse overexpression studies. Mamm Genome, 26 (11-12), pp. 598-608. | Show Abstract | Read more

For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.

Lakhal-Littleton S, Wolna M, Carr CA, Miller JJJ, Christian HC, Ball V, Santos A, Diaz R, Biggs D, Stillion R et al. 2015. Correction. Proc Natl Acad Sci U S A, 112 (14), pp. E1812. | Read more

Lakhal-Littleton S, Wolna M, Carr CA, Miller JJJ, Christian HC, Ball V, Santos A, Diaz R, Biggs D, Stillion R et al. 2015. Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. Proc Natl Acad Sci U S A, 112 (10), pp. 3164-3169. | Show Abstract | Read more

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Liu KX, Edwards B, Lee S, Finelli MJ, Davies B, Davies KE, Oliver PL. 2015. Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis. Brain, 138 (Pt 5), pp. 1167-1181. | Show Abstract | Read more

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder characterized by the progressive loss of spinal motor neurons. While the aetiological mechanisms underlying the disease remain poorly understood, oxidative stress is a central component of amyotrophic lateral sclerosis and contributes to motor neuron injury. Recently, oxidation resistance 1 (OXR1) has emerged as a critical regulator of neuronal survival in response to oxidative stress, and is upregulated in the spinal cord of patients with amyotrophic lateral sclerosis. Here, we tested the hypothesis that OXR1 is a key neuroprotective factor during amyotrophic lateral sclerosis pathogenesis by crossing a new transgenic mouse line that overexpresses OXR1 in neurons with the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Interestingly, we report that overexpression of OXR1 significantly extends survival, improves motor deficits, and delays pathology in the spinal cord and in muscles of SOD1(G93A) mice. Furthermore, we find that overexpression of OXR1 in neurons significantly delays non-cell-autonomous neuroinflammatory response, classic complement system activation, and STAT3 activation through transcriptomic analysis of spinal cords of SOD1(G93A) mice. Taken together, these data identify OXR1 as the first neuron-specific antioxidant modulator of pathogenesis and disease progression in SOD1-mediated amyotrophic lateral sclerosis, and suggest that OXR1 may serve as a novel target for future therapeutic strategies.

Lyon TD, Farber NJ, Chen LC, Fuller TW, Davies BJ, Gingrich JR, Hrebinko RL, Maranchie JK, Taylor JM, Tarin TV. 2015. Total Psoas Area Predicts Complications following Radical Cystectomy Advances in Urology, 2015 | Show Abstract | Read more

© 2015 Timothy D. Lyon et al. Purpose. To determine whether total psoas area (TPA), a simple estimate of muscle mass, is associated with complications after radical cystectomy. Materials and Methods. Patients who underwent radical cystectomy at our institution from 2011 to 2012 were retrospectively identified. Total psoas area was measured on preoperative CT scans and normalized for patient height. Multivariable logistic regression was used to determine whether TPA was a predictor of 90-day postoperative complications. Overall survival was compared between TPA quartiles. Results. 135 patients were identified for analysis. Median follow-up was 24 months (IQR: 6-37 months). Overall 90-day complication rate was 56% (75/135). TPA was significantly lower for patients who experienced any complication (7.8 cm2/m2 versus 8.8 cm2/m2, P=0.023) and an infectious complication (7.0 cm2/m2 versus 8.7 cm2/m2, P=0.032) than those who did not. On multivariable analysis, TPA (adjusted OR 0.70 (95% CI 0.56-0.89), P=0.003) and Charlson comorbidity index (adjusted OR 1.34 (95% CI 1.01-1.79), P=0.045) were independently associated with 90-day complications. TPA was not a predictor of overall survival. Conclusions. Low TPA is associated with infectious complications and is an independent predictor of experiencing a postoperative complication following radical cystectomy.

Correa AF, Theisen K, Ferroni M, Maranchie JK, Hrebinko R, Davies BJ, Gingrich JR. 2015. The Role of Interferon in the Management of BCG Refractory Nonmuscle Invasive Bladder Cancer Advances in Urology, 2015 | Show Abstract | Read more

© 2015 Andres F. Correa et al. Background. Thirty to forty percent of patients with high grade nonmuscle invasive bladder cancer (NMIBC) fail to respond to intravesical therapy with bacillus Calmette-Guerin (BCG). Interferon-α2B plus BCG has been shown to be effective in a subset of patients with NMIBC BCG refractory disease. Here we present a contemporary series on the effectiveness and safety of intravesical BCG plus interferon-α2B therapy in patients with BCG refractory NMIBC. Methods. From January of 2005 to April of 2014 we retrospectively found 44 patients who underwent induction with combination IFN/BCG for the management of BCG refractory NMIBC. A chart review was performed to assess initial pathological stage/grade, pathological stage/grade at the time of induction, time to IFN/BCG failure, pathological stage/grade at failure, postfailure therapy, and current disease state. Results. Of the 44 patients who met criteria for the analysis. High risk disease was found in 88.6% of patients at induction. The 12-month and 24-month recurrence-free survival were 38.6% and 18.2%, respectively. 25 (56.8%) ultimately had disease recurrence. Radical cystectomy was performed in 16 (36.4%) patients. Conclusion. Combination BCG plus interferon-α2B remains a reasonably safe alternative treatment for select patients with BCG refractory disease prior to proceeding to radical cystectomy.

Loeb S, Bayne CE, Frey C, Davies BJ, Averch TD, Woo HH, Stork B, Cooperberg MR, Griebling TL, Eggener SE. 2015. Updated Survey of Social Media Use by Members of the American Urological Association Urology Practice, 2 (3), pp. 138-143. | Show Abstract | Read more

© 2015 American Urological Association Education and Research, Inc. Introduction: We performed a more detailed, updated analysis of social media use by AUA members. Specifically we sought to characterize the frequency of and reason for using different social media platforms as well as barriers to social media use. Methods: From November to December 2013 we sent a 21-item survey on social media use to 16,376 AUA members with a valid email address. A total of 1,114 members (6.8%) completed the survey. Responses were tallied and statistical analysis was performed to evaluate use patterns based on demographic characteristics. Results: Overall 71% of AUA members who responded to the survey currently had a social media account. The most popular social media platform was Facebook® (89% of respondents), followed by LinkedIn® (59%), YouTube™ (54%), Twitter® (48%) and Google+™ (35%). All platforms except LinkedIn were used primarily for personal reasons. Fewer than a third of respondents had viewed an AUA social media site and 35% of physician respondents participated in a physician-only social media community. Among respondents who did not use social media the most common reasons were no perception of added value and privacy concerns. Conclusions: Although most AUA respondents are involved in social media, they primarily use social media for personal reasons. There remains significant potential for growth and education on the usefulness of social media for urologists in the professional setting.

Jacobs BL, Davies BJ. 2015. Radical versus partial nephrectomy. Health Aff (Millwood), 34 (5), pp. 881. | Read more

Hortin N, Diaz R, Biggs D, Preece C, Brown W, Davies B. 2014. Integrases for genome engineering TRANSGENIC RESEARCH, 23 (5), pp. 855-855.

Davies B, Hatton E, Hussin J, Altemose N, Pratto F, Moralli D, Gupta A, Biggs D, Brick K, Green C et al. 2014. Reprogramming meiotic recombination in the mouse TRANSGENIC RESEARCH, 23 (5), pp. 854-854.

Pascal LE, Ai J, Masoodi KZ, Wang Y, Wang D, Eisermann K, Rigatti LH, O'Malley KJ, Ma HM, Wang X et al. 2013. Development of a reactive stroma associated with prostatic intraepithelial neoplasia in EAF2 deficient mice. PLoS One, 8 (11), pp. e79542. | Show Abstract | Read more

ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2(-/-) mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2(-/-) mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2(-/-) animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.

Xu Z, Thomas L, Davies B, Chalmers R, Smith M, Brown W. 2013. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol, 13 (1), pp. 87. | Show Abstract | Read more

BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. CONCLUSIONS: We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

Zeron-Medina J, Wang X, Repapi E, Campbell MR, Su D, Castro-Giner F, Davies B, Peterse EFP, Sacilotto N, Walker GJ et al. 2013. A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell, 155 (2), pp. 410-422. | Show Abstract | Read more

The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.

Sacilotto N, Monteiro R, Fritzsche M, Becker PW, Sanchez-Del-Campo L, Liu K, Pinheiro P, Ratnayaka I, Davies B, Goding CR et al. 2013. Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development. Proc Natl Acad Sci U S A, 110 (29), pp. 11893-11898. | Show Abstract | Read more

The mechanisms by which arterial fate is established and maintained are not clearly understood. Although a number of signaling pathways and transcriptional regulators have been implicated in arterio-venous differentiation, none are essential for arterial formation, and the manner in which widely expressed factors may achieve arterial-specific gene regulation is unclear. Using both mouse and zebrafish models, we demonstrate here that arterial specification is regulated combinatorially by Notch signaling and SoxF transcription factors, via direct transcriptional gene activation. Through the identification and characterization of two arterial endothelial cell-specific gene enhancers for the Notch ligand Delta-like ligand 4 (Dll4), we show that arterial Dll4 expression requires the direct binding of both the RBPJ/Notch intracellular domain and SOXF transcription factors. Specific combinatorial, but not individual, loss of SOXF and RBPJ DNA binding ablates all Dll4 enhancer-transgene expression despite the presence of multiple functional ETS binding sites, as does knockdown of sox7;sox18 in combination with loss of Notch signaling. Furthermore, triple knockdown of sox7, sox18 and rbpj also results in ablation of endogenous dll4 expression. Fascinatingly, this combinatorial ablation leads to a loss of arterial markers and the absence of a detectable dorsal aorta, demonstrating the essential roles of SoxF and Notch, together, in the acquisition of arterial identity.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K et al. 2013. A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia. Cell Rep, 3 (5), pp. 1440-1448. | Show Abstract | Read more

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Lakhal-Littleton S, Biggs D, Diaz R, Santos A, Preece C, Davies B, Robbins P. 2013. THE ROLES OF HEPCIDIN AND FERROPORTIN IN AUTOCRINE REGULATION OF IRON LEVELS AMERICAN JOURNAL OF HEMATOLOGY, 88 (5), pp. E49-E49.

Davies B, Davies G, Preece C, Puliyadi R, Szumska D, Bhattacharya S. 2013. Site specific mutation of the Zic2 locus by microinjection of TALEN mRNA in mouse CD1, C3H and C57BL/6J oocytes. PLoS One, 8 (3), pp. e60216. | Show Abstract | Read more

Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.

Chen C-M, Bentham J, Cosgrove C, Braganca J, Cuenda A, Bamforth SD, Schneider JE, Watkins H, Keavney B, Davies B, Bhattacharya S. 2012. Functional significance of SRJ domain mutations in CITED2. PLoS One, 7 (10), pp. e46256. | Show Abstract | Read more

CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.

Oliver PL, Finelli MJ, Edwards B, Bitoun E, Butts DL, Becker EBE, Cheeseman MT, Davies B, Davies KE. 2011. Oxr1 is essential for protection against oxidative stress-induced neurodegeneration. PLoS Genet, 7 (10), pp. e1002338. | Show Abstract | Read more

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease.

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Scopus

Chen CM, Krohn J, Bhattacharya S, Davies B. 2011. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiC31 integrase mediated cassette exchange approach in mouse es cells PLoS ONE, 6 (8), | Show Abstract | Read more

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells. © 2011 Chen et al.

Chen C-M, Krohn J, Bhattacharya S, Davies B. 2011. A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS One, 6 (8), pp. e23376. | Show Abstract | Read more

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells.

Marin TM, Keith K, Davies B, Conner DA, Guha P, Kalaitzidis D, Wu X, Lauriol J, Wang B, Bauer M et al. 2011. Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation. J Clin Invest, 121 (3), pp. 1026-1043. | Show Abstract | Read more

LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11(Y279C/+) (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

Davies B, Kirchhoff C. 2011. Adhesion-GPCRs in the Male Reproductive Tract. Adv Exp Med Biol, 706 pp. 179-188. | Show Abstract | Read more

The male reproductive tract expresses a diverse array of adhesion-GPCRs, many in a highly specific and regulated manner. Despite this specificity of expression, little is known about the function of this receptor family in male reproductive physiology. Insights into function are beginning to emerge with the increasing availability of genetically modified mice harbouring mutations in these genes. Gpr64 is the best characterised of the adhesion-GPCRs in the male reproductive system and the phenotype of Gpr64 knock-out mice implicates this receptor in the regulation of fluid absorption in the efferent ducts and proximal epididymis. This chapter summarizes recent data concerning this receptor and other family members in the male reproductive system.

Smaldone MC, Davies BJ. 2010. BC-819, a plasmid comprising the H19 gene regulatory sequences and diphtheria toxin A, for the potential targeted therapy of cancers. Curr Opin Mol Ther, 12 (5), pp. 607-616. | Show Abstract

BC-819 (DTA-H19), in development by BioCancell Therapeutics Inc, under license from the Hebrew University of Jerusalem, is a double-stranded DNA plasmid carrying the gene for the A subunit of diphtheria toxin under the regulation of the H19 gene promoter. H19, a paternally imprinted, oncofetal gene, encodes an RNA that acts as a riboregulator. Expressed at substantial levels in embryonic and malignant tissues, but minimally or not expressed in adult tissues, elevated H19 RNA expression has been observed in over 30 malignancies prompting investigation into its utility as a targeted therapeutic agent. While most in vivo studies have investigated BC-819 for the treatment of bladder cancer, recent studies have also yielded encouraging results in NSCLC,colon, pancreatic and ovarian cancers. A phase I/IIa clinical trial in patients with non-muscle invasive bladder cancer receiving intravesical BC-819 reported mild local toxicity and complete and partial response rates of 22 and 44%, respectively. At the time of publication, a phase IIb trial was ongoing in patients with bladder cancer, while phase I/II clinical trials in patients with ovarian and pancreatic cancer were accruing participants. This review provides a focused summary of the existing experimental evidence demonstrating the effectiveness of the plasmid construct, early clinical outcomes and a discussion of the potential role of BC-819 as a targeted cancer therapy.

Eisenberg ML, Davies BJ, Cooperberg MR, Cowan JE, Carroll PR. 2010. Letter to the editor European Urology, 58 (3), | Read more

Dolatshad H, Ahuja A, Hammett M, Preece C, Davies B. 2010. Site specific insertion of genomic transgenes via the PhiC31 integrase TRANSGENIC RESEARCH, 19 (2), pp. 327-327.

Eisenberg ML, Davies BJ, Cooperberg MR, Cowan JE, Carroll PR. 2010. Prognostic implications of an undetectable ultrasensitive prostate-specific antigen level after radical prostatectomy. Eur Urol, 57 (4), pp. 622-629. | Show Abstract | Read more

BACKGROUND: The prognostic meaning of an undetectable ultrasensitive prostate-specific antigen (USPSA) level after prostatectomy remains unclear. OBJECTIVE: To determine whether an undetectable USPSA level obtained after surgery is a predictor of biochemical recurrence (BCR)-free survival. DESIGN, SETTING, AND PARTICIPANTS: From the Urologic Oncology Database at the University of California San Francisco, 525 men were identified who had a USPSA measurement 1-3 mo postoperatively with at least 2 yr of follow-up. All preoperative and pathologic criteria were recorded. MEASUREMENTS: Patients were stratified based on their initial USPSA level. We defined an undetectable USPSA level at ≤0.05 ng/ml. Recurrence was defined as two consecutive prostate-specific antigen (PSA) levels ≥0.2 ng/ml or secondary treatment. RESULTS AND LIMITATIONS: We found that 456 patients (87%) had undetectable USPSA and 69 patients (13%) had detectable USPSA immediately postprostatectomy. A 5-yr recurrence-free rate of 86% was found in the undetectable USPSA group compared with 67% in the detectable USPSA group (p<0.01). For patients with pT3 disease, men with an undetectable USPSA had a 5-yr BCR-free survival rate of 78% compared with 40% for men with a detectable USPSA (p<0.01). A multivariable analysis confirmed that patients with an undetectable USPSA were 67% less likely to recur (hazard ratio: 0.33; 95% confidence interval: 0.20-0.55). As the detection level of PSA is lowered, the false-positive rate of BCR necessarily increases. A limitation of the study is its retrospective nature. CONCLUSIONS: An undetectable USPSA after radical prostatectomy is a prognostic indicator of BCR-free survival at 5 yr and may aid in predicting outcome in higher risk patients.

Waller-Evans H, Prömel S, Langenhan T, Dixon J, Zahn D, Colledge WH, Doran J, Carlton MBL, Davies B, Aparicio SAJR et al. 2010. The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse. PLoS One, 5 (11), pp. e14047. | Show Abstract | Read more

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

Dall'Era MA, Cooperberg MR, Chan JM, Davies BJ, Albertsen PC, Klotz LH, Warlick CA, Holmberg L, Bailey DE, Wallace ME et al. 2008. Active surveillance for early-stage prostate cancer: review of the current literature. Cancer, 112 (8), pp. 1650-1659. | Show Abstract | Read more

The natural history of prostate cancer is remarkably heterogeneous and, at this time, not completely understood. The widespread adoption and application of prostate-specific antigen (PSA) screening has led to a dramatic shift toward the diagnosis of low-volume, nonpalpable, early-stage tumors. Autopsy and early observational studies have shown that approximately 1 in 3 men aged >50 years has histologic evidence of prostate cancer, with a significant portion of tumors being small and possibly clinically insignificant. Utilizing the power of improved contemporary risk stratification schema to better identify patients with a low risk of cancer progression, several centers are gaining considerable experience with active surveillance and delayed, selective, and curative therapy. A literature review was performed to evaluate the rationale behind active surveillance for prostate cancer and to describe the early experiences from surveillance protocols. It appears that a limited number of men on active surveillance have required treatment, with the majority of such men having good outcomes after delayed selective intervention for progressive disease. The best candidates for active surveillance are being defined, as are predictors of active treatment. The psychosocial ramifications of surveillance for prostate cancer can be profound and future needs and unmet goals will be discussed.

Smaldone M, Sweeney D, Davies BJ. 2008. Recurrent urinary tract infections in children: Risk factors and association with prophylactic antimicrobials Southern Medical Journal, 101 (4), pp. 338. | Read more

Akhavan A, Jackman SV, Costa G, Davies B, Wu T, Bond G, Abu-Elmagd K. 2007. Urogenital disorders associated with gut failure and intestinal transplantation. J Urol, 178 (5), pp. 2067-2072. | Show Abstract | Read more

PURPOSE: Intestinal transplantation has been increasingly performed in patients with short bowel syndrome and irreversible gut failure with successful outcomes. In parallel a common association was observed between gut failure and different urological disorders. To our knowledge this study is the first to address such an important clinical observation with special reference to the underlying disease. MATERIALS AND METHODS: The medical records of 175 consecutive adult intestinal recipients were reviewed. Data were compiled and described according to the documented urogenital disorder, cause of intestinal failure and treatment modality, including transplantation. RESULTS: Of the patients 43 experienced a total of 53 urogenital disorders for an overall incidence of 25%, including 24 (56%) who had the disorder before referral and 19 (44%) in whom the morbidity developed as a result of transplantation. Interestingly hypercoagulability, pseudo-obstruction, Crohn's disease and Gardner's syndrome were the underlying urogenital and intestinal disorder pathologies in 6.3% of the study patients. Treatment for prior urogenital disorders, including malignancy, precipitated intestinal failure in 3.4% of the referred patients. Reciprocally surgical treatment for the primary intestinal disease and management of gut failure by total parenteral nutrition, followed by transplantation, resulted in different urogenital complications in 17.7% of the total population with an 8.6% incidence of chronic renal failure. CONCLUSIONS: Gut failure and intestinal transplantation are commonly associated with different urogenital disorders. Accordingly a designated urological service should be considered part of the multidisciplinary team required for treating this unique population.

Baumann C, Davies B, Peters M, Kaufmann-Reiche U, Lessl M, Theuring F. 2007. AKR1B7 (mouse vas deferens protein) is dispensable for mouse development and reproductive success. Reproduction, 134 (1), pp. 97-109. | Show Abstract | Read more

AKR1B7 (aldo-keto reductase family 1, member 7; also known as mouse vas deferens protein) is a member of the AKR superfamily, and has been suggested to play a role in detoxifying processes on account of its preferred substrates, 4-hydroxynonenal and isocaproaldehyde. High levels of protein expression were found in the vas deferens and the adrenal gland, where sustained expression is dependent on androgen or ACTH respectively. Recently, a remarkable induction of AKR1B7 expression has been reported in the ovary following exogenous injections of LH. In the present study, we confirm this regulation physiologically during the estrous cycle, observing Akr1b7 expression to be restricted to the theca and stromal cells of the proestrus ovary. To further investigate the role of this detoxifying enzyme in both male and female reproduction, we generated knockout mice deficient in AKR1B7. Although AKR1B7 expression in the vas deferens is considerable and tightly regulated in the ovary of wild-type animals, homozygous mutant animals were found to be viable and no reproductive phenotype was observed. Ovarian follicle maturation and spermatozoa parameters remained normal in the absence of this protein. The determination of serum progesterone revealed an increase in hormone concentration in metestrus, while progesterone was found to be decreased in the estrus phase of the cycle in knockout females.

Davies BJ, Konety B. 2007. Critical evaluation and risk: Elderly patient undergoing Contemporary Urology, 19 (6), pp. 30-37. | Show Abstract

Elderly patients have traditionally been viewed as poor candidates for urologic surgery. However, a review of bladder and prostate cancer literature supports what most urologists know intuitively: properly selected elderly patients are safe to operate on and can potentially gain survival benefits from surgery. What is needed is an accurate set of risk assessment tools that facilitate proper patient selection.

Davies B, Behnen M, Cappallo-Obermann H, Spiess A-N, Theuring F, Kirchhoff C. 2007. Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption. Mol Reprod Dev, 74 (5), pp. 539-553. | Show Abstract | Read more

Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates.

Davies B, Noh P, Smaldone MC, Ranganathan S, Docimo SG. 2007. Paratesticular rhabdomyoma in a young adult: case study and review of the literature. J Pediatr Surg, 42 (4), pp. E5-E7. | Show Abstract | Read more

Paratesticular rhabdomyoma is a rare benign tumor, which usually presents as a painless mass in the scrotum or groin. We report the first case of a locally invasive paratesticular rhabdomyoma in a 17-year-old male teenager who presented with chronic scrotal pain. We further review the current literature of this rare tumor.

Davies BJ, Konety BR. 2007. Does periprostatic nerve block with local anesthetic reduce pain during prostate biopsy?: Commentary Nature Clinical Practice Urology, 4 (2), pp. 76-77. | Read more

Akhavan A, Baker K, Cannon GM, Davies B, Horton JA, Docimo SG. 2007. Pilot evaluation of functional questionnaire for predicting ability of patients with tetraplegia to self-catheterize after continent diversion. J Spinal Cord Med, 30 (5), pp. 491-496. | Show Abstract | Read more

BACKGROUND/OBJECTIVES: Creation of a continent catheterizable stoma has dramatically improved the ability of the patient with spinal cord injury to perform clean intermittent catheterization (CIC). However, not all patients are good candidates for this procedure. To aid in patient evaluation, we propose the use of a functional questionnaire to predict a candidate's ability to negotiate a continent catheterizable stoma. METHODS: A published functional questionnaire was adapted to assess the self-perceived ability to perform upper extremity tasks similar to those involved in the manipulation of catheter. Tetraplegic patients who had undergone creation of a content catheterizable stoma were given the questionnaire and asked to describe demographics, method of catheterization, motivational factors, and satisfaction with the procedure. RESULTS: Subjects varied in age (23-36 years) and level of impairment (C4-C6). Functional scores correlated with level of injury. Of the 4 women and 1 man who responded, only 2 were able to self-catheterize before diversion. Their catheterization times decreased significantly after creation of a continent stoma. Two patients unable to perform CIC preoperatively were able to perform CIC postoperatively. The patient with the lowest score was unable to perform CIC preoperatively or postoperatively. All were satisfied with outcome after diversion. CONCLUSIONS: In our small cohort, a low functional score was associated with inability to perform CIC after continent diversion. Administration of this questionnaire to a larger spinal cord injury population should aid in selecting appropriate candidates for the creation of a continent catheterizable stoma.

Kirchhoff C, Obermann H, Behnen M, Davies B. 2006. Role of epididymal receptor HE6 in the regulation of sperm microenvironment. Mol Cell Endocrinol, 250 (1-2), pp. 43-48. | Show Abstract | Read more

HE6 (GPR64) is a highly conserved, tissue-specific heptahelical receptor of the human epididymis. The seven transmembrane (TM7) domains are a hallmark of G-protein-coupled receptors (GPCRs) which have a proven history of being excellent therapeutic targets. Of all currently marketed drugs, >30% are modulators of specific heptahelical receptors, emphasizing the potential of HE6 as a target for pharmaceutical intervention. Targeted mutation of the mouse HE6 counterpart resulted in male infertility, further emphasizing its role as a candidate target for male contraception. However, the precise function of HE6, together with its potential ligand(s), and signal transduction pathways have remained largely unknown. On the basis of shared sequence motifs within the TM7 region, HE6 has been grouped into the B class of GPCRs. Within this class, HE6 belongs to the 'large N-termini' family-B seven-transmembrane (LNB-TM7) receptors, also termed the adhesion-GPCRs. Members of this subgroup are 'orphan' receptors, and they all seem to be cleaved within a conserved GPCR proteolytic site (GPS) domain. The biological significance of the two-subunit architecture is still unknown. Clues to the function of HE6 within the epithelium of male excurrent ducts may come from its co-localisation with the apical actin cytoskeleton and from the down-regulation in "knockout" male mice of various proteins specific to the initial segment.

Gottwald U, Davies B, Fritsch M, Habenicht UF. 2006. New approaches for male fertility control: HE6 as an example of a putative target. Mol Cell Endocrinol, 250 (1-2), pp. 49-57. | Show Abstract | Read more

Reversible contraceptive methods for males are still not available. During the last few years several marketing studies have clearly shown that men and women would welcome a situation where men could assume responsibility for family planning. Schering AG and Organon are currently collaborating to develop a hormonal method for male fertility control based on the combination of etonogestrel as gestagenic component and testosterone undecanoate. To further optimize male contraceptives in terms of improved efficiency, rapid onset, reversibility, fewer side effects and a convenient method of application, a search for innovative non-hormonal approaches was started. During the last few years, numerous proteins were identified which play a specific role in male fertility. These proteins have first to fulfil a set of indication-specific criteria before a drug discovery process can be initiated. The most important criteria for a putative target protein are tissue-selective expression, crucial biological function in fertility, drugable properties and feasibility of assay development for high-throughput-screening and lead optimization. The G-protein-coupled receptor HE6 was selected as target and the above selection criteria were applied. HE6 displays a preferred epididymis-specific expression pattern and belongs to the superfamily of GPCRs, which are well known to be drugable with small molecules. A knockout mouse was generated which revealed an infertility phenotype with the onset occurring 6 weeks after initiation of spermatogenesis at the latest. Surprisingly, no epididymis-specific phenotype was observed. Instead, the reabsorption of testicular fluid along the efferent ducts was strongly affected. No further obvious side effects were observed in male or female mice. This study with HE6 exemplifies how targets for male contraception have to be validated before drug development can start.

Bert B, Fink H, Hörtnagl H, Veh RW, Davies B, Theuring F, Kusserow H. 2006. Mice over-expressing the 5-HT(1A) receptor in cortex and dentate gyrus display exaggerated locomotor and hypothermic response to 8-OH-DPAT. Behav Brain Res, 167 (2), pp. 328-341. | Show Abstract | Read more

The serotonin 1A (5-HT(1A)) receptor is one of the best described receptor subtypes of the serotonergic system. Due to the complex distribution pattern, the pre- and postsynaptic localisation, the impact on various monoamines, as well as the influence on a wide range of physiological functions, the contribution of 5-HT(1A) receptors to behavioural outcomes is difficult to define. In this study, we present a new transgenic mouse model with a prominent over-expression of the 5-HT(1A) receptor in the outer cortical layers (I-III) and the dentate gyrus. Behavioural studies revealed a slight decrease in baseline motor activity of homozygous mice during the open field test. Moreover, core body temperature of male transgenic mice was significantly lower than that of wild-type mice. Pharmacological studies with the 5-HT(1A) receptor agonist 8-OH-DPAT (0.1-2.5 mg/kg, i.p.) revealed an exaggerated drug response in mutant mice. 8-OH-DPAT led to a drastic decrease in motor activity in the open field and elevated plus maze test. This significant effect on motor activity became more apparent by investigating the serotonergic syndrome induced by 8-OH-DPAT. Concentration as low as 0.5 mg/kg 8-OH-DPAT caused immobility in transgenic mice for 30 min, head weaving behaviour, and backward walking, whereas in wild-type animals, typical behaviours of the serotonin syndrome were first observed at concentrations of 1.5 mg/kg and more. In addition, the 8-OH-DPAT induced hypothermia was more pronounced in mutant mice than in wild-type animals. Therefore, these genetically modified mice represent a promising model for further investigations of the role of 5-HT(1A) receptors.

Kusserow H, Davies B, Hörtnagl H, Voigt I, Stroh T, Bert B, Deng DR, Fink H, Veh RW, Theuring F. 2004. Reduced anxiety-related behaviour in transgenic mice overexpressing serotonin 1A receptors. Brain Res Mol Brain Res, 129 (1-2), pp. 104-116. | Show Abstract | Read more

Serotonergic neurons play a major role in the modulation of emotion and behaviour. Especially knockout studies have revealed a role for the serotonin(1A) (5-HT(1A)) receptor in anxiety related behaviour. Mutant animals exhibit enhanced anxiety-like responses, possibly resulting from impaired autoinhibitory control of midbrain serotonergic neurons. To further elucidate the role of the 5-HT(1A) receptors in affective behaviour, a complementary approach has been used and transgenic mice overexpressing this receptor subtype have been generated. The expression of the active 5-HT(1A) receptor protein as indicated by autoradiography was transiently increased during early postnatal development (P1.5) as compared to wild-type mice. Within the next 2 weeks, the increase in receptor binding vanished and was also not apparent in adult animals indicating adaptive changes in the regulation of 5-HT(1A) receptor expression. Although no evidence for increased receptor binding in the brains of adult homozygous mice was found by autoradiography, typical phenotypic changes indicative of 5-HT(1A) receptor overactivity were apparent. Transgenic mice revealed a reduced molar ratio of 5-hydroxyindoleacetic acid to serotonin in several brain areas and elevated serotonin values in the hippocampus and striatum. Moreover, anxiety-like behaviour was decreased in male and female transgenic mice and body temperature was lowered in male transgenic mice in comparison with heterozygous and wild-type mice. These findings further underline the pivotal role of 5-HT(1A) receptors in the homeostasis of anxiety-like behaviour and the crucial importance of stimulation of the 5-HT(1A) receptor during the early postnatal development for normal anxiety-like behaviour throughout life.

Davies B, Baumann C, Kirchhoff C, Ivell R, Nubbemeyer R, Habenicht U-F, Theuring F, Gottwald U. 2004. Targeted deletion of the epididymal receptor HE6 results in fluid dysregulation and male infertility. Mol Cell Biol, 24 (19), pp. 8642-8648. | Show Abstract | Read more

Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B(2)) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family.

Ansorge M, Tanneberger C, Davies B, Theuring F, Kusserow H. 2004. Analysis of the murine 5-HT receptor gene promoter in vitro and in vivo. Eur J Neurosci, 20 (2), pp. 363-374. | Show Abstract | Read more

The expression level of the 5-HT(1A) receptor gene (htr1a) in the central nervous system (CNS) is implicated in the aetiology and treatment of anxiety disorders and depression. Previous studies of the murine htr1a have revealed that its proximal promoter is GC rich and TATA-less. Several functional transcription factor binding sites, including MAZ and SP1 recognition sequences, have been identified. To further analyse the promoter of this receptor gene, additional upstream sequence information extending to -5.5 kb of the murine htr1a was generated and promoter fragments extending to -20 kb were analysed for activity in cell culture and transgenic animals. Promoter fragments greater than 4.5 kb in length were active in 5-HT(1A) receptor mRNA positive cells and inactive in 5-HT(1A) receptor mRNA negative cells. Smaller fragments were not able to confer this specificity. In agreement, using additive transgenesis to drive LacZ expression in vivo, CNS specific reporter gene expression was found with these longer constructs. Transgene expression in the 4.5- and 20-kb mouse lines resembled the endogenous htr1a expression pattern, whereas the 5.5-kb mouse lines surprisingly revealed strongly reduced expression. None of the three constructs was prone to confer ectopic expression, however, variation of expression between the transgenic lines was observed. Using colocalization studies we analysed the degree of concurrence of transgenic and endogenous htr1a expression brought about by these three different constructs. The highest degrees of colocalization were observed in mice harbouring the 20-kb construct, suggesting a large promoter fragment is required to faithfully direct transgene expression in a 5-HT(1A) receptor like pattern.

Chung SY, Stein RJ, Landsittel D, Davies BJ, Cuellar DC, Hrebinko RL, Tarin T, Averch TD. 2004. 15-year experience with the management of extrinsic ureteral obstruction with indwelling ureteral stents. J Urol, 172 (2), pp. 592-595. | Show Abstract | Read more

PURPOSE: We assessed the success of retrograde placement of indwelling ureteral stents in the management of ureteral obstruction due to extrinsic compression. MATERIALS AND METHODS: Between July 1987 and December 2002 adequate followup was available for 101 patients who underwent primary retrograde ureteral stenting for extrinsic ureteral obstruction. Mean age at presentation was 61.4 years (range 33 to 90). Chart review was performed on all patients for primary diagnosis, symptomatology, degree of hydronephrosis, creatinine levels (baseline, treatment and posttreatment), location of compression, size and number of stents used, progression to percutaneous nephrostomy tube (PNT), stent failure, days to stent failure, post-stent therapy and status at last followup. RESULTS: Mean length of followup was 11 months (range 1 to 127). In 101 patients 138 ureteral units (UU) were stented. Total stent failure occurred in 41 (40.6%) patients and 58 (42.0%) UU. A total of 40 (29.0%) UU required PNTs at a mean of 40.3 days (range 0 to 330) with 18 PNTs placed in less than 1 week. Cases of stent failure that did not undergo PNT placement included 18 (13.0%) UU at a mean of 52.4 days (range 3 to 128). A total of 90 (89.1%) patients had metastatic cancer at stenting with 32.2% dead at 5.8 months (range 1 to 32). Univariate and multivariate analyses identified cancer diagnosis, baseline creatinine greater than 1.3 mg/dl and post-stent systemic treatment as predictors of stent failure. Proximal location of compression and treatment creatinine greater than 3.11 mg/dl were marginal predictors of failure on univariate analysis, while proximal location of obstruction was also marginally significant on multivariate analysis. No predictors were identified for early stent failure (less than 1 week). CONCLUSIONS: At almost 1 year followup stent failure due to extrinsic compression occurred in nearly half of treated patients. Analysis of data revealed a diagnosis of cancer, baseline mild renal insufficiency and metastatic disease requiring chemotherapy or radiation as predictors of stent failure. Managing extrinsic compression by retrograde stenting continues to be a practical but guarded decision and should be tailored to each patient.

Davies BJ, Chung SY, Nelson JB. 2004. Delayed intraoperative hydration limits blood loss during radical retropubic prostatectomy Urology, 64 (4), pp. 712-716. | Show Abstract | Read more

To evaluate the effects of limiting hydration during prostate mobilization on intraoperative blood loss. The patient records of 519 consecutive men undergoing radical retropubic prostatectomy by a single surgeon from January 2000 through April 2003 were reviewed. In the initial 328 cases, intravenous fluids were not limited throughout the case (constant hydration group). In the next 189 cases, intravenous fluids were limited to a target of 1500 mL during prostate dissection (delayed hydration group). After the prostate was removed, hydration was brisk for an additional target of 3500 mL. The patient characteristics, perioperative events, and postoperative recovery were evaluated. Delayed hydration resulted in a statistically significant reduction in estimated blood loss compared with the constant hydration group, averaging 700 mL versus 965 mL, respectively. The immediate postoperative hematocrit values were also significantly greater in the delayed hydration group (31.5%) than in the constant hydration group (30.2%). Furthermore, the delayed hydration group had significantly fewer cases of blood loss greater than 1500 mL and fewer patients needed intraoperative transfusions. No statistically significant difference was found in total intravenous fluids given and no increased morbidity occurred with delayed hydration. Delayed hydration appears to reduce blood loss during radical retropubic prostatectomy. In the hemodynamically stable patient, limiting intravenous fluids before complete dissection of the prostate is feasible without increasing morbidity. © 2004 Elsevier Inc.

Leng WW, Davies BJ, Tarin T, Sweeney DD, Chancellor MB. 2004. Delayed treatment of bladder outlet obstruction after sling surgery: association with irreversible bladder dysfunction. J Urol, 172 (4 Pt 1), pp. 1379-1381. | Show Abstract | Read more

PURPOSE: Our urethrolysis cohort demonstrated an unusual delay time to surgical treatment of bladder outlet obstruction. We determined whether urethrolysis outcomes, ie persistent bladder symptoms, were associated with time between sling and urethrolysis surgeries. MATERIALS AND METHODS: Retrospective analysis of all patients who underwent urethrolysis for post-sling voiding dysfunction between June 1997 and June 2002 was performed. We excluded from study 6 patients with a known history of overactive bladder symptoms, neurogenic bladder dysfunction and use of anticholinergic pharmacotherapy before stress incontinence surgery. The remaining 15 patients were stratified into 2 outcomes groups based upon the absence or presence of post-urethrolysis bladder storage symptoms. Patients (7) in group 1 have no current bladder symptoms. Patients (8) in group 2 still require anticholinergic drug therapy for significant bladder symptoms of frequency and urgency. Data collected for the 2 groups included mean age, existence of urinary retention before urethrolysis, mean time to urethrolysis in months, urethrolysis outcome based upon subjective bladder symptoms and followup duration. For comparison of mean age between groups the standard t test was used. Fisher's exact test was used to compare frequency of urinary retention before urethrolysis between groups. Lastly the Mann-Whitney U test was conducted to compare time to urethrolysis between groups. All statistical analyses were conducted using the SPSS software package (SPSS, Inc., Chicago, Illinois). RESULTS: There was no statistically significant difference between the groups with respect to age or frequency of urinary retention before urethrolysis. Time to urethrolysis for the whole cohort ranged from 2 to 66 months. Mean followup after urethrolysis was 17.3 +/- 22.9 months. Comparison of mean time between incontinence and urethrolysis surgeries between group 1 (9.0 +/- 10.1 months) and group 2 (31.25 +/- 21.9 months) demonstrated a statistically significant difference (p = 0.01). CONCLUSIONS: This urethrolysis population demonstrated an unusual delay time to surgical treatment of bladder outlet obstruction. We categorized the cohort according to absence or presence of persistent bladder storage symptoms, and found a strong association between persistent bladder symptoms and greater delay to urethrolysis.

Davies BJ, Bastacky S, Chung SY. 2003. Large cerebellar lesion as original manifestation of transitional cell carcinoma of the bladder. Urology, 62 (4), pp. 749. | Show Abstract | Read more

Patients with transitional cell carcinoma of the bladder classically present with irritative voiding symptoms or painless hematuria. Common sites of vascular metastases include the liver (38%), lung (36%), bone (27%), adrenal glands (21%), and intestine (13%). Vascular metastasis to the brain, without a prior history of chemotherapy, is extremely rare. To our knowledge, this is the first report of a patient with bladder transitional cell carcinoma whose original presentation was from a symptomatic, metastatic, cerebellar lesion.

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European Pubmed Central

Davies B, Kearns IR, Ure J, Davies CH, Lathe R. 2001. Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity. J Neurosci, 21 (18), pp. 6993-7000. | Show Abstract | Read more

Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult.

Pickard BS, Davies BJ, Rose KA, Stapleton G, Steel M, Lathe R. 1999. Chapter 2.2.3 Brain region-specific genes: the hippocampus Techniques in the Behavioral and Neural Sciences, 13 (C), pp. 212-224. | Read more

Davies BJ, Pickard BS, Steel M, Morris RG, Lathe R. 1998. Serine proteases in rodent hippocampus. J Biol Chem, 273 (36), pp. 23004-23011. | Show Abstract | Read more

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

Hinch AG, Zhang G, Becker PW, Moralli D, Hinch R, Davies B, Bowden R, Donnelly P. 2019. Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm. Science, 363 (6433), pp. 1300-+. | Show Abstract | Read more

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

Davies B, Brown LA, Cais O, Watson J, Clayton AJ, Chang VT, Biggs D, Preece C, Hernandez-Pliego P, Krohn J et al. 2017. A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability. Hum Mol Genet, 26 (20), pp. 3869-3882. | Show Abstract | Read more

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Cebrian-Serrano A, Davies B. 2017. CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools. Mamm Genome, 28 (7-8), pp. 247-261. | Show Abstract | Read more

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases.

Hanssen LLP, Kassouf MT, Oudelaar AM, Biggs D, Preece C, Downes DJ, Gosden M, Sharpe JA, Sloane-Stanley JA, Hughes JR et al. 2017. Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo. Nat Cell Biol, 19 (8), pp. 952-961. | Show Abstract | Read more

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

Vieira JM, Howard S, Villa Del Campo C, Bollini S, Dubé KN, Masters M, Barnette DN, Rohling M, Sun X, Hankins LE et al. 2017. BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease. Nat Commun, 8 pp. 16034. | Show Abstract | Read more

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin β4 (Tβ4) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with Tβ4 and is recruited by CCAAT/enhancer-binding protein β (C/EBPβ) to discrete regulatory elements in the Wt1 locus. BRG1-Tβ4 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair.

Cebrian-Serrano A, Zha S, Hanssen L, Biggs D, Preece C, Davies B. 2017. Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models. PLoS One, 12 (1), pp. e0169887. | Show Abstract | Read more

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply.

Davies B, Hatton E, Altemose N, Hussin JG, Pratto F, Zhang G, Hinch AG, Moralli D, Biggs D, Diaz R et al. 2016. Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature, 530 (7589), pp. 171-176. | Show Abstract | Read more

The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

Dolatshad H, Biggs D, Diaz R, Hortin N, Preece C, Davies B. 2015. A versatile transgenic allele for mouse overexpression studies. Mamm Genome, 26 (11-12), pp. 598-608. | Show Abstract | Read more

For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.

Lakhal-Littleton S, Wolna M, Carr CA, Miller JJJ, Christian HC, Ball V, Santos A, Diaz R, Biggs D, Stillion R et al. 2015. Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. Proc Natl Acad Sci U S A, 112 (10), pp. 3164-3169. | Show Abstract | Read more

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Xu Z, Thomas L, Davies B, Chalmers R, Smith M, Brown W. 2013. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol, 13 (1), pp. 87. | Show Abstract | Read more

BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. CONCLUSIONS: We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

Zeron-Medina J, Wang X, Repapi E, Campbell MR, Su D, Castro-Giner F, Davies B, Peterse EFP, Sacilotto N, Walker GJ et al. 2013. A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell, 155 (2), pp. 410-422. | Show Abstract | Read more

The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.

Davies B, Davies G, Preece C, Puliyadi R, Szumska D, Bhattacharya S. 2013. Site specific mutation of the Zic2 locus by microinjection of TALEN mRNA in mouse CD1, C3H and C57BL/6J oocytes. PLoS One, 8 (3), pp. e60216. | Show Abstract | Read more

Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.

CRISPR-activation and inhibition - manipulating gene expression within the stem cell

CRISPR/Cas9 is a programmable site-specific nuclease that can be used to manipulate the sequence of DNA in a precise and efficient manner. Using these “genome editing” approaches, genes can be knocked out of the genome to allow an assessment of their function in both stem cell and animal models. Furthermore, single nucleotide variations which have been found to associate with a particular disease progression or susceptibility can also be introduced into these systems, allowing models of human ...

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Re-engineering the DNA binding characteristics of a speciation gene

In most species, including humans, all genetic variation is generated by two interlinked processes: mutation, which generates new variable positions, and recombination, which randomly shuffles mutations into new combinations, which then drive genetic differences in our disease risk, are acted upon by natural selection, and provide information on our genetic ancestry. Recombination is probably why sexual reproduction exists. It is also essential for human fertility, while errors in recombination ...

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Epigenetic targets for immunomodulatory therapy in infectious disease and cancer

This project aims to identify and validate potential epigenetic drug targets for use as immunomodulators. Epigenetic mechanisms play a key role in an effective, coordinate immune response with dysregulation resulting in, or contributing to, diverse disease states. There is growing interest in the therapeutic use of epigenetic inhibitors in infectious disease and cancer but further advances are needed to validate targets and understand potential therapeutic indications.Human genetics provides ...

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