Castration-resistant prostate cancer (CRPC) is now the second most common cause of male cancer-related mortality. Although docetaxel has recently been shown to extend the survival of patients with CRPC in two large randomised phase III studies, subsequent treatment options remain limited for these patients. A greater understanding of the molecular causes of castration resistance is allowing a more rational approach to the development of new drugs and many new agents are now in clinical development. Therapeutic targets include the adrenal steroid synthesis pathway, androgen receptor signalling, the epidermal growth factor receptor family, insulin growth factor-1 receptor, histone deacetylase, heat shock protein 90 and the tumour vasculature. Drugs against these targets are giving an insight into the molecular pathogenesis of this disease and promise to improve patient quality of life and survival. Finally, the recent discovery of chromosomal translocations resulting in the upregulation of one of at least 3 ETS genes  may lead to novel agents for the treatment of this disease. Despite the significant health burden of advanced prostate cancer, the underlying biology of this disease is only now being unravelled. A wealth of new laboratory data is fuelling rational, molecularly targeted drug development programmes  regulates transcription of a diverse range of target genes involved in prostate cell proliferation, differentiation and apoptosis. Drugs that reduce circulating levels of androgens or that competitively inhibit the action of androgen at the AR (antiandrogens) remain central to the treatment of prostate cancer. Gonadotrophin-releasing hormone (GnRH) agonists, such as goserelin and leuprorelin, inhibit luteinising hormone (LH) secretion and suppress testicular testosterone generation . However, the duration of response to castration is short (12–33 months) and in almost all patients, is followed by the emergence of a castration-resistant phenotype . Antiandrogens  can be used in combination with GnRH agonists to achieve ‘maximum androgen blockade’. However, although low levels of androgens remaining after castration are thought to play a role in the onset of CRPC, this combination strategy has not proven to prolong survival. In addition, up to 30% of patients experience a drop in prostate specific antigen (PSA) after discontinuing antiandrogens . High do−2 and daily 10 mg prednisolone has been shown to confer a survival advantage of a median of 2.5 months and improve quality of life in patients with CRPC when compared to mitoxantrone and is now regarded as the standard of care in patients for whom chemotherapy is indicated  PSA secretion and the presence in tumour samples of androgens and AR mRNA expression at levels associated with active AR signalling strongly suggest that reactivation of the AR and AR-responsive pathways is one mechanism by which tumours become resistant to androgen deprivation  and histone deacetylases (HDAC) whose role is in maintaining the function and stability of the AR.While androgens are pivotal for the growth of CRPC, mutations in the AR lead to its activation by nonandrogenic steroid molecules and antiandrogens . The majA number of signal transduction processes have been implicated in activation of downstream AR signalling that is independent of ligand binding to the AR. These include the epidermal growth factor receptor family, the insulin growth factor-1 receptor, and the phosphoinositide 3-kinase pathway .One of the most important mechanisms in the development of CRPC is the induction of a bypass pathway independent of the AR that can overcome apoptosis induced by androgen depletion. By bypassing the AR completely, prostate cancer cells survive independent of ligand-mediated or nonligand-mediated AR activation . One sucα2β1 and CD133 and are being screened for unique characteristics that will allow their selective targeting with novel therapeutic agents (Prostate cancer stem cells (PCSC) are rare, undifferentiated cells that do not express the AR and are not dependent on androgens for survival. Prostate cancer stem cells are thought to be responsible for maintaining the tumour and as they are able to survive androgen deprivation therapy (ADT), they could result in relapse with castration-resistant disease several months to years after an initial response. PCSC have been isolated from tumour samples based on high surface expression of integrin With our increasing understanding of the genes and pathways that drive CRPC, therapeutic agents can be developed that act on the molecular targets defined by the key genetic and molecular abnormalities responsible. With a plethora of new agents becoming increasingly available for CRPC, it is critical that the drug development process is optimised to ensure the cost and length of time required for the approval of successful drugs is reduced. Critical to this process is the use of pharmacokinetic (PK) and pharmacodynamic (PD) end points, allowing a pharmacological ‘audit trail’ to be constructed. PK end points provide information on how much drug enters the body, and ideally into target tissues; PD end points allow understanding of what the drug does with respect to modulation of the molecular target and biochemical pathway, and whether this translates into achieving the desired biological effect (e.g. apoptosis). This ensures that all of the key stages in prostate cancer drug development – from preclinical models testing and drug administration in patients, through to the biological effect and the clinical outcome – can be monitored and interpreted . The audKetoconazole, an azole antifungal agent that weakly inhibits several cytochrome P450 enzymes involved in adrenal steroid synthesis has been utilised to treat CRPC. Twenty to 35% of patients who progress on antiandrogens have a short-lived PSA response to ketoconazole and on progression postresponse have a significant rise in plasma adrenal androgens  and androstenedione. Its inhibition causes a reduction in testosterone levels  is a potent, orally bioavailable and irreversible inhibitor of 17in vitro and in vivo, and phase I trials have now been completed  and suggest clinically relevant antitumour activity.Histone deacetylase inhibitors can also inactivate the HSP90 chaperone and hence can deplete key client proteins such as AR. LAQ824 and SAHA have demonstrated activity in preclinical models, with depletion of AR through HSP90 inactivation  (ASCENT) were presented at ASCO 2005 by Beer et al (Abstract 4516). Patients received weekly docetaxel for 3 weeks of a 4-week cycle and either oral DN-101 or placebo. At a median of 18 months follow-up, survival data from 250 patients favours DN-101 (23.5 months) over placebo (16.4 months) . Low calcaemic vitamin D analogues, such as seocalcitol (EB1089), have demonstrated antiproliferative effects in preclinical models and may allow improved dose delivery and antitumour activity.Calcitriol , the principal active metabolite of vitamin D, demonstrates significant antineoplastic activity in preclinical models of prostate cancer. The vitamin D receptor, a member of the nuclear steroid hormone receptor superfamily, mediates transcriptional activation of cyclin-dependent kinase inhibitors causing G0/G1 cell-cycle arrest. Calcitriol also modulates growth factor signalling, induces apoptosis through downregulation of the antiapoptotic protein Bcl-2 and is antiangiogenic ), ErbB2 (HER2/neu), and ErbB3 (HER3)), the insulin-like growth factor receptor (IGF-1R), and the platelet-derived growth factor receptor (PDGF-R).The development of agents to target specific upstream genes and proteins potentially deregulated in prostate cancer also provides an opportunity for anticancer therapy for CRPC. A number of potential therapeutic targets have been elucidated for CRPC. The most common genetic alteration in CRPC is probably loss of the in vitro data suggest that overexpression and activation of AKT can trigger prostate cancer androgen escape via altered sensitivity and activation of AR (The phosphoinositide 3-kinase (PI3K) pathway regulates many key cellular processes. There is now overwhelming evidence implicating the PI3K/AKT/mTOR pathway as a regulator in the malignant progression of prostate cancer. Functional loss of PTEN (which is the negative regulator of PI3K) is thought to occur in up to half of all prostate cancers, and is associated with increased activation of AKT and the downstream kinase mTOR, which is involved in regulating protein synthesis. Loss of PTEN and increased AKT-1 phosphorylation is typically associated with higher Gleason grading, advanced stage and poorer prognosis  proteins and activation of intracellular signalling pathways, including PI3K and MAPK. The activated receptor is thus able to induce AR signalling in the absence of AR ligand activation. IGF-1R expression has been reported to alter as prostate cells progress from a normal to a malignant phenotype and IGF-1R is implicated in resistance to therapy  family and is a candidate gene that can block apoptosis. Survivin has been associated with phenotypically aggressive prostate carcinoma and with resistance to antiandrogen therapy (Science 2005). Bevacizumab, a humanised murine monoclonal antibody to VEGF, has resulted in clinical benefit in a number of tumour types, including colorectal, non-small-cell lung and breast cancer, and is currently being evaluated in CRPC in a randomised double-blinded, placebo-controlled phase III study administering docetaxel with or without bevacizumab (CALGB 90401).An alternative strategy is targeting neovascularisation. Progression of prostate cancer has been associated with increased angiogenesis, at least in part associated with upregulation of proangiogenic factors, such as members of the VEGF family (Endothelin-1, via the endothelin-A receptor, inhibits apoptosis, stimulates proliferation of prostate cancer cells and osteoblasts and induces neovascularisation in response to hypoxia, making this effector pathway a promising therapeutic target. Atrasentan, a selective oral ET-A receptor antagonist, has been tested in patients with CRPC but its antitumour activity has been insufficient to warrant regulatory approval for its inclusion in the treatment of advanced CRPC with bone metastasis (18F-fluoro-2-deoxy-D-glucose or FLT 18F-fluoro-3′deoxy-3′-L-fluorothymidine may also be valuable tools to assess CRPC and may allow early assessment of disease response. These surrogates could become primary end points in efficacy trials expediting future advances and drug approval.It is envisioned that the next decade will result in significant changes in the treatment of prostate cancer . A key c


GSTpi is a marker of prostate cancer, and global patterns of histone marking appear to be linked to cancer prognosis with levels of acetylated histones H3K9, H3K18, and H4K12, and of dimethylated H4R3 and H3K4, dividing low-grade prostate cancer (Gleason 6 or less) into two prognostically separate groups. Elevated levels of several components of the polycomb group protein complex, EZH2, BMI1, and RING1, can also act as biomarkers of poor clinical outcome. Many components of the epigenetic machinery, including histone deacetylase (whose expression level is linked to the TMPRSS2:ERG translocation) and the histone methylase EZH2, are potential therapeutic targets. The recent discovery of the role of small RNAs in governing the epigenetic status of individual genes offers exciting new possibilities in therapeutics and chemoprevention.Substantial evidence now supports the view that epigenetic changes have a role in the development of human prostate cancer. Analyses of the patterns of epigenetic alteration are providing important insights into the origin of this disease and have identified specific alterations that may serve as useful diagnostic and prognostic biomarkers. Examination of cancer methylation patterns supports a stem cell origin of prostate cancer. It is well established that methylation of  The term ‘epigenetic’ refers to mechanisms of inherited change of gene expression that do not involve changes in DNA sequence or copy number. Conventionally, this includes CpG methylation, histone modification or marking, and gene imprinting, but recent evidence shows that small RNAs could also play a critical role in directing epigenetic silencing. In cancers, when alterations in the normal epigenetic portrait arise, they are subjected to the same selective pressures as genetic alterations. However, epigenetic alterations have the potential to cause changes in the expression of individual or groups of genes at considerably elevated rates when compared with their genetic counterparts. A view is emerging that epigenetic disruption of progenitor cells may be an initial step in cancer development, leading to a polyclonal precursor population within which subsequent genetic and epigenetic events may occur. There is considerable interplay between different classes of epigenetic modification, but the mechanisms of interaction are poorly understood, and the hierarchical order of epigenetic alterations during unscheduled gene silencing in cancer is unknown. Here, following a summary of the evidence supporting the role of epigenetic change in prostate cancer development, we review new concepts and technologies emerging in this field that may have particular relevance to the clinical management of prostate cancer.5-position of cytosine in CpG sequences and cancer development is well established. Cancer formation is accompanied by dramatic changes in the cellular methylation profile such that global demethylation of the genome occurs in parallel with CpG hypermethylation at specific genes strongly linked to their transcriptional inactivation. A list of genes that are hypermethylated at CpG islands in prostate cancer is shown in INK4a, RASSF1a and APC, exhibit tumour suppressor functions whose inactivation associated with hypermethylation of CpG islands in 5′ regulatory regions occurs during cancer development. Inappropriate gene hypermethylation catalysed by DNA methylases (DNMTs) may represent an early event in cancer development, possibly linked to ageing. Methylation of GSTpi was absent in normal epithelium and present in 6.4% of proliferative inflammatory atrophy, in 70% of high-grade PIN and in 90% of prostate cancer  gene, whose downregulation has been suggested to play a role in cancer metastasis, has also been documented in prostate cancer  are inherited as epigenetic variations and linked to gene activation or silencing depending on the precise nature and position of the modification . Promoters of expressed genes are commonly associated with the active marks, such as H3 lysine 4 dimethylation (H3K4me2) and H3 lysine 9 acetylation (H3K9acetyl). In contrast, in transcriptionally silenced genes, these are replaced by repressive marks, including H3K27me3, H3K9me2, and H3K9me3. Changes in histone marks arise during cancer development and appear to have prognostic potential. DAB2IP  protein complexes PRC1–PRC4 play a key role in controlling transcriptional silencing. Polycomb group protein complex-1 contains BMI1 and RING1, whereas PRC2–PRC4 contain EZH2. Expression of all three proteins  is associated with adverse pathological features in prostate cancer, but only BMI1 provides additional prognostic power in multivariate analysis. EZH2 is the component of the PRC2 complex responsible for catalysing methylation of both H3K27 and H1K26, but this protein can also become associated with DNMTs, and has a role in the induction and targeting of DNA methylation. Following the action of EZH2, the H3K27me3 mark attracts PRC1, which contains other proteins, including BMI1, which is involved in maintaining gene silencing. One of the target genes for EZH2 in prostate cancer cells is Formation of acetyl histone marks that are associated with transcriptional activation, is catalysed by histone acetyl transferases (HATs). In leukaemia, the HAT TIP60 directly interacts with, and is a co-activator for, the translocation activated transcription factor C/EBPa. The co-activator function is acetyltransferase dependent, with acetylation of histones 3 and 4 associated with activation of downstream genes. Histone acetyl transferases involved in histone marking may also acetylate non-histone proteins, although the relationship of these activities to epigenetic marking is unclear. For example, treatment of prostate cancers with di-hydrotestosterone induces acetylation at a lysine residue in the hinge region of the androgen receptor (AR) by the HATs p300, P/CAF, and TIP60. Acetylation enhances transactivation of the AR by coactivators  and increases access to androgen-responsive elements. Similarly, TIP60 also has a role in modulating DNA repair: TIP60 acetylates p53, and activation of the DNA damage sensor ATM depends on TIP60. Haploinsufficiency of TIP60 has been linked to breast cancer development  is that inactive histone marks are associated with DNA hypermethylated promoters, whereas active marks are normally associated with hypomethylated promoters. Recent data from IGF-II and H19 alleles. These observations may be of particular relevance to prostate cancer because alterations both of insulin-like growth factors (IGFs) and of their binding proteins (IGFBPs) have been implicated in development of this disease. Biallelic expression of IGF-II, implying reactivation of the maternal allele, was observed in prostate cancers removed at radical surgery for localised adenocarcinoma as well as in samples of apparently normal adjacent tissue. In contrast, biallelic expression was absent from virtually all BPH specimens  of prostate cancer , histone methyltransferase (SUV39H), histone acetylases , and histone deacetylases (HDAC2).A key question is whether the different classes of epigenetic changes discussed above are responsible for driving cancer development, or whether some of the alterations are a secondary consequence of the development of this disease. If the former is true, defects in the machinery controlling DNA-methylation and chromatin structure should be linked to cancer. This is indeed the case, although current supporting evidence is entirely restricted to cancer types other than prostate. It is not our intention to carry out a systematic review of this area apart from noting that mutations linked to cancer development in humans or mice have been found in genes encoding DNA methyltransferases, methyl-CpG-binding proteins, chromatin remodelling proteins (TMRSS2–ERG gene fusion) in novel therapeutic and chemopreventive approaches.Small RNAs may have a key role in controlling the epigenetic state. It is well established that siRNAs can direct post-transcriptional silencing, but it is now becoming apparent that promoter-targeted siRNAs can also direct both the activation and repression of gene transcription through orchestrating epigenetic changes in a process that requires RNA polymerase II and Argonaute 1 and 2. The targeted promoter may be rendered transcriptionally silent, exhibiting the repressing marks H3K9me2 and H3K27me3 . Han et A popular view is that cancer stem cells or dividing precursor cells are the point of origin of individual cancers. In support of this hypothesis, cancers often exhibit alteration in pathways known to be involved in the preservation of stem cells. New evidence indicates that particular chromatin patterns controlling important regulatory genes in stem cells may leave these same genes vulnerable to DNA methylation during cancer development. SFRP5, and where the pattern of histone methylation had changed to resemble more closely that found in cancer cells.Direct support for the idea that genes held in this ‘bivalent’ state are susceptible to methylation was provided by analyses of individual clones of stem cells containing ectopically overexpressed BMI1 . Clones of stem cells were identified that exhibited increased methylation of the Wnt antagonist gene de novo methylation  and dihydrocoumarins (found in sweet clover) inhibit SirT1. Prostate cancer frequently progresses very slowly, and for many cases, treatments that delay the development or progression of this disease, even marginally, could have a significant clinical impact. If epigenetic alterations occur gradually through ageing or under the influence of dietary factors as the first step in cancer development, they may provide an excellent target for preventative strategies. In this respect, it is worthy of note that the potential chemopreventive agents sulphoraphane, butyrate, and diallyl disulphide are all known to act as inhibitors of HDACs. The antioxidant EGCG, present in green tea, is a DNMT inhibitor. Support for the idea that chemoprevention strategies can be successful comes from the study of patients with genetically determined hyperhomocysteinemia where treatment by folate supplementation can restore the normal DNA methylation and expression patterns of specific genes (Axin(Fu) allele, and epidemiological evidence (Epigenetic marks may not always be erased by passage through the germ line, and in some cases can be inherited from one generation to the next. The influence of maternal diet in this process is well documented. It is, however, now emerging that paternal transmission may also occur. This has been described in mice for the Whole genome analysis of histone modifications and of proteins involved in epigenetic modulation can be performed using ChiP-based approaches . For exaTMPRSS2:ERG fusion where HDAC1 is overexpressed (Enzymes controlling epigenetic status and involved in cross talk between epigenetic systems are potential targets for cancer therapy. The enzymes include HDACs, HATs, co-activators including TIP60, PcG proteins including EZH2, and the DNMTs. In principle, enzymes that modulate the activities of these proteins are also potential drug targets, as are proteins that bind methylated CpG sequences. One strategy would be to try to use inhibitors of these proteins to reprogramme cells returning their epigenetic status to that reminiscent of a normal cell. A complication with this targeting strategy is that the effects of each individual targeted protein are most likely to be complex and difficult to predict. Several HDAC inhibitors have activity in the nanomolar range and have been demonstrated to inhibit growth of prostate cancer. Clinical trials have, however, been largely restricted to cancer types other than prostate (Epigenetics is now accepted as mainstream area of cancer research. However, many challenges remain both in understanding its importance in cancer development and in applying new knowledge to the benefit of prostate cancer patients. There is an urgent need to comprehensively assess epigenetic alterations on a genomic scale in a broad variety of normal cells, stem cells, and in corresponding cancer cells and to assess the importance of the role of small RNAs and their therapeutic potential.


Noroviruses accounted for 12% of severe gastroenteritis cases among children <5 years of age. We conducted a systematic review of studies that used reverse transcription–PCR to diagnose norovirus (NoV) infections in patients with mild or moderate (outpatient) and severe  diarrhea. NoVs accounted for 12%  of severe gastroenteritis cases among children <5 years of age and 12% (95% CI 9%–15%) of mild and moderate diarrhea cases among persons of all ages. Of 19 studies among children <5 years of age, 7 were in developing countries where pooled prevalence of severe NoV disease (12%) was comparable to that for industrialized countries (12%). We estimate that each year NoVs cause 64,000 episodes of diarrhea requiring hospitalization and 900,000 clinic visits among children in industrialized countries, and up to 200,000 deaths of children <5 years of age in developing countries. Future efforts should focus on developing targeted strategies, possibly even vaccines, for preventing NoV disease and better documenting their impact among children living in developing countries, where >95% of the deaths from diarrhea occur. Despite improved safety of food, water, and sanitation and aggressive promotion of noninvasive interventions  and prevention strategies , diarrhea remains a common cause of illness worldwide. It accounts for ≈1.8 million annual deaths in children <5 years of age (Norovirus and Sapovirus) in the family Caliciviridae and are collectively referred to as human caliciviruses (Noroviruses (NoVs) and sapoviruses are genetically and antigenically diverse single-stranded RNA viruses that belong to 2 different genera  study duration was >1 year, and 2) study used RT-PCR to diagnose caliciviruses (NoV and sapovirus) or NoV in patients with diarrhea. We included studies that tested for caliciviruses, even when they did not differentiate between NoV and sapovirus. For these studies, we multiplied the proportion of caliciviruses detected in each study by the mean proportion of caliciviruses that were NoV among studies that differentiated between NoVs and sapoviruses to yield the estimated NoV prevalence in each study. We excluded studies that did not provide a denominator  or that only conducted molecular analysis using a fraction of the fecal samples  as NoV infection; however, we also present data on mixed infections, when available. Pooled proportions and 95% confidence intervals (CIs) of NoV-positive cases were calculated by using the random effects models . For the studies that included fecal testing on concurrent diarrhea-free controls, the pooled proportions were based on absolute difference in NoV detection rate between cases and controls, thus only including the fraction of cases attributable to NoVs. The Cochran Q statistic and degrees of freedom (df) are presented as a measure of heterogeneity among studies. Analyses were conducted with StatsDirect version 2.5.7 (StatsDirect Ltd).We stratified studies into 2 settings: community or clinic-based (mild or moderate diarrhea) and hospital-based including emergency department and inpatients (severe diarrhea). We counted cases in which NoV was detected in the presence of To calculate the number of outpatient NoV episodes and hospitalizations for children living in industrialized countries (where 23 of 31 studies in our review were conducted), we multiplied the total number of estimated diarrhea episodes in each clinical setting by the pooled proportion attributable to NoV based on the studies we reviewed to yield the number of NoV cases in each setting  . Five stTwenty-three studies  evaluated NoV disease among hospitalized diarrhea case-patients in whom the proportion of NoV disease ranged from 3% to 31%; pooled proportion was 11% (95% CI 8%–14%). Most (n = 19) of these studies of severe diarrhea cases focused on children <5 years of age, and the pooled proportion of NoV disease in these studies was 12% (95% CI 10%–15%) . In the Most (>95%) of the world’s diarrheal deaths occur in low-middle– and low-income countries. In our review, 7 of 19 studies assessing prevalence of severe NoV disease among children <5 years of age were conducted in these countries  of the controls . This finding, combined with the fact that most studies only assessed for NoV among samples that previously tested negative for other bacterial and viral pathogens, suggests that our overall pooled proportion attributable to NoVs is unlikely to be much lower. In addition, for the studies that enrolled diarrhea-free controls, we subtracted control prevalence from the case prevalence of NoV disease when calculating the overall pooled estimate. Lastly, other unmeasured factors underestimating disease prevalence that could not be accounted for, such as inefficient primers, low virus shedding, delays in specimen collection, and lack of a sensitive case-definition are also likely to exist in these studies.The heterogeneity in the NoV literature is evident and should be considered when interpreting the results of this review. Our systematic approach and strict inclusion criteria likely reduced heterogeneity but do not eliminate biases in the original studies, diversity in study design and population, and publication bias. Nonetheless, the findings of this review suggest that NoVs are a frequent cause of mild and severe sporadic gastroenteritis among children in high- and middle-income countries. In addition, hospital studies in our review only assessed patients admitted with diarrhea. Because of the highly infectious nature of NoVs, substantial additional health and economic effects would also occur from nosocomial disease and outbreaks in healthcare facilities, as previously identified by Lopman et al (The increasing evidence documenting the magnitude of the NoV disease prevalence provides support for considering targeted interventions, such as vaccines, for reducing the extent of this illness among young children. However, if one considers that NoV frequently causes both sporadic and epidemic gastroenteritis and can affect all age groups, some other potential targets for vaccination may include elderly persons in nursing homes, who are vulnerable to severe complications, and military recruits, in whom sporadic and epidemic NoV disease is known to incur substantial illness and financial costs from work disruption . The devCarefully designed epidemiologic studies that evaluate NoV prevalence in children with diarrhea and a suitable comparison group and that use sensitive molecular assays will help further define target groups that would benefit from vaccines and other interventions. A particularly pressing need exists for better quantifying the extent of severe norovirus disease among children in developing countries and identifying prevention strategies to help reduce the prevalence of deaths from diarrhea in the poorest countries.References Excluded from ReviewReferences Used in Review but Not Cited in Article


In this essay, Abigail Shearin and Elaine Ostrander discuss the proposed genomic mechanisms for the extraordinary level of phenotypic variation observed in the domestic dog and the evidence detailing the variants responsible for the many shapes, sizes, textures, and colors of man's best friend. As a result of domestication, selection for desirable phenotypes, and breed propagation, the domestic dog is unmatched in its diversity as a land mammal. Exhibiting extraordinary levels of both interbreed heterogeneity and intrabreed homogeneity, evidenced in part by the extensive linkage disequilibrium observed in many breeds, the dog provides an as-yet unrealized opportunity to uncover the molecular mechanisms that govern natural variation across mammalian species. We herein discuss recent advances in canine genomics that have made exploration of genetic mechanisms controlling breed-specific differences possible. We consider some examples where molecular mechanisms controlling simple traits have been uncovered. Finally, we reveal how combinations of genes produce complex phenotypes that can be revealed through studies of dog breeds featuring specific traits.As Darwin himself noted, the domestic dog displays a remarkable level of phenotypic diversity Canis familiaris, and possess a 2.8 Gb genome featuring 38 autosomes and the sex chromosomes, similar in size to the 3 Gb human genome. Dogs of any breed can, for the most part, be crossed to produce fertile offspring. Breeds were developed largely during the Victorian era, with special selection for both morphologic traits based on size, proportion, coat, etc., as well as behavior. To be a registered member of a breed, both of a dog's parents have to be registered members of the same breed, and their parents in turn must be registered members of the breed. Thus, each breed is effectively a closed breeding population that offers many statistical advantages for doing genetics beyond what can be done in studies of human populations There are over 300 dog breeds identified worldwide, with nearly 170 recognized in the United States by the American Kennel Club (AKC) In this essay we consider some of the features of the canine genome relevant for successful studies of selected traits. We discuss current hypotheses regarding the development and maintenance of genetic variation in dogs today. We consider examples in which identified genes account for unique, and sometimes complex, phenotypes. Finally, we consider the implications of these findings for studies of true complex traits, such as those associated with behavioral genetics.−4, which is essentially the same high level of nucleotide diversity reported in the human population. As expected, however, the level of genetic diversity within any single breed is considerably less than the species as a whole The canine genome was sequenced to both 2× The loss of diversity reported by Gray et al. The extensive LD that characterizes dogs means that genome-wide association studies (GWAS) can be done in the dog with as few as 20,000–30,000 single nucleotide polymorphisms (SNPs), compared to the million needed for the more outbred human population To aid in the selection of breeds for any given study we recently did a cluster analysis of 132 dog breeds and showed that breeds divide into five major groups: Asian and ancient dogs; hunting and gun; mastiff and terrier; herding and sight hound; and a mountain group The closest relative to the domestic dog is undeniably the gray wolf, from which the dog differs by only 0.04% in nuclear coding-DNA sequence Alx-4 gene, which is postulated to be responsible for their characteristic rear digit polydactyly Alx-4 allele, while one Great Pyrenees who lacked rear digit polydactyly did not carry the variant allele The degree to which new mutations have played a role in the development of the modern dog still requires intense scrutiny, but three major sources of genomic variation have been proposed as contributors to the high levels of phenotypic variation observed in today's domestic dogs. The first is variability associated with microsatellites or simple sequence repeats (SSRs). Fondon and Gardner hypothesized that repeat length polymorphisms, particularly those occurring in regulatory regions, were an important source of morphologic variation, in part because they occur at a mutation rate 100,000× greater than SNPs Elaborating on this theme, the same investigators found that members of the Canidae family possessed elevated genome-wide basal slippage rates, the rate at which DNA replication machinery creates new alleles due to errors in replicating repeat elements, compared to humans, non-human primates, and other members of the Carnivora order  Another mechanism that clearly accounts for a subset of diversity between breeds is carnivore-specific short-interspersed nuclear elements (SINEs) fibroblast growth factor-4 (fgf4) retrogene, a gene copied by reverse transcriptase from processed mRNA and inserted into the genome, which we demonstrated is associated with chondrodysplastic breeds displaying disproportionately short limbs FGF4 but none of the introns or regulatory machinery. Expression studies revealed that the adjacent genes were expressed in neonatal chondrocytes, as was the retrogene. However, the retrogene was not expressed in the cartilage of mature dogs. Although expressed retrogenes are common in insects, this is the first example we are aware of in which alleles of a retrogene segregate in a mammalian species such that they are a major source of morphological variation Other possible mechanisms of variation in the dog are common to many species and include mutational hotspots, chromosomal fission, and gene duplications. The latter are particularly interesting. For instance, duplication of a 133-Kb region spanning three fibroblast growth factor genes was shown to be associated with the appearance of a characteristic ridge on the back of the Rhodesian ridgeback breed The first large-scale genetic studies of canine skeletal morphology were done by Chase et al., in the Portuguese Water Dog (PWD) insulin like growth factor-1 (IGF-1) gene IGF-1 haplotype was shared among all small dogs, suggesting that a single ancestral mutation had been selected for in the development of all small dog breeds studied. Two distinct IGF-1 haplotypes segregated in large dogs, suggesting a more complicated scenario for enlarging breed size We showed that the primary signal for body size (PC1) was a four million base pair (bp) locus spanning several genes on CFA15 cis-acting mechanisms, although the critical mutation remains to be found.Additional skeletal traits studied include PC2, which defines leg length versus width in the original study of Chase et al., R-spondin-2 (RSPO2) gene is strongly associated with wire hair and “furnishings”, the latter being the moustache and eyebrows characteristically seen, for instance, in the schnauzer fibroblast growth factor-5 (FGF5) gene. Curly versus straight fur is associated with a coding SNP within the keratin71 (KRT71) gene Some of the most exciting progress in understanding the genetics of variation in dogs relates to the complex traits of coat texture and color. We recently showed that variation in canine pelage, including pattern, length, curl, and texture (smooth versus wire), are controlled by combinations of alleles at only three genes RSPO2, FGF5, and KRT71 and thus possesses long, curly hair with furnishings  pathway. Variants result from mutations in the microphthalmia-associated transcription factor (MITF), which is crucial for melanocyte migration Several dog breeds exhibit complete or partial absence of pigmentation. For instance, Karlsson et al. mapped a locus for white-spotting to a 102-kb haplotype on CFA 20 in a region that spans a single gene; The identification of genetic variants controlling morphology in the dog population has reached an exciting juncture. The current set of available molecular tools allows us to finally address the critical questions. For instance, the striking morphological variation observed between breeds of dogs provides us with unique opportunities to study the genetic basis of both evolution and domestication. A deeper understanding of the genomics and variation in wild canids would enhance our ability to pursue these questions.Several hypotheses have been proposed as to why the dog, as opposed to any other domestic land mammal or any other domesticated creature, displays such extremes of morphologic variation (Box 1). Each theory has its champions, and most likely a combination of mechanisms contribute with the strong artificial selection imposed by man being the most important. The question remains: if under the same intense artificial selection for novel morphological traits, would other domestic creatures exhibit equivalent variation? As scientists continue hunting for the genes and tracking the mutations that control morphologic variation in the domestic dog, we expect still more secrets will be revealed regarding the genetic basis of man's extraordinary best friend.Microsatellites or simple sequence repeatsHigh levels of repeat purityThe abundance and location of SINEC_Cf elements in the canine genomeCommon sources of variation: mutational hotspots, chromosomal fissions, and gene duplicationsIntense artificial selectionRapid perpetuation of new mutations


Streptococcus pyogenes become hypervirulent in mice due to selection of mutants in the covRS regulatory genes, we set out to dissect the impact of these mutations in vitro and in vivo from the impact of other adaptive events. Using a murine subcutaneous chamber model to sample the bacteria prior to selection or expansion of mutants, we compared gene expression dynamics of wild type (WT) and previously isolated animal-passaged (AP) covS mutant bacteria both in vitro and in vivo, and we found extensive transcriptional alterations of pathoadaptive and metabolic gene sets associated with invasion, immune evasion, tissue-dissemination, and metabolic reprogramming. In contrast to the virulence-associated differences between WT and AP bacteria, Phenotype Microarray analysis showed minor in vitro phenotypic differences between the two isogenic variants. Additionally, our results reflect that WT bacteria's rapid host-adaptive transcriptional reprogramming was not sufficient for their survival, and they were outnumbered by hypervirulent covS mutants with SpeB−/Sdahigh phenotype, which survived up to 14 days in mice chambers. Our findings demonstrate the engagement of unique regulatory modules in niche adaptation, implicate a critical role for bacterial genetic heterogeneity that surpasses transcriptional in vivo adaptation, and portray the dynamics underlying the selection of hypervirulent covS mutants over their parental WT cells.The onset of infection and the switch from primary to secondary niches are dramatic environmental changes that not only alter bacterial transcriptional programs, but also perturb their sociomicrobiology, often driving minor subpopulations with mutant phenotypes to prevail in specific niches. Having previously reported that M1T1  Group A streptococci (GAS) are human pathogens that infect over 700 million children and adults each year hi/SpeA−/Sda1low, irreversibly switch to the hypervirulent SpeB−/SpeA+/Sda1high phenotype after ≥3 days in vivo covRS genetic locus hasABC), the streptolysin S operon (SLS or sagA-H), and the streptokinase gene (ska) We previously reported that virulent representatives of M1T1 GAS, with the phenotype SpeBcovS are thus expected to affect CovR phosphorylation status differentially in vitro and in vivo (under stress conditions), and to consequently modulate CovRS signaling-regulation circuits in a complex manner that remains largely unexplored. This complexity is further magnified by the reported strain-specific differences in the impact of CovS on pathogenesis covS mutations, albeit clustered in its histidine kinase domain, might have different effects on expression of CovR-regulated genes covS mutations generate hypervirulent isolates associated with invasive forms of streptococcal infection Mutations in covS mutations and the emergence of the hypervirulent phenotype of M1T1 strains, it is unclear whether this increased virulence can be entirely attributed to the modulation of the CovR regulon or if other networks are also perturbed in vivo directly, indirectly, or independently of the CovRS system. Additionally, the effects of covS mutations on bacterial niche adaptability are still undetermined because the in vivo transcriptomes of the wild type (WT) and animal-passaged (AP) bacteria have not been compared under the same experimental conditions.Despite the association between S. pyogenes niche adaptation and pathogenesis. In addition, this approach offered a rare transcriptional snapshot of the SpeBhi/SpeA−/Sda1low population prior to its extinction in vivo.To address these issues and improve our understanding of the gene regulatory impact of mutational and adaptive events contributing to the hypervirulent phenotype of M1T1 strains, we analyzed differences in growth requirements, transcriptome profiles, and regulatory circuits of the virulent (WT) and hypervirulent (AP) phenotypes of the M1T1 strain both in vitro and during initial in vivo infection. Such comprehensive analyses highlighted the behavior of genomic subsystems that may be involved directly or indirectly in ® Phenotype Microarrays (PM) to screen 1900 different growth conditions, including a large set of different carbon and nitrogen sources, pH values and salt concentrations, as well as different concentrations of various antimicrobial agents . Minimal media are not optimal for the expression of GAS proteins, which may explain why GAS failed to grow at many of the PM conditions /ml vs. ∼2×109 CFU/ml inoculum. We extracted RNA from these bacteria immediately after their recovery from mouse chambers with no additional culturing and used the extracted RNA for transcriptome profiling as detailed in In this study, we designed and performed in vivo passage experiments in which equal loads of in vitro-grown WT and AP bacteria were separately inoculated into murine subcutaneous chambers To gain biologically relevant knowledge from the transcriptome studies without compromising statistical significance, we interrogated the data using multiple strategies that had been developed for microarray analysis and visualization, taking into consideration the strengths and limitations of each strategy.coexpression clusters, CCs , By clustering normalized expression values from different biological replicates in all data sets, we generated an overall “pathovivogram” that includes 276 genes in ten ers, CCs . This paers, CCs , CC1-CC3The patterns in clusters CC1-CC3 describe those gene sets whose transcription was turned on (CC1-CC2) or off (CC3) to drive the bacterial adaptation to the host environment, but are not primarily related to bacterial pathogenesis. Many of these genes encode metabolic enzymes , ribosomal proteins (mostly downregulated), and sugar or peptide transport systems, reflecting the transition from a carbohydrate-rich laboratory medium to a protein-rich, oxygen-poor subcutaneous tissue. Such transition is bound to have major downstream effects on the bacterial virulence gene expression While the expression patterns in clusters CC4, CC5, CC8, and CC9 match the previously described in vitro differences between WT and AP strains P value <0.05, and moving to thresholds that are more conservative. At all significance cutoffs, the fewest observed transcriptional changes were those differentiating WT and AP bacteria in vitro compared to other pairs of conditions. By contrast, the most dramatic transcriptional reprogramming was that exhibited by the WT bacteria in their attempt to adapt to the in vivo environment . Alternatively, the mutation in  strains ; however strains  suggestiOne last factor that adds to the complexity of data integration analysis and that may explain unexpected transcriptional patterns is that regulators are often controlled by sensitive feedback mechanisms. Inactivation or downregulation of one regulator may eventually perturb the entire system, and several other regulators are likely to become engaged in compensatory mechanisms to maintain cellular homeostasis.covS gene in AP bacteria It is now well established that bacterial populations often consist of heterogeneous communities rather than genetically identical cells with synchronized gene expression profiles In vitro, the growth of these two variants of the M1T1 bacteria was comparable and, although 137 (5.9%) of their genes were differentially expressed , in vitrsic, emm, scp); hyaluronic acid capsule-encoding genes (hasABC); and genes encoding the toxins SpeA, SclA, SLO, and the streptodornase Sda1 , is also suggestive of immune evasion. Among the upregulated genes are dipepetide and sucrose-specific transporters, whose upregulation supports the hypothesis that the bacteria are switching diets Several phage genes are split between the upregulated and downregulated gene clusters; this may indicate a stress-dependent reprogramming of prophage induction and gene expression that needs to be explored in future studies.covS mutation in AP bacteria are only manifested in vivo. For example, several gene sets  were upregulated in WT bacteria in vivo, but were mostly silenced in AP bacteria in vivo , we ran qPCR to validate the microarray findings, focusing on virulence genes that are biomarkers of the bacterial switch to a hypervirulent phenotype. These include SpeA, SpeB, Sda1, M protein, and SIC Importantly, we did not base our analysis on single measurements of gene expression, nor did we focus on individual genes; rather, we used gene expression data from biological replicates to rule out biological variability  and to look for changes in gene clusters, operons, and pathways. In doing so, we were able to assess trends across experiments rather than absolute numerical values that could vary due to technical rather than biological factors. We believe that this approach provided more confidence in the final assessment of which genes/pathways were expressed similarly or differentially in both WT and AP bacteria in vitro and in vivo. It also allowed us to dissect changes related to niche adaptation from those reflecting the selection of the fittest members of the bacterial community when faced with different environments and conditions.The use of multiple microarray analysis strategies and the integration of their results strengthened this study since each strategy has advantages and disadvantages. For example, coexpression clustering—one of the earliest developed microarray analysis tools Besides these two clustering methods, expression ratios provided pairwise comparison, thereby allowing the quantification of the impact of each individual change of condition on overall gene expression  as well Having used multiple strategies and integrated the PM data with the in vitro and in vivo microarray data, we propose the following hypotheses about streptococcal niche adaptation and switch to hypervirulence:covS mutation ((1) Exposure to the in vivo environment perturbs a substantial number of GAS genes and regulatory networks. This has been demonstrated by the larger number of genes affected during in vivo adaptation of both M1T1 variants compared to those affected by the mutation . Howevermutation ; yet, thcovS mutation in AP bacteria seems to be preferentially modulating virulence mechanisms, as about 25% of the known genes perturbed by the mutation belong to virulence subsystems ((2) The bsystems  and manybsystems . In addibsystems  are morebsystems . This fiIt is noteworthy that SpeB expression has a dominant effect, since even if secreted in low amounts by a minor subpopulation, this broad-spectrum protease would still be able to degrade, fully or partially, many GAS virulence proteins, including Sda1, thereby rendering the bacteria vulnerable to different effector mechanisms of the host's innate immune system including neutrophil killing (3) The GAS genome has at least 13 two-component regulatory systems covS mutation goes beyond the defined CovR regulon. This finding is in accordance with previous observations that CovR and CovS are not committed to each other, as CovR could be phosphorylated in the absence of a functional CovS covS mutations, albeit clustered in the histidine kinase domain, might have different transcriptional effects (4) We also show that the impact of the covS sensor kinase. We believe that our results offer a proof of principle that in vivo-extracted RNA can provide transcriptional profiles that better reflect the complexity of heterogeneous bacterial communities, and, as shown in this study, can provide a transcriptional snapshot of a bacterial population right before its extinction. The use of in vivo-driven RNA in understanding virulence has been appreciated in streptococcal research In conclusion, we have established a model that allowed us to resolve two sets of complex transcriptional events: (i) those occurring in response to the host environment and (ii) those caused by a mutation in This study is a first step towards exploring the sociomicrobiology of invasive GAS in vivo. Having captured snapshots of different transcriptional programs within the same bacterial community, we plan to follow with single cell studies All animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the institutional animal care and use committees at the University of Cincinnati, OH, USA and the VA Medical Center, Memphis, TN and Cincinnati, OH, USA.covS mutant We used the clinical isolate, GAS 5448 both runs are reported as significant.Both bacterial strains GAS 5448 WT and 5448 AP were analyzed by the Biolog® Phenotype Microarray (PM) technology. The analysis and data processing were performed by the Biolog® team . Duplicate arrays were run, and the average of the two runs was calculated. Only results that were significantly different in Oligomers (70-mers) of the M1-based microarrays were obtained from Dr. Kevin McIver and Dr. June Scott, and printed in the Molecular Resource Center, UTHSC by the use of MicroGrid II . Each array consists of 2,346 oligomers that represent all open reading frames (ORFs) in M1 GAS strain SF370  and used a FastPrep instrument (QBiogene) to shear the bacterial walls. Then, we extracted total RNA from the sheared bacteria using RNeasy kits , treated it with DNase Turbo  for 1 h to remove contaminating genomic DNA, and further purified the samples using RNeasy columns (Qiagen). Sometimes we used RNeasy MinElute columns to concentrate RNA when the yield was low, typically in case of in vivo-recovered RNA. We confirmed the absence of genomic DNA in the samples by running 40-cycle PCR reactions using GAS For in vivo experiments, we used the subcutaneous murine Teflon chamber model developed in our laboratory and described earlier 8 CFU/mouse (100 ul of a 2×109 CFU/ml culture). After 24 h, we used the bulk of the recovered bacteria for immediate RNA extraction (as detailed above) with no additional culturing, and kept a small aliquot intact to verify retention of SpeB phenotype using protease screens on casein-agar plates. RNA from bacteria homogeneously expressing or not expressing proteolytic activity was used for transcriptome profiling while RNA from the few populations that exhibited mixed SpeB+ and SpeB− phenotypes was excluded.To recover enough RNA for downstream experiments, we inoculated the mouse chambers with 2×10http://www.genisphere.com/array_detection_900.html), which use the dendrimer technology to amplify the fluorescent signal of cDNA. Following the manufacturer's protocol, we tagged each sample with the proprietary capture reagent, mixed equal amounts from both tagged samples , and used the mixed samples to hybridize with the probes on the microarray slides overnight. After the first hybridization, we washed the slides, incubated them with equal amounts of the fluorescent dyes  for 3 h, washed them again, and immediately scanned them. Alternatively, we used DyeSaver (Genisphere) to coat the array slides and protect the dyes from fading when immediate scanning was not possible.To convert the bacterial RNA to labeled cDNA ready for hybridization, we used the 3DNA Array 900TM kits  http://seed-viewer.theseed.org) to allow the visualization of array results. Additional clustering, statistical analysis, and generation of gene lists and Venn diagrams were performed by multiple tools in the GeneSpring suite (Agilent Technologies). All genome annotations and subsystems data used in this study were obtained from the NMPDR database http://host-pathogen.net/publications/Aziz_2010_Arrays/microarrays).We scanned the arrays using GenePix 4000B scanner  and we performed the primary analysis using the GenePixPro 4.0 software (Axon Instruments/Molecular Devices) The primary analysis included spot finding, alignment and adjustment, fluorescence normalization, flagging out poorly hybridized spot, and background subtraction. We performed subsequent analyses using multiple tools, including Microsoft Excel, GeneSpring , as well as custom-written Perl scripts that are integrated in the NMPDR  analysis chart generated from two sets of 20 plates that were run twice for each the wild type (WT) and animal-passaged (AP) bacteria over a 48 h time period. Each well of each plate represents a different condition . The red curves represent AP and the green ones represent WT growth curves. Yellow color is the result of superimposition of red and green areas.(6.26 MB TIF)Click here for additional data file.Figure S2A higher resolution version of (25.80 MB TIF)Click here for additional data file.Figure S3Microarray results summary statistics.(3.60 MB TIF)Click here for additional data file.Figure S4A higher resolution version of (0.70 MB PNG)Click here for additional data file.Table S1All array data used to generate the pathovivogram  and S2.(0.30 MB XLS)Click here for additional data file.Table S2Gene lists reflecting significant (P<0.05) differential expression ratios between each pair of conditions.(0.28 MB XLS)Click here for additional data file.