To optimize appropriate antimicrobial use in a university hospital and identify barriers hampering implementation strategies, physicians were interviewed regarding their opinions on antimicrobial policies. Results indicated that effective strategies should include regular updates of guidelines that incorporate the views of relevant departments and focus on addressing senior staff and residents because residents do not make independent decisions in a teaching-hospital setting. In an era of increasing bacterial resistance and the availability of a plethora of antimicrobial agents, hospitals have developed policies to promote prudent antimicrobial prescribing  the guideline, 2) physicians’ characteristics, and 3) characteristics of the institution. Interviewing and analysis were partly simultaneous, which is consistent with the grounded theory approach . All physicians, except for one supervisor, began the patient’s treatment with broad-spectrum antimicrobial agents. Residents were hesitant to streamline initial therapy, fearing that such changed therapy might be clinically less effective. Infectious disease consultants and supervisors streamlined therapy based on gram-stain results only.Our findings support earlier study findings that an intensive implementation strategy is needed for physicians to make their prescribing practices consistent with guideline recommendations. Residents are more open to using the guidelines; they are willing to adopt the recommendations because it helps them in their learning process, making them ideal candidates for interventions. For them, the barrier to be addressed is whether streamlining is safe. One way of affirming this is facilitating a better understanding of culture and sensitivity tests, for example, through infectious disease consultants’ support (In an institutional context where residents are not independent decision makers, any implementation plan should combine strategies aimed at both residents and supervisors. For residents who change departments regularly, a generally adopted hospitalwide guideline facilitates a consistent learning environment and increase their rational decision making. Addressing the role model function of supervisors for residents may be one more way to motivate them to use the guideline, in view of the impact that supervisors have on residents (The limited number of physicians interviewed in this study is in line with a qualitative research approach aimed at generating hypotheses (In conclusion, intervention strategies should focus on improving dissemination and credibility of the recommendations, focusing on both supervisors and residents, although each group needs a tailored approach. Active outreach, as in face-to-face educational visits, may be the best approach to tackling the various barriers in one intervention program aimed at optimizing antimicrobial use.


It is widely accepted that systemic neoplastic spread is a late event in tumour progression. However, sometimes, rapidly invasive cancers are diagnosed because of appearance of metastatic lesions in absence of a clearly detectable primary mass. This kind of disease is referred to as cancer of unknown primary (CUP) origin and accounts for 3-5% of all cancer diagnosis. There is poor consensus on the extent of diagnostic and pathologic evaluations required for these enigmatic cases which still lack effective treatment. Although technology to predict the primary tumour site of origin is improving rapidly, the key issue is concerning the biology which drives early occult metastatic spreading. This review provides the state of the art about clinical and therapeutic management of this malignant syndrome; main interest is addressed to the most recent improvements in CUP molecular biology and pathology, which will lead to successful tailored therapeutic options. Cancer of unknown primary (CUP) origin defines metastatic tumour detected when the site of primary origin cannot be identified based on clinical history, complete physical examination, routine laboratory tests, imaging and radio-metabolic techniques and careful review of histological specimens. Although this malignant syndrome accounts for 3-5% of all cancer diagnosis, the majority of patients still lacks effective therapeutic regimens  CUP clinThere is poor consensus on the extent of diagnostic evaluations in front of metastatic cancers without a primary mass. At the present immunohistochemistry (IHC) is often the only standard method by which a putative primary origin can be postulated. Nevertheless, recent improvements in molecular diagnostics will allow a most appropriate pathological classification of early metastases. However the most relevant approach to CUP is related to the understanding of the biology which drives precocious metastatic spreading.This review summarizes the current knowledge on pathological features, diagnostic tools and biological behaviour of CUPs, mainly focusing on the next future therapeutic implications.Almost one third of advanced tumours presents with metastases at time of diagnosis. In the majority of cases the organ site of primary lesion becomes shortly evident after clinical, pathological and radio-metabolic evaluations. The other cases can be roughly defined as metastases of unknown primary origin. Indeed this definition includes two groups of tumours: 1) the cases in which the primary site might be postulated at least by their ICH profile; 2) the metastatic lesions which remain really 'orphan' even after an exhaustive IHC screening. Although the separation into two groups based on the IHC profile could be somehow artificial, it may be important for patient management and the choice of initial therapeutic regimen.It is generally reported that the incidence of CUPs is about 3-5% of all cancer diagnosis . The annhttp://www.nccn.org,http://www.esmo.org), all the patients who present with a metastatic cancer suspected to be of unknown primary should have an accurate physical examination, complete laboratory tests and a whole body imaging study (Computed Tomography - CT - scan and Positron Emission Tomography - PET). Besides, female patients have to undergo mammography and vaginal ultrasound (US) scan, whereas prostate US scan is required in males. Various endoscopic procedures  should be ordered in selective cases, based on several clinical factors such as patients' relevant symptoms and/or signs, physical examination findings, sites of metastases, occult blood in the stool, laboratory findings as well as any other factor which would prompt endoscopies. More than 50% of CUP patients present with multiple sites of involvement while the rest have a single site, most commonly liver, bone, lung or lymph nodes , Rosetta Genomics and Prometheus (RT-PCR for microRNA), and bioTheranostics (RT-PCR for mRNA). All tests claim prediction accuracies in known primary cancers between 80% and 90% . HoweverSurgical management of metastases of unknown primary is generally related to limited/single disease. Nevertheless, a recent report demonstrated for the first time that surgery in case of multiple sites of spinal disease did not influence survival; whereas the presence of extraspinal disease had a negative impact . HoweverThe continuing development of new therapies targeted to the various cancer types makes mandatory the identification of the primary tumor and--as discussed above--many efforts are now directed to integrate molecular based medicine and clinical practice to assign a primary as soon as possible after diagnosis of metastases of occult primary origin. However in case of CUP diagnosis, therapy regimes lack to be specific and, in the vast majority of cases, chemotherapy regimens include platinum, an alkylating agent which is effectively used for the management of patients with the most common solid tumors. In those settings, treatment may only help to control metastatic disease for a time and to improve patient symptoms. Overall clinical prognostic markers include: patient's age, gender, weight loss and performance status; histopatology and tumor burden, location and number of metastatic sites; as well as serum biomarkers . In addiFor adequate therapeutic guidance CUP entities should be categorized into favorable or unfavorable subsets . In the Carboplatin plus paclitaxel combination chemotherapy has been reported to be effective in patients with predominantly nodal/pleural metastases of unknown primary carcinoma and in women with peritoneal carcinomatosis but showed lower benefits in patients affected by liver, bone or multi-metastatic disease . SimilarRadiotherapy is a conventional therapeutic approach in case of cervical lymphnodes metastases of unknown origin, not only for unresectable disease but for a comprehensive treatment of the whole neck. Indeed neck dissection is not always necessary; for instance, it is not necessary for N1 disease . IntensiMetastatic spread is generally considered the final step of tumor progression and it clinically sets up the worse level of the cancer staging system. According to this model, the early cancer diagnosis is the one able to detect a single localized tumor mass which is still susceptible of surgical exeresis. However, as discussed above, in 3-5% of all human cancers distant dissemination arises at an early stage of tumor progression, so that unexpected metastatic phenotype can overcome on the primary lesion's growth. In some of those cases the putative primary can be detected by routine IHC stains or at least through gene expression profiling. On the contrary, in some instances morphology, imaging and molecular predictive assays are 'non-contributory': only these samples could be correctly defined as CUPs.Thus, there are at least two different hypotheses which can be involved in CUP biology: i) the first suggests that CUPs are heterogeneous group of site-specific tumors which share the properties of the small primary from which derive; ii) the second regards to CUP as to a distinct entity with a specific genetic asset. It is clearly evident that the biological and genetic enigma that could be related to CUPs is enclosed in the molecular mechanisms that speed up distant metastatization so that to confer to the primary lesion a status comparable to dormancy. In other words, it is conceivable that CUPs are a distinct biological entity involving specific genetic and phenotypic alterations, although at the present there are no known and validate molecular features to clearly distinguish these cancers. This observation is further supported by a recent epidemiological analysis performed on a CUP cohort from the Swedish Family Cancer Database. This study showed that CUPs cluster with families of kidney, lung, and colorectal cancers and suggested a marked genetic basis in the onset of the syndrome .A wide variety of chromosomal abnormalities have been described in CUPs: aberration of chromosome 1, 6, 7 and 11 are the most frequently reported . AneuploWith respect to the oncogenic molecular asset of CUP, several studies have evaluated the expression and the mutational status of both oncogenes and tumor suppressor genes. Unexpectedly--although few studies are available--lesions of the key players know to drive the vast majority of human cancer cannot be documented in CUPs. Expression of c-myc, KRAS, HER2 has been studied by IHC in a series of 26 CUP cases and was present in less than one third of samples . NeverthInterestingly immunohistochemistry studies demonstrated that EGFR is frequently expressed in CUPs, whereas c-KIT and HER2/neu are infrequently activated. Notably no significant association has been established between EGFR expression level and patients prognosis .http://www.sanger.ac.uk/cosmic) in CUP whereas the intron 1 cytosine-adenosine (CA) repeat has been reported [The EGFR oncogene seems to be infrequently mutated .Metastasis follows the inappropriate activation of a genetic program termed 'invasive growth' , which is a physiological process that occurs during embryonic development and post-natal organ regeneration . BurgeonSeveral strategies to block the activation of MET are under development, such as the use of tyrosine kinase inhibitors or monoclonal antibodies and some of these compounds have already been used in clinical trials .vs. the 1-3% of the general cancer population), in the absence of high mutational background. Nucleotide changes found clustered either in the kinase domain or in the extracellular semaphorin domain. Mutated receptors were functional and sustained the transformed phenotype, suggesting that MET activating mutations are genetic markers associated with the CUP syndrome. Therefore, MET mutation may as well reflect either differentiation grade and/or organ of origin. In this respect a preferential expression of MET in cancer stem cells has been postulated [We have recently demonstrated , by a scstulated .Metastases are defined as macroscopic lesions which develop far, through blood or lymphatic vessels from the primary tumor: they are generally resistant to therapy and eventually lead patients to death. The molecular and cellular mechanisms which drive the metastatic spread are the topic of constant debate and scientific research due to the potential implications for cancer patients' prognosis. The process of metastases might begin before the growth of the primary mass. Recent evidence suggests that tumor cells might start conditioning for distant tissues colonization through the establishment of a so called 'pro-metastatic' niche . Early mThis ideal of personalized oncology has not been reached for most patients and the clinical reality at this time is that at least some CUP patients may be treated with more effective by recognizing their tissue of origin. Patients with advanced cancer whether the primary site is known or not, may eventually be treated based upon specific genomic abnormalities that are discovered in their cancer cells. This is already occurring in some instances. However, knowing the tissue of origin remains an immediate important factor in determining therapy for most patients with advanced cancer.Nevertheless, further studies should be addressed to deeper investigate the molecular profile of CUPs. Preliminary results suggest a role for MET somatic mutations as driving force in CUP syndrome and put MET among the most promising therapeutic targets. Main address should be also focused on the potential role of the cancer stem cells compartment. Data should be collected to characterize the role played by undifferentiated cancer cells in tumor dissemination and their interaction with surrounding stroma to define molecular markers able to detect cancers displaying occult disseminative potential. This approach will lead to the creation of a new sensitive platform that might select an otherwise heterogeneous group of patients, such as that of patients presenting with metastatic cancers of unknown primary site of origin.Fluorodeoxy-glucose; ToO: Tissue of origin; RT-PCR: Reverse transcriptase polymerase chain reaction; RNA: Ribonucleic acid; FFPE: Formalin-fixed paraffin-embedded; CK: Cytokeratin; TTF: Thyroid transcription factor; ER: Estrogen receptor; CA125: Cancer antigen 125; VIM: Vimentin; PSA: Prostate specific antigen; CCP-CUP: Colon cancer profile--cancer of unknown primary origin; IMRT: Intensity modulated radiation therapy; K-RAS: v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog; EGFR: Epidermal growth factor receptor; HIF: Hypoxia inducible factor; COX: Cyclooxygenase; GluT: Glucose transporterCUP: Cancer of unknown primary origin; IHC: Immunohistochemistry; CT: Computed tomography; MRI: Magnetic resonance imaging; PET: Positron emission tomography; FDG: The authors declare that they have no competing interests.GMS, RS and PC were involved in the conception of the manuscript; AC and MR contribute to collect and analyze bibliographic references; GMS, RS and PC structured and wrote the manuscript. All the authors approved the final version of the manuscript.


New applications of evolutionary biology are transforming our understanding of cancer. The articles in this special issue provide many specific examples, such as microorganisms inducing cancers, the significance of within-tumor heterogeneity, and the possibility that lower dose chemotherapy may sometimes promote longer survival. Underlying these specific advances is a large-scale transformation, as cancer research incorporates evolutionary methods into its toolkit, and asks new evolutionary questions about why we are vulnerable to cancer. Evolution explains why cancer exists at all, how neoplasms grow, why cancer is remarkably rare, and why it occurs despite powerful cancer suppression mechanisms. Cancer exists because of somatic selection; mutations in somatic cells result in some dividing faster than others, in some cases generating neoplasms. Neoplasms grow, or do not, in complex cellular ecosystems. Cancer is relatively rare because of natural selection; our genomes were derived disproportionally from individuals with effective mechanisms for suppressing cancer. Cancer occurs nonetheless for the same six evolutionary reasons that explain why we remain vulnerable to other diseases. These four principles—cancers evolve by somatic selection, neoplasms grow in complex ecosystems, natural selection has shaped powerful cancer defenses, and the limitations of those defenses have evolutionary explanations—provide a foundation for understanding, preventing, and treating cancer. Our understanding of cancer is in the midst of a major transition. Extraordinary recent progress in genetics and cell biology is revealing details about cancer that undermine prior conceptions, and highlight the value of an evolutionary perspective. The naïve notion that cancer is one entity, with one cause, for which we can find a single cure, is fading as the complex and dynamic nature of cancer is becoming better understood Gatenby . The emeThe utility of evolutionary medicine Co-evolution with fast-evolving pathogens Constraints on what selection can do Trade-offs Reproductive success (RS) at the expense of health Defenses with costs as well as benefits .These four principles—somatic evolution of neoplasms, ecological analysis of tumor environments, selection for mechanisms that suppress cancer, and evolutionary explanations for their limits—provide an evolutionary foundation for understanding, preventing, and treating cancer. Research making use of these principles is well underway, but still at an early stage. The sections below illustrate the opportunities and some directions forward.Cancer is far from a single, well-defined disease. It is highly diverse, in ways more subtle than the obvious differences between cancers originating from different organs or tissues. For example, subtypes of breast cancer each have different risk factors, different phenotypic and genotypic characteristics, different effective treatment regimes, and different recurrence risks  leads to longer survival than the traditional high dose chemotherapy in a study of mice injected with ovarian cancer cells   might slow development of therapeutic resistance to chemotherapeutic agents Pepper . AlthougResource availability for a cell is influenced by both its own characteristics and by interactions with neighboring cells. For example, cancer cells that coordinate and regulate angiogenic signaling (for blood vessel growth) may induce greater blood flow to the tumor. Cells that do not coordinate angiogenic signaling may induce leaky vessels that temporarily increase blood flow , suggests close attention to processes such as habitat destruction Kareva , competiEcological theory offers tools and perspectives that help make sense of the complexities of cancer. Closer attention to the environment in which cancer evolves may suggest ways to slow cancer progression, enhance the effectiveness of treatment, or otherwise prolong life. These approaches include limiting the availability of resources for cancer cells, altering the social signals and context of neoplastic cells, and shaping interactions with microbes in ways that limit the fitness of neoplastic cells. They are parallel to approaches for understanding and limiting the abilities of pathogens to establish niches that allow them preferential access to resources (such as oxygen and glucose) and protection from threats (such as circulating immune cells). In the gastrointestinal tract “especially” perspectives from ecology, evolution, and microbiology intersect in ways with profound implications for understanding, preventing, and treating cancer.The story of cancer begins about one billion years ago at the dawn of multicellularity. Before the transition to metazoans, natural selection shaped one-celled organisms for whatever traits maximized their representation in future generations, especially maximal proliferation, invasion of adjacent spaces, and transmission to open niches. The transformation from unicellular to multicellular life was possible only when cells that cooperated by inhibiting their replication gained a selective advantage over those that went it alone  rates of cancer than small, shorter lived animals , one might expect that fast and effective wound healing to be a relatively stronger selective pressure than cancer suppression. Within a species, individuals with faster wound healing may be more vulnerable to cancer. Even within individuals, exposure to injuries or physical threats could conceivably shift physiological systems toward faster wound healing despite the increased risk of cancer. This hypothesis predicts that variations in the prevalence of injury among species  may be associated with faster wound healing and higher cancer risk. These admittedly speculative suggestions can be tested using the comparative method, and they gain some support from evidence that aggression is associated with cancer risk in fish  is crucial to this process, but leaves organisms vulnerable to cancer . Mice with supernumerary copies of p53 are protected from cancer, likely because they exhibit an enhanced response to DNA damage  notes that high levels of nutrition in modern Western cultures increases both investment in mate competition and testosterone levels. Subpopulations where competition is especially physical and desperate have higher testosterone levels and increased mating success, but at the cost of increased rates of prostate cancer. This thesis receives support from comparative studies showing higher rates of prostate cancer in human polygamous societies compared with monogamous cultures living adjacent in similar circumstances in Africa  and reproductive effort  Stearns . In enviCancer suppression is as ancient as multicellular life. Multicellular organisms must have mechanisms to suppress cancer effectively. As evolution shaped larger and longer lived species, the problem of cancer suppression became more challenging, and the solutions for suppressing cancer became remarkably effective. They can never be perfect, however, for the same six evolutionary reasons that other bodily traits remain vulnerable to disease.The benefits of using evolutionary principles to understand cancer provide a specific example of the benefits of evolutionary medicine more generally. An evolutionary approach can help us understand why cancer exists and how it progresses (somatic evolution), how cancer cells interact with environments , why it is not more common , and why cancer suppression mechanisms can never be perfect . Evolution is essential for understanding cancer. It provides a framework for studying the evolutionary origins and progression of cancer that is parallel and complementary to the Hallmarks of Cancer framework for studying the mechanisms of cancer.The importance of an evolutionary understanding cancer is not just an academic pursuit; it has great clinical utility that remains largely untapped. Evolutionary theory and methods have led to critical advances that promise to improve how we understand and treat cancer. For example, the finding that diversity in the premalignant biopsies predicts progression to cancer (Maley et al. In retrospect, it is remarkable that the evolution of cells within tumors was not recognized until the 1970s with Nowell's  paper ‘T


Advances in genome sequencing are providing unprecedented resolution of rare and private variants. However, methods which assess the effect of these variants have relied predominantly on information within coding sequences. Assessing their impact in non-coding sequences remains a significant contemporary challenge. In this review, we highlight the role of regulatory variation as causative agents and modifiers of monogenic disorders. We further discuss how advances in functional genomics are now providing new opportunity to assess the impact of rare non-coding variants and their role in disease. The extent of which was highlighted by two recent exome-sequencing that uncovered as much as 95% of protein-coding variation is rare (MAF ≤ 5%) and among these rare variants are the majority of sites predicted to be deleterious  mapping requires three important steps:Data normalization:Normalization is critical to mitigating the influence of technical artifacts or “batch effects” . Generally, the expression trait is assumed to be additive where each allele contributes equivalently. For population data, the most commonly used approach is a single-marker to single-trait linear or non-parametric regression; this analysis can be performed by statistical packages like R or statistical genetics tools such as PLINK . However, the size of the selected interval and the number of genes tested can be as influential as differences in sample size and number of variants tested. Complementary approaches have aimed to increase statistical power by integrating information from genes sharing regulatory pathways  analyses, many of the same limitations as present in GWA studies are equally applicable – common covariates such as ethnicity, relatedness, age and sex are also confounding factors in eQTL studies and should be appropriately accounted.Despite the availability of tools and simple statistical methods that support eQTL discovery, there remain important caveats in their utilization. For instance, considerable statistical power advantages can be achieved by restricting tests to variants nearby a gene  or by generating synthetic genotypes and phenotypes . Using a permutation approach, a false-discovery rate can be estimated.Given the large numbers of tests and a typically small sample size, multiple testing correction is critical for eQTL mapping. Commonly used methods are Bonferroni correction, FDR/q-value   can provide a comprehensive survey of transcription factor binding sites across the genome. ChIP-seq studies comparing related and unrelated individuals . However, robust calls of ASE typically require heterozygous sites to be identified through genotyping or DNA sequencing. Genotypes may be directly observed from RNA data, however due to systematic variation of read depth of RNA (since transcripts are expressed at different levels and have different sizes) and reference genome mapping biases (due to unobserved variants or homologous sequences) such approaches are error prone (Degner et al., cis-eQTL (Sun, Allele-specific expression has also been shown to be a heritable trait under genetic control through family studies (Yan et al., QTL Sun,  and causQTL Sun, . For proMost early attempts to connect expression to phenotypic traits in families have involved using standard Pearson correlation between the expression and trait measurements. However, despite the statistical simplicity of this methodology, it can inflate significance when there are few families and the trait and expression variance has a large between family component, and can reduce significance if there is a large within family component and no correlation between families. To address this, expression trait concordance tests which account for family structure have been reported (Kraft et al., When combining genetic data to search for concordance of expression and trait association, an advantage is that the component of expression due to technical or environmental perturbation can be better controlled. We have previously reported methods that connect genetic effects on gene expression in unrelated samples to trait values by assessing if the genetic perturbation on expression is similar to the distribution of association scores for a trait (Nica et al., de novo mutations on the correct haplotype background and aid in investigations of genetic interaction. Furthermore, advances in ChIP-Seq and RNA-Seq will better aid in characterization of genetic effects on transcription factor binding and isoform expression and will ultimately unlock more complex functional interactions that underlie the etiology of diverse traits.As genome studies have been identifying large numbers of rare variants, it is expected that new methods and data will be required to uncover the impact of these variants and their involvement in diseases and traits. One of the most promising future technologies which will aid in interpreting the effect of rare regulatory variants in different tissues and developmental stages will be through the generation of induced pluripotent cells where gene expression can be assayed in multiple stages and differentiated cell types. Complementing this will be further advances in sequencing methods which provide phasing information – these advances will position rare and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


More healthful eating patterns can help people achieve recommended nutrient intakes, control calorie intake, and reduce the risk of some of the leading causes of chronic illness and death in the United States such as cardiovascular disease, diabetes, and certain types of cancer . Making Preventing Chronic Disease presents findings from the first 7 of those 39 communities to publish results from their CPPW retail food environment initiatives. Three communities focused their efforts on improving access in corner stores. The other 4 communities chose to improve access at farmers markets in low-income areas via incentive programs or increased acceptance of food assistance benefits.This collection of articles in Improving access does not necessarily mean adding large, full-service grocery stores like supermarkets and supercenters to areas with low levels of access. Although these types of stores frequently offer a wider selection of high-quality, affordable, and more healthful options , new stoEach of 3 highlighted CPPW initiatives working to improve access via corner stores used qualitative or quantitative baseline assessments to identify potential barriers, engage community residents, understand neighborhood context, and develop solutions to increase access. In Pitt County, North Carolina, store assessments indicated that the pricing and quality of food items in rural corner stores were similar to that of food items in urban corner stores, but the rural corner stores were more likely to carry healthful foods . QualitaQuantitative and qualitative baseline assessments by the second of the 3 CPPW awardees in Nashville, Tennessee, focused more narrowly on 4 areas researchers identified as providing limited access, based on existing data . PhysicaIntroducing or altering inventory in corner stores is only one way of increasing access to more healthful food options. Farmers markets, farm stands, and other retail venues that sell fresh farm produce can increase access to high-quality fresh produce, be set up in a variety of locations, and be implemented at a low cost –16. TheyEach of the 4 highlighted CPPW initiatives working to improve access at farmers markets in low-income areas was able to demonstrate that increasing affordability of fruits and vegetables through a bonus incentive program is a successful strategy for increasing Supplemental Nutrition Assistance Program (SNAP) use or sales or both at farmers markets. Two CPPW initiatives were also able to demonstrate increases in fruit and vegetable consumption. In 9 farmers markets in lower-income regions of King County, Washington, SNAP/EBT (electronic benefit transfer) acceptance rates increased by 79% for market stalls after introduction of subsidized (EBT) terminals for processing SNAP cards . AnalyseAlthough people who live in areas with greater access to retailers of more healthful foods may have a better diet than those with limited access, dietary quality is still fairly low even in areas of high access . InterveEnsuring that all Americans have easy access to retail venues that offer affordable, healthful foods is an important step toward supporting more healthful choices and a high-quality diet in communities. Each of the 7 CPPW awardees highlighted in this commentary implemented an environmental change and has prospectively evaluated or plans to evaluate its effect. The 4 awardees improving access via farmers markets demonstrated that financial incentives increased sales at farmers markets; 2 of them also demonstrated that incentives increased fruit and vegetable intake. The 3 awardees improving access via corner stores each engaged the community to develop a context-specific approach to improving access and established a baseline. Postevaluation in each is under way. Each of the prospective program evaluations in these communities can help others understand how they can implement retail environment changes that may prompt changes in behavior. Continued evaluation efforts that prospectively evaluate retail environment changes like CPPW and stronger research study designs that can rule out alternative explanations for observed associations  can help


Asynchronous replication of the genome has been associated with different rates of point mutation and copy number variation (CNV) in human populations. Here, our aim was to investigate whether the bias in the generation of CNV that is associated with DNA replication timing might have conditioned the birth of new protein-coding genes during evolution. We show that genes that were duplicated during primate evolution are more commonly found among the human genes located in late-replicating CNV regions. We traced the relationship between replication timing and the evolutionary age of duplicated genes. Strikingly, we found that there is a significant enrichment of evolutionary younger duplicates in late-replicating regions of the human and mouse genome. Indeed, the presence of duplicates in late-replicating regions gradually decreases as the evolutionary time since duplication extends. Our results suggest that the accumulation of recent duplications in late-replicating CNV regions is an active process influencing genome evolution. Not all genes in a genome accumulate mutations and evolve at the same rate , a phenoThe dynamics of replication seems to be largely driven by the configuration of chromatin within the nucleus, whereby more open, physically connected chromosome territories rich in transcriptionally active genes replicate earlier than more tightly packed ones . We alsoWhereas point mutations might shape the function of existing genes, the birth of novel genes generally requires mechanisms that generate new genomic regions. Structural changes, such as copy number variants (CNVs), represent one of the main sources of intra- and inter-specific nucleotide differences between individuals . CNVs tyCNV formation in ancestral species might have led to genomic amplification of regions that contain genes. Later fixation of these regions in the population may occur when a percentage of individuals in a given species harbor a genomic region with an extra gene copy. Although further deletion or pseudogenization might often prevent such genes from becoming fixed , the accFork stalling and template switching (FoSTeS) or Microhomology-mediated break-induced replication (MMBIR)  of (macro or micro) homologous tracks, or non-homologous (NH) repair mechanisms that are at play during replicative stress (e.g.  (MMBIR) ). In hum (MMBIR) . A relat (MMBIR) . Further (MMBIR) , and tha (MMBIR) . Signifi of CNVs .In this study we aimed to elucidate the possible relevance of the association of CNV regions with later DNA replication times on gene birth and evolution . Having demonstrated a clear association between CNVs and gene duplication, we studied the distribution of CNV genes in different evolutionary duplication ages and we found that recent PDGs are clearly enriched in CNV-genes (−150). Indeed, most PDGs duplicated since the primate ancestor were in CNV regions (61%), while most of the genes older than the Eutheria phylostratum (97%) seem to have completely fixed their copy number, which no longer varied in the human population  are rarely implicated in CNV formation.The asynchronous DNA replication that occurs in the genome is related to different patterns of DNA damage, replicative stress and genome rearrangements . Most prorder of replication of each gene from the genome-wide RT profiles, as a relative measure of the moment of replication of each human protein-coding gene (see −15). More interestingly, the association of CNV and late replication is distinct for singletons and PDGs. While CNV-PDGs replicate clearly later than non-CNV PDGs (−15), we did not observe such an association in singleton genes , a trend that disappears completely for old PDGs . Indeed, we observed a very different behavior for these two age groups , whereby−112). Thus, we explored the association between gene duplication age and RT in detail, comparing RT in different phylostrata. Strikingly, we observed a clear correlation between RT and gene phylogeny, whereby younger genes gradually became more likely to be replicated later in the S phase . This trend is robust, even when we used the RT profiles of human lymphoblasts .It was evident from our previous analyses  that genhoblasts  or fibrohoblasts  obtainedMus musculus . In this way, we again found that the younger mouse PDGs in the mouse genome tend to be late replicating . Therefore, the association of gene duplication age and RT appears to be highly significant in Primates and Rodents.To determine how widespread this correlation was in other mammals, we performed an independent analysis of 14,677 mouse PDGs using mouse ESC RT maps . FollowiPericentromeric and subtelomeric regions have previously been described as hotspots of gene duplication  and thus−38, chi-squared test) and mouse  chromosomes. We did not observe a significant enrichment of PDGs duplicated during Primate or Rodent evolution in subtelomeric regions. However, both regions in human are enriched in CNV PDGs, with a 1.44 fold enrichment in subtelomeric regions  and 2.35 fold enrichment in pericentromeric regions . These observations are in agreement to previous estimates . We also performed a similar analysis for mouse genes and the association between gene age and RT was also significant for the three chromosomal regions (−255) in interstitial regions, although it was also significant in the pericentromeric and subtelomeric regions. These observations highlight the prevalence of the association between RT and gene duplication, irrespective of the chromosomal regions where these evolutionary clades concentrate their gene birth events.We next analyzed the RT of the PDGs in each of the three regions of human chromosomes. The correlation between gene RT and evolutionary age remains statistically significant when human pericentromeric, interstitial and subtelomeric PDGs are analyzed separately , althoug regions . In the In conclusion, the younger the PDG is in evolutionary terms, the later it tends to replicate during S-phase in dividing cells. This surprising temporal parallel can be observed in different mammalian lineages and in different genomic regions. These data reinforce the view of RT as a fundamental element in the organization of the mammalian genome. Remarkably, this relationship can still be detected in the PDGs duplicated at different periods before the mammalian split , suggesting that difficulties associated with late replication (such as replicative stress) might have exerted a strong influence on the evolution of new functions from the earliest stages in the evolution of multicellular organisms.Primate ancestor were seen to be much more likely to be located in human CNV regions, suggesting that copy number variation in current populations and the fixation of new PDGs are two extremes of a continuous process.We have shown here that protein-coding genes duplicated in evolution (PDGs) are preferentially located in CNV regions. These CNV PDGs are prone to replicate later than non-CNV PDGs, suggesting a link between CNVs, gene duplication and late replication in human cells. We performed a precise phylostratification analysis to determine the ancestral species in which each human PDG was duplicated for the last time. PDGs duplicated after the common Primate CNV PDGs replicate even later than Primate non-CNV PDGs. This tendency was not observed for older PDGs, which tend to replicate early even if they are located in CNV regions. These results also suggest that copy number formation in gene coding regions is affected distinctly by two mechanisms recently associated to RT. Accordingly, early replicating CNVs are frequently linked to recombination mechanisms such as NAHR, while late-replicating CNVs are more frequently associated to non-homology (NH) based mechanisms  is clearly enriched in the late S-phase. These observations suggest that there is an active process causing newborn duplicated genes to progressively accumulate in the late-replicating genomic regions. Although we propose that gene duplication associated to structural variations such as CNVs may be an important factor explaining this trend, retropositions have also been shown to be a source of gene duplicates . Given tThe regular trends observed at distinct evolutionary ages indicate that this process might have been in operation since ancient periods of metazoan evolution. Moreover, this association clearly persists when we analyze pericentromeric, interstitial and subtelomeric regions separately . These the chromatin that replicates late  and mouse NCBIM37 (mm9) genome builds. We used the Ensembl assembly converter to update the human data in NCBI36 to GRCh37.p2 and the mouse data in NCBIM36 to 37.We used accurate gene copy number variation data from a recent study performed on 159 human genomes (including 15 high coverage genomes ). In thiWe established an analytical pipeline to perform precise phylostratification  in a manHomo sapiens  represents that of the ancestral species in which the duplication event that led to the generation of the extant gene was detected. For this purpose, we only considered duplication events showing a consistency score above 0.3 . When th sapiens ; Table 1 sapiens  Euarchon sapiens , we deci sapiens . Given tWe retrieved the probe log-ratios of the processed and normalized replication times for four human ESCs  from the GEO  dataset,Processed and normalized log-ratios of murine RT correspond to GSE17983 , which chttp://www.ensembl.org/info/docs/api/index.html). The data transformations and file parsing needed to run our gene birth dating pipeline were performed using perl (http://www.perl.org). All statistical analyses and plots were carried out using R basic functions (http://cran.r-project.org) and all our code is available upon request.ENSEMBL databases were accessed using the ENSEMBL Perl API Core and Compara (


The biological consequences of exposure to explosive blast are extremely complex. Serum protein biomarkers in blast-induced traumatic brain injury (bTBI) can aid in determining injury severity, monitoring progress, and predicting outcome. Exposure to blast results in varying degrees of physical injury. Explosive blast can also induce psychological stress that can contribute to or amplify the extent of physical damage. Given the complexity, scale of injury, and variety of symptoms, bTBI may be best described as a spectrum disorder. In this focused review, we summarize the status of serum protein biomarkers in bTBI in the context of the classification and pathological changes of other forms of TBI. Finally, we recommend specific and easily implementable measures to accelerate serum protein biomarker discovery and validation in bTBI. Serum protein biomarkers have long held promise in the treatment of traumatic brain injury (TBI). They can aid in diagnosing the disease, monitoring progress, predicting outcome, and providing pertinent molecular information about ongoing pathological changes for designing evidence-based therapeutic interventions  estimates that ∼1.7 million Americans sustain TBI every year  is an especially common form of TBI among military populations. According to the Joint Theater Trauma Registry, IEDs were the source of about 80% of all casualties of veterans from Iraq and Afghanistan between October 2001 and January 2005  or closed head injury. Penetrating head injury not only causes substantial direct tissue damage, but also instantaneously breaks down existing biological barriers, generating massive pathological responses to toxic molecules and cellular debris. In contrast, closed head injury typically causes metabolic changes and axonal damage of various degrees. In response to the typically short-lasting primary injury mechanism, there is a second wave of long-lasting pathological changes called the secondary injury mechanism. These pathologies include metabolic changes, neuroinflammation, axonal injury, vascular abnormalities, and neuronal and glial cell death  but were not injured had no increase in the serum levels of the previously mentioned protein biomarkers in both experimental and clinical studies in order to enable the longitudinal analysis of changes in serum levels of protein markers in bTBI. This approach can reveal temporal patterns of changes that may be of vital diagnostic and therapeutic value. Second: changes in the serum levels of a whole panel of proteins rather than a single protein need to be analyzed (at multiple time points). Multiplex assays, already in use in cancer biology, can provide substantially improved diagnostic precision, and especially so if combined with a systems biology analysis. Third: changes in serum protein biomarkers should be analyzed in relation to functional and neurobehavioral changes in both clinical and experimental settings. Such a combined analysis would radically improve the diagnostic and prognostic value of serum protein biomarkers by facilitating a much more direct understanding of how serum changes relate to functional deficits.In addition to proteins, there are other potential biomarkers such as microRNAs. While microRNAs have some advantages  compared to proteins, there is currently much less known about their functions. However, as our knowledge increases about their involvement in the various pathological processes their value as serum biomarkers will also increase.In summary, serum-based protein biomarkers have the enormous potential to fundamentally change our understanding of bTBI and ultimately can – and will – be of major help in designing evidence-based treatments for individuals suffering from the consequences of blast injury.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gasterosteus aculeatus, using size-manipulated females of both ecotypes. Specifically, we asked if male courtship preferences are present, and if they are based on relative body size, non-size aspects of ecotype, or other traits. Because male behaviors were correlated with each other, we conducted a principal components analysis on the correlations and ran subsequent analyses on the principal components. The two male ecotypes differed in overall behavioral frequencies, with stream-resident males exhibiting consistently more vigorous and positive courtship than anadromous males, and an otherwise aggressive behavior playing a more positive role in anadromous than stream-resident courtship. We observed more vigorous courtship toward smaller females by  stream-resident males and the reverse pattern for (relatively large) anadromous males. Thus size-assortative male courtship preferences may contribute to reproductive isolation in this system, although preferences are far from absolute. We found little indication of males responding preferentially to females of their own ecotype independent of body size.Studies of mating preferences and pre-mating reproductive isolation have often focused on females, but the potential importance of male preferences is increasingly appreciated. We investigated male behavior in the context of reproductive isolation between divergent anadromous and stream-resident populations of threespine stickleback,  Speciation research increasingly points toward divergent or disruptive ecological selection as a key cause of speciation. Such selection may drive the evolution of behavioral reproductive isolation either through pleiotropy as a byproduct of ecological adaptation or as an adaptation itself, through reinforcement or a similar process Gasterosteus aculeatus. The stickleback species pairs are now well established as an important model system for the study of speciation Here we investigate the potential role of male behavior in reproductive isolation between divergent anadromous and stream-resident populations of the threespine stickleback, In a large-scale study, McKinnon et al. All work was approved by the University of Wisconsin-Whitewater Institutional Animal Care and Use Committee  and carried out in strict accordance with national guidelines.The general experimental design, including collection and rearing methods, is presented elsewhere Mating trials were ‘no choice’ tests involving one male and one female paired in a 96 liter aquarium and allowed to interact freely .10 transformations (of the raw data plus one) to improve normality. Most male behaviors were correlated with each other  the four male behaviors were significantly correlated with each other  with onlIn a principal component analysis, the first principal component (PC1) accounted for 55.1% of the variance. The variables loading most strongly on PC1, as indicated by the eigenvectors, were nest work (0.606), zig-zags (0.544) and direct leads (0.510); bite-bumps showed a lower loading at 0.277. We interpret PC1 as being associated with unambiguous, vigorous courtship in which males display to the female, try to lead her to the nest to spawn, and work on the nest in preparation for spawning; because PC1 is both readily interpretable and accounted for more than 55% of variance, it is emphasized in subsequent analyses. PC2, accounting for 24.5% of variance, was much more strongly associated with bite-bumps (0.883) and negatively associated with direct leads (−0.461) and nest work (−0.066); zig-zags essentially did not load on PC2 (0.057). We interpret PC2 as characterizing either rejection of the female or a distinct, perhaps more aggressive aspect of courtship not closely associated with preparation for spawning.We analyzed male courtship behavior using a full factorial ANOVA with the independent variables male ecotype (anadromous or stream-resident), female ecotype (anadromous or stream-resident), female region (British Columbia or Japan) and female size class . For the dependent variable PC1, the most significant independent variable was male ecotype : stream-We found no evidence of males responding preferentially to females of their own ecotype independent of body size. The male ecotype by female ecotype term was nonsignificant . Higher order interaction terms involving male and female ecotype were also all nonsignificant, as were all other terms in the ANOVA .When a measure of female preference and courtship success, whether or not the female inspected the male’s nest at some point in the full trial For the second principal component (PC2), which was associated mainly with more frequent bite-bumps and less frequent leads, there was no overall difference between stream and anadromous males  and all significant effects in the preceding analysis remained so, suggesting that PC2 is not so clearly closely associated with courtship success as PC1, overall also see , 4. WhenTo further assess the relationship between female body size and male courtship behavior for males of each ecotype, we conducted additional, somewhat simplified analyses for each principal component of the relationship between male ecotype, female body size, and their interaction. Rather than categorizing females simply as large or small based on the size manipulation, as for the ANOVA’s, this GLM is based on actual standard lengths of individual females and thus takes into account all variation in female body size . As in the previous analyses of PC1, male ecotype was highly significant  owing to higher scores for stream males. Female standard length had no consistent relationship with PC1  but the interaction between male ecotype and female standard length was significant , owing to the tendency of anadromous males to respond more strongly to larger females and stream males to respond more strongly to smaller females as in , confirmIn an effort to more fully elucidate the different relationships between female size class and male behaviors summarized by PC1 and PC2, we analyzed the correlations among male behaviors separately for males of each ecotype , Table 3For bite-bump, the most anomalous behavior in these analyses, we also conducted univariate analyses of its relationships  with female body size and nest inspection, for each male ecotype. In ANOVA’s analogous to those above but conducted separately for each male ecotype , the effect of female size class was consistent for both male ecotypes, with males bite-bumping large females more frequently in both cases ; all interaction terms were non-significant for both male ecotypes , so the effect of female size class was not complicated by other variables. When nest inspection was added to the analyses, its effect was significant for anadromous males, who bite-bumped at a higher frequency in trials that resulted in a nest inspection ; moreover, the effect of size class was rendered non-significant  with inclusion of nest inspection, indicating a strong link between female size class and nest inspection in this context. In contrast, the nest inspection term was non-significant when added to the analysis for stream males , whereas the female size class term remained significant .Thus anadromous males who are zig-zagging, readying their nest for the female and attempting to lead her to their nest are also likely to be frequently bite-bumping her; however there is no relationship between such vigorous, successful courtship and bite-bumps for stream males. Consequently, the increase in pooled analysis PC2 toward large females may occur for different reasons in stream and anadromous males, given that it is dominated by the bite-bump behavior.Male sticklebacks in this study showed more vigorous courtship toward females that were manipulated to be similar to them in body size than to females manipulated to be different in size. Because courtship was assayed early in each experimental trial, the influence of female behavior on males should have been limited. While male preferences could potentially have been based on correlated aspects of female phenotype, correlations between size and other traits should have been minimized through the size-manipulation of females from four different populations. Raising fish on similar diets and in generally similar conditions may also have minimized differences in phenotypically plastic traits.The preferential courtship of relatively large females by anadromous males is not surprising given previous studies The superficially similar elevated behavioral response of both stream and anadromous males to large size-manipulated females, in terms of the second (pooled data) behavioral principal component, PC2, is of interest given that it may have evolved for different reasons in each ecotype and reflect basic differences in courtship behavior. Based on the supplementary analyses exclusively of anadromous male behavior, it appears that when anadromous males pursue a courtship destined to be successful, and presumably reflective of strong motivation to spawn, they include a high frequency of bite-bump; this behavior can also be aggressive but appears to be a typical part of anadromous courtship also see . Thus thConversely, the high pooled PC2 scores of stream males toward large females contrast with their lower scores toward such females on PC1. For stream males, the supplementary ecotype-specific analyses suggest that bite-bumps, which load heavily only on PC2 and do not correlate positively with other behaviors, are not associated with vigorous, positive courtship. Thus high PC2 scores for stream males, either in the pooled or ecotype-specific analysis, may represent aggressive rejection, or possibly very tentative courtship, of large females. The trend toward an association for stream males between failed courtship and pooled PC2 was not significant but certainly there was no positive correlation with courtship success.Our results support and extend the findings of earlier studies of male courtship in sticklebacks from the Salmon River and the nearby Little Campbell River. In previous experiments involving paired female presentations, Salmon River stream males from the area of sympatry with anadromous sticklebacks courted and spawned with relatively small stream females preferentially, whereas males from an upstream allopatric site spawned more often with large females. Moreover, the sympatric stream males showed more frequent aggressive behavior and reduced zig-zagging toward relatively large females Based on data from experiments with limnetic and benthic sticklebacks, Kozak et al. Stream males responding less positively to large females in nature would tend to mate assortatively by ecotype, since anadromous females are larger. This preference may have arisen as a result of either reinforcement or direct selection, since the populations of the males tested here are sympatric. It is also possible that this preference is a byproduct of divergence in body size or some other trait, but given the benefits of fathering the large clutches produced by large females and the fact that body size differences were not extreme , together with earlier results on spawning success of Salmon River sticklebacks in choice tests Imprinting has recently been shown to play a role in female preference development, and assortative mating (or lack thereof) in benthic-limnetic pairs . FemalesIn conclusion, our results suggest that stream-resident and anadromous male sticklebacks, from a site where the forms are sympatric, preferentially court females relatively similar in size to themselves when correlations between size and other traits have been minimized through a manipulative experiment. Because male courtship differs between the ecotypes, however, interpreting patterns in male behavior is not trivial. These preferences may contribute to reproductive isolation in a natural setting but at present we can draw no strong conclusions as to which evolutionary forces are responsible for male size-preferences.Table S1Number of trials of each male-female combination.(DOC)Click here for additional data file.


Patients who survive either ventricular fibrillation or sustained ventricular tachycardia carry a high risk of further episodes, which could be fatal. Anti-arrhythmic drugs were the standard treatment for patients with malignant ventricular arrhythmias, but despite using the best appropriate medical treatment, arrhythmia recurrence rates remain high at 40-50% at five years. Implantable cardioverter defibrillators (ICD) are effective at treating ventricular arrhythmias and there has been clear evidence that they reduce mortality. Cardiac resynchronization therapy (CRT) improves symptoms and reduces morbidity and mortality in patients with advanced heart failure. In addition, many patients have indications for combined ICD and CRT therapy.While the number of ICD/CRT implantations in the developed world has increased, device utilization rates are less in low- and middle-income countries, despite having higher incidence of risk factors for sudden cardiac death and heart failure. The primary obstacle to the growth of this therapy in developing nations is its cost - ranging from $8000 to $16,000 depending on the model and the features available. Due to its cost, this therapy is currently accessible only to the affluent in developing countries, whereas the subset of patients who requires this therapy is increasing rapidly. According to available data, only a few hundreds of patients in India which has a population of over 120 million receive ICD implants every year, whereas in developed countries with much smaller population the number is at least in thousands.In this context, devices donated for use by charitable organizations, industry or devices harvested by physicians/industry for reuse, becomes extremely important to patients in low- and middle-income countries. Reuse of devices poses risks such as infection, device malfunction and premature battery depletion. But, recently published studies have shown that there is no increase in morbidity or mortality associated with device reuse, if implanted with good technique and proper sterile precautions. However, there is no proper data or registry available regarding the availability of devices with potential for reuse in the developing or even in the developed world.The HRS guidelines recommend that physicians seek patients' consent for post-mortem device retrieval while they are alive. In routine practice, informed consent for device implants rarely includes a discussion regarding post-mortem handling of devices. A survey of patient preferences showed that 87% of patients did not know how devices were handled after their death despite the fact that majority were willing to have their devices retrieved for reuse . MoreoveIn an interesting study published in this issue, Iyer IR and Mackall J tried to unravel veterans' preferences regarding post-mortem handling of devices and also to create some data on reuse potential of these expensive devices . In thisThis is an important study considering that data regarding reuse or reuse potential of these expensive life saving devices are so scarce and its implications especially in developing countries are many. Major limitations of this study are small sample size and retrospective nature for assessing reuse potential of devices. Devices and battery technology analyzed in this study are different from currently used devices; recent generation devices show longer battery life and therefore may offer greater reuse potential. A larger study utilizing large datasets maintained by device manufacturers and multiple centers may give a better estimate of number of devices with significant reuse potential in order to formulate a health policy on this rather important issue.


Yersinia species, as well as many other Gram-negative pathogens, use a type III secretion system (T3SS) to translocate effector proteins from the bacterial cytoplasm to the host cytosol. This T3SS resembles a molecular syringe, with a needle-like shaft connected to a basal body structure, which spans the inner and outer bacterial membranes. The basal body of the injectisome shares a high degree of homology with the bacterial flagellum. Extending from the T3SS basal body is the needle, which is a polymer of a single protein, YscF. The distal end of the needle serves as a platform for the assembly of a tip complex composed of LcrV. Though never directly observed, prevailing models assume that LcrV assists in the insertion of the pore-forming proteins YopB and YopD into the host cell membrane. This completes a bridge between the bacterium and host cell to provide a continuous channel through which effectors are delivered. Significant effort has gone into understanding how the T3SS is assembled, how its substrates are recognized and how substrate delivery is controlled. Arguably the latter topic is the least understood; however, recent advances have provided new insight, and therefore, this review will focus primarily on summarizing the current state of knowledge regarding the control of substrate delivery by the T3SS. Specifically, we will discuss the roles of YopK, as well as YopN and YopE, which have long been linked to regulation of translocation. We also propose models whereby the YopK regulator communicates with the basal body of the T3SS to control translocation. Yersinia species cause human diseases ranging from relatively mild intestinal disease for Yersinia pseudotuberculosis and Yersinia enterocolitica  that has become a well-established model system for this form of protein secretion. Though first described in Yersinia, type III secretion is a conserved virulence factor amongst many human pathogens such as enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), Salmonella sp., Pseudomonas aeruginosa, Shigella flexneri, and Chlamydia sp., which collectively cause significant healthcare costs annually   , and YscD and YscJ form a ring in the inner membrane (the MS ring). Together these proteins create a scaffold anchored within the peptidoglycan, and therefore, they will be referred to as scaffold proteins  as well as proteins embedded within or connected to the scaffold: export apparatus  is severely compromised  , secretion of late substrates is largely prevented due to the action of the YopN-TyeA-YscB-SycN complex, also referred to as the calcium plug , which include LcrV, YopB, and YopD. These proteins are secreted into the extracellular milieu during tissue culture infection and during in vitro growth in calcium-replete medium  can be isolated from host membranes, and LcrV (IpaD in Shigella) is required for insertion of YopB/YopD (IpaB/IpaC)  are now delivered directly into host cells.Though a complete conduit between the needle and the host cell has never been observed, there is evidence from Y. pseudotuberculosis cells in calcium-replete medium, indicating that bacteria are coated with Yops prior to host cell contact  is present in all three pathogenic Yersinia spp. with a high degree of sequence homology  reporter  in host cells more rapidly than wild type, while overexpression of YopK resulted in a lack of cytotoxicity , in host cells  gave rise to higher levels of effector translocation, it was hypothesized that the phenotype may be related to a change in translocation pore size. As a result, several studies were undertaken to investigate this possibility.Shortly before the characterization of YopK by Holmstrom and colleagues, a contact-hemolytic assay, previously utilized for pore-forming toxins  release is a common cytotoxicity assay that is often used to measure membrane disruption and/or cell death by detecting the concentration of cytosolic LDH that is released into the extracellular medium. Several groups have used this assay to test lytic pore formation as correlate of translocation regulation. In contrast to the contact-hemolysis assay, the ΔIt is not yet clear how the observed changes in “pore” size (as determined by lytic pore formation assays) correlate with changes in translocation levels. If the translocation pore is indeed larger, then is the internal diameter of the needle larger to accommodate increased flow of effectors? Considering prevailing models in which the translocation pore complex is connected to the basal body through the needle, a change in the pore should trigger compensatory changes throughout the injectisome. Another possibility is that the assays which measure pore formation do not directly measure the pores that are actively translocating, and instead, the results could simply reflect an altered conformation that is perhaps less stable or more flexible, and therefore appears larger. If the pore size is not physically larger, it may be that the altered conformation of the pore, and by extension the rest of the injectisome, triggers a change in the export apparatus and/or ATPase complex such that secretion substrates are recognized and initiated through the channel at a faster rate.Y. pestis and a ΔyopK mutant initiate injection with similar efficiency  does not complement a ΔY. pseudotuberculosis, providing the first evidence of a direct interaction between YopK and the pore  abolishes the ability of YopK to control the rate of late Yop delivery but is still able to inhibit injection of middle Yops  YopK binds to the translocation pore to trigger conformational changes that influence substrate recognition and/or transport by the basal body  was evaluated. Although YopER144A was translocated efficiently, it could not prevent phagocytosis by HeLa cells  actin regulation. Because yersinae proliferate extracellularly, it is essential to block phagocytosis after contact with host cells. Y. pseudotuberculosis ΔyopE mutant released more LDH from HeLa cells than did a wild type infection, and that the phenotype was dependent on the GAP domain of YopE (Viboud and Bliska, Y. pseudotuberculosis ΔyopE caused hemoglobin release similar to wild type infection (Holmstrom et al., yopK mutants also yielded contrasting results, but in the case of yopK, it was the hemolysis assay that showed a phenotype. The difference in phenotypes may be related to differences in the cytoskeleton of each cell type, as RBCs and epithelial cells have different actin cytoskeletal networks (Nans et al., Another facet of YopE's function is the ability to inhibit the formation of lytic pores. Several groups have shown that infection with a yopE mutant and resulted in high LDH release (Viboud and Bliska, yopE mutant (Viboud and Bliska, Clostridium difficile ToxB, an inhibitor of Rho, Rac, and Cdc42 GTPases (Mejia et al., To further analyze the role of YopE GAP activity in lytic pore formation, Viboud et al. transfected HeLa cells with constitutively active forms of RhoA or Rac1 that cannot hydrolyze bound GTP. When HeLa cells were transfected with activated GTPases before infection, ectopic expression of YopE could no longer complement the yopE mutant strains have been shown to over-inject Yops into host cells during infection. Fractionation of infected cells has shown that a Y. pseudotuberculosis ΔyopE mutant injects higher levels of YopH, and the corresponding YopER144A GAP mutant over-injects both YopH and YopE, (Aili et al., Y. pestis, YopE was shown to regulate translocation using the YopM-Bla reporter (Dewoody et al., yopE mutant showed an approximately 50% increase in YopM-Bla injection compared to infection with wild type. This phenotype is dependent on YopE's GAP ability as ectopic expression of YopER144A could not complement the ΔyopE parent strain (Dewoody et al., ΔYersinia infection: anti-phagocytosis, repression of lytic pore formation, and translocation regulation. Each of these processes depends on small Rho GTPase deactivation and thus inhibition of actin polymerization. What has yet to be determined is whether one of these roles is the major function of YopE or if each is essential for infection. Furthermore, since there is no evidence for YopE binding directly to the translocation pore complex, any affect it has on the injectisome pore and translocation must be indirect. Finally, direct evidence for connections between YopE, the actin cytoskeleton, and changes to the injectisome have yet to be revealed.In summary, YopE plays a role in three major aspects of Y. enterocolitica as an effector that was translocated into host cells where it disrupted actin stress fiber and caused cytotoxicity (Iriarte and Cornelis, Y. enterocolitica and Y. pestis strains, but only a portion of Y. pseudotuberculosis strains (Viboud and Bliska, Y. enterocolitica, and a ΔyopT mutant shows a slight increase in virulence (Iriarte and Cornelis, Y. pseudotuberculosis infection, but can partially restore the virulence of a ΔyopE mutant, suggesting some degree of redundancy between the two cytotoxins (Viboud et al., YopT was first discovered in Y. enterocolitica strains expressing YopT have shown that activated RhoA is released from the host membrane (Zumbihl et al, yopT mutants do not have a virulence defect in vivo (Iriarte and Cornelis, YopT is a cysteine protease and shares the conserved invariant C/H/D residues necessary for proteolytic function (Shao et al., Y. pseudotuberculosis YopT alone is able to significantly reduce LDH release and lytic pore formation of infected HeLa cells (Viboud et al., yopE mutant, but not when activated Rho or Rac are expressed in host cells (Viboud and Bliska, Due to its ability to regulate host actin regulation, it is not surprising that YopT has a role in preventing phagocytosis and lytic pore formation. Expression of yopEHJT Y. pseudotuberculosis background resulted in a small decrease in phagocytosis when expressed at native levels and larger inhibition when overexpressed (Viboud et al., Y. enterocolitica ΔyopT mutant resulted in phagocytosis well above that of wild type and not significantly different from a ΔyopE mutant (Grosdent et al., yopE mutant had no affect on phagocytosis while the ΔyopT mutant showed significantly more internalization (Grosdent et al., yopK and yopE mutants, the host background plays a confounding role in the functions of these proteins. Given that different eukaryotic cell types lead to different phenotypes for these mutants, future work may be able to utilize this knowledge to gain insight into the underlying mechanisms.Inhibition of phagocytosis by YopT was measured using both a gentamicin protection assay and double immunofluorescence staining. Expression of YopT in a ΔTranslocation of effectors by the T3SS is tightly regulated to optimize the infectious process of disease. It is well-established that YopN functions prior to cell contact in order to prevent the premature release of late Yops into the medium, thereby providing a measure of fidelity to substrate recognition by the T3SS. New data discussed here demonstrates that YopK also acts as a regulator of fidelity by preventing the export of middle Yops into host cells. How YopN and YopK coordinate these regulatory activities is unclear. Future endeavors will need to determine whether YopN and/or YopK are physically associated with the basal body and if so, with which proteins do they interact and are those interactions indeed required to regulate substrate specificity before and after cell contact? Likewise, YopK and YopE are both major regulators of translocation that function after they are injected into the host cell. YopE appears to exert its effect via manipulation of the actin cytoskeleton, while YopK associates with the translocation pore complex. How cytoskeletal changes influence the T3SS function, and whether these changes are channeled through the YopK-pore complex interaction is a mystery. Understanding the hierarchy and mechanisms of translocated regulatory proteins is an exciting new component of T3SS research.in vitro (Darveau et al., Yersiniaouter-membrane proteins. It has also been shown that host cells can take up YopH-Bla coated on the surface of a ΔyopH Y. pseudotuberculosis mutant (Akopyan et al., Yersinia field and to the research presented herein. Several questions remain unanswered. Do both methods of Yop delivery occur, and if so, which one is predominant in vivo? If the needle is not connected to the host as a continuous channel, then how is cell contact sensed in order to provide the critical signal to release the YopN regulatory blockade on late Yop secretion? If the injectisome is not involved in direct translocation of effectors into host cells, how are Y. pestis effectors YopK and YopE transmitting signals back to the bacteria? Do complexes of effector-Yops and translocator-Yops form pores that are subject to regulation by YopK, YopE, and YopT? It is difficult to envision a scenario in which a two-step delivery method would be compatible with the regulatory roles that YopK, YopE, and YopT seem to have within host cells. Data presented herein strongly implicate these bacterial effectors in generating a feedback signal that originates within host cells, which in turn implies that there is a continuous channel whose ends are able to communicate and exact precise control over the timing, specificity, and amplitude of Yop delivery. Of course such a channel remains hypothetical, and future work needs to place an emphasis on developing tools to allow visualization of actively translocating injectisomes docked onto host cells, as well as a more detailed view of the pore complex architecture. Such technological advances are crucial for providing insight into these key aspects of injectisome assembly and function.A new model for Yop translocation has been suggested recently: the “two step” translocation model (Akopyan et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression. Complimentary, the continuous technological developments in the field of metabolomics per se has further demonstrated its potential as a very suitable readout to unravel metabolite-mediated signaling processes. As foundation for these studies here we outline and discuss recent advances in elucidating retrograde signaling molecules and pathways with an emphasis on metabolomics and systems biology driven approaches.A tight coordination of biological processes between cellular compartments and organelles is crucial for the survival of any eukaryotic organism. According to cellular requirements, signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear gene expression in a process called retrograde signaling. Whilst many research efforts have been focused on dissecting retrograde signaling pathways using biochemical and genetics approaches, metabolomics and systems biology driven studies have illustrated their great potential for hypotheses generation and for dissecting signaling networks in a rather unbiased or untargeted fashion. Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of  Biological systems rely on complex interactions of heterogeneous small- and macro-molecules to execute cellular functions required for their growth, survival, and propagation . To mainIn contrast to bacteria, the eukaryotic cell comprises a large number of diverse subcellular compartments and organelles which are usually delineated by a lipid bilayer to maintain specific microenvironments . Along wWhile the different subcellular compartments are involved in storage, detoxification, and synthesis of a variety of specific and important compounds , plastidArabidopsis have resulted in identifying the genomes uncoupled (gun) mutants, which block Mg-proto mediated retrograde signaling by affecting its accumulation  components of tetrapyrrole biosynthetic pathway intermediates , (ii) remulation . Those emulation . Howevermulation . These fmulation . Innovatmulation . As the Whilst much research efforts have been focused on dissecting signaling pathways using biochemical and genetics approaches, the number of yet discovered mechanisms of retrograde signaling are likely not sufficient to explain the tight regulation and interdependence of organelle functions. For instance, the cellular metabolism is tightly correlated with processes occurring in chloroplasts and mitochondria. It is widely believed that changes in the concentration of metabolites triggered by the organelles are likely sensed in the cytosol and thus, might regulate nuclear gene expression . TherefoOver the past decades a number of analytical methods, such as gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis (CE) coupled to mass spectrometry (MS) or nuclear magnetic resonance (NMR), have been developed and improved to accurately and sensitively analyze small molecules from complex sample mixtures . As thesceh1 mutant, which displayed changes in expression of stress-related genes associated to salicylic acid response and resistance to the pathogen Pseudomonas. Ceh1 is caused by a mutation in the HDS gene responsible for the conversion of MEcPP to hydroxymethylbutenyl diphosphate (HMBPP) in the methylerythritol phosphate (MEP) pathway. Metabolite profile analysis of MEP pathway intermediates by LC-MS revealed an accumulation of the intermediate metabolite MEcPP in ceh1. MEcPP is a specific and critical retrograde signaling metabolite that acts as a stress sensor by triggering the expression of specific stress-responsive nuclear encoded plastidial proteins  and confirm its role as a retrograde signal  nanosensor makes use of a recognition element (a protein that binds with the metabolite of interest) fused to a report element (a fluorophore pair). Changes in protein conformation triggered by ligand (metabolite)-recognition element binding leads to the emission of fluorescent light via the report element  and short-term chemical treatments  involved in plant mitochondrial dysfunction has been carried out to identify targets and pathways involved in mitochondrial retrograde signaling  which has been recently identified as a regulator of the sulfur status in Arabidopsis using computational analysis of time-series experiments and inducible transgenic plants revealing conditional increased OAS levels (Another important issue for the identification of signals is to distinguish between “true” primary targets from tertiary targets . SeveralS levels . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Management of orofacial clefts requires a comprehensive approach to address the multifaceted aspects of possible aesthetic and functional deficits. Many children with cleft palate with or without cleft lip continue to have speech abnormalities that can affect socialization, education, and childhood development. An interdisciplinary cleft team ideally manages most orofacial clefts and can include members from the fields of facial plastic surgery, otolaryngology, speech and language pathology, pediatrics, nursing, audiology, genetics, oral surgery, orthodontics, dentistry, and social work. These teams follow a relatively established timeline and algorithm for each of the discrete steps of orofacial cleft treatment. Cleft lip repair is often performed after 3–5 months of age. Palatoplasty is delayed after 8–14 months of age. Speech and language pathology assessment and therapy are targeted as the vocabulary develops (2–5 years old).Nonlinear dynamic analysis of vowels in cleft palate children with or without hypernasality” reports on cephalometric differences between the skull base and velopharyngeal anatomy in patients with unilateral cleft lip and palate compared to normal controls. These data contribute to the existing literature of abnormal skull base anatomy and inclination in children with velocardiofacial syndrome, which often associated with VPD. This special issue deals with the cleft surgeon and speech therapists role in treatment of velopharyngeal dysfunction (VPD), which is often related to velopharyngeal insufficiency (VPI). This lack of coordinated closure of the soft palate to the posterior pharyngeal wall is often a sequela of cleft palate, even after primary repair. One paper from this issue titled “Noncleft velopharyngeal insufficiency: etiology and need for surgical treatment” analyzes those children with VPD without obvious orofacial clefting, and assesses the etiologies and potential treatment options. This allows for comparison to the traditional orofacial cleft-related VPD management. The next paper titled “Nonlinear dynamic analysis of vowels in cleft palate children with or without hypernasality” assesses the relationship between two speech assessment tools, Lyapunov exponents, and Nasalance scores in the presence of hypernasal speech. Furthermore, an additional paper titled “Assessment of single-word production for children under three years of age: comparison of children with and without cleft plate” presents a case-control testing of a single word assessment tool in children less than 3 years of age with cleft palate, which indentified speech differences in error patterns, consonants, articulation, and accuracy between children with and with clefts. The velopharyngeal workup is often a coordinated assessment by the cleft surgeon and speech pathologists. It can include subjective assessment, which is the subject of the next two papers. First, the next paper titled “Objective assessment of hypernasality in patients with cleft lip and palate with the nasalView system: a clinical validation study” assesses the validity and reliability of a new tool for hypernasality testing called NasalView. Speech and language therapy is instituted to address typical compensatory speech patterns seen in children with cleft palate .  Objective assessment of VPD also can include either fluoroscopic speech examination or nasopharyngoscopy to characterize the pattern of VPD and target a surgical goal. The next paper titled “ If hypernasal speech is not responsive to speech and language therapy, secondary speech surgery is warranted to address hypernasality in this time period. Surgical options may include a superiorly based pharyngeal flap, dynamic sphincter pharyngoplasty, or posterior pharyngeal wall augmentation. Occasionally, a palate-lengthening procedure (Furlow double-opposing Z-plasty) is performed. These procedures can limit nasal air emissions, but potential obstructive sleep apnea from excessive nasal obstruction is a risk.Speech outcomes after tonsillectomy in patients with known velopharyngeal insufficiency”. The risk of sleep apnea after surgically treating the velopharynx may be decreased if the tonsils and adenoids are addressed preemptively. Before pharyngeal flap surgery, some surgeons perform tonsillectomy to limit the potential variability of speech and airway changes that comes with fluctuating tonsillar hypertrophy, tonsillitis, or upper respiratory infections. The use of tonsillectomy in the face of VPI in children with cleft palate is somewhat controversial and is the topic of the last paper “ The guest editors are pleased to present a collection of papers dealing with management of the speech-related difficulties in children with orofacial clefts. In this era of the expanding role of evidenced-based medicine, the effectiveness of treatments can only be monitored and improved by assessment with valid and consistent outcome measures, which are reviewed in this issue.Travis T. TollefsonTravis T. TollefsonDavid WhiteDavid WhiteJames BrookesJames BrookesSteven GoudySteven Goudy


Homo life history. Rather, broader cooperative networks within and between the sexes provide the primary basis for our unique life history.Derived aspects of our human life history, such as short interbirth intervals and altricial newborns, have been attributed to male provisioning of nutrient-rich meat within monogamous relationships. However, many primatologists and anthropologists have questioned the relative importance of pair-bonding and biparental care, pointing to evidence that cooperative breeding better characterizes human reproductive and child-care relationships. We present a mathematical model with empirically-informed parameter ranges showing that natural selection favors cooperation among mothers over a wide range of conditions. In contrast, our analysis provides a far more narrow range of support for selection favoring male coalition-based monogamy over more promiscuous independent males, suggesting that provisioning within monogamous relationships may fall short of explaining the evolution of  Human life history is puzzling. Despite producing relatively large, altricial newborns that grow expensive tissues (e.g. our brain) Ardipithecus radius, a human ancestor who lived 4.4 million years ago, suggests to some that males and females pair-bonded, making monogamy and biparental care the ancestral condition in earlier HomoHowever, some have attributed this shift to male provisioning in monogamous relationships. The lack of sexual dimorphism in the bipedal ape Yet empirical studies suggest that humans have evolved to engage in broadly cooperative food-sharing and infant care networks Cebus capucinus), females regularly nurse one another’s infants, with a higher rate of allo-nursing among low-ranking mothers Cebus nigritus) has been hypothesized to serve significant social benefits Papio hamadryas ursinus) form stable, close, persistent social bonds known to enhance fitness, despite not engaging in cooperative infant care Microcebus murinus), mothers recognize their own infants but will allow other female’s infants to suckle in communal sleeping nests Mungos mungo) tradeoff protective babysitting roles and often give birth synchronously, which maximizes pup survival by reducing asymmetry in pup competitiveness and inhibiting infanticide Mus domesticus), pairs of females cooperatively nurse each other’s pups and defend the shared nest. These cooperative relationships between house mice females are not necessarily contingent on relatedness, are established before conception, and can persist across reproductive events Females from a broad range of mammalian taxa often directly or indirectly care for offspring that are not their own . Among wWhen males or coalitions of males contribute parental effort it may be a byproduct of actions that could be classified as mating effort or efforts to build or maintain coalitions. For example, male chimpanzees engage in cooperative territorial defense The cohesive picture that emerges from these empirical studies is that humans have evolved to engage in broadly cooperative food-sharing and infant care networks, rather different from the behavior observed in strongly pair-bonded primate species such as gibbons and siamangs In the model we consider three female and two male provisioning and reproductive strategies. The fitness of each strategy is determined by the infant care roles played between females, between males, and between females and males. One primary goal of the model is to relate its formulation to the empirical knowledge of primate behavior and life history. Therefore the model is developed to describe the reciprocal behavioral interactions among females and males. We assume the benefits of cooperation between females to be improved foraging efficiency of females when not encumbered by infants such as has been observed in some other primates a) an Independent Mother, (IM) who strictly provisions her own children, (b) a Cooperative Mother (CM), who engages in allo-parenting with another CM, and (c) a Opportunistic Mother (OM), who accepts allo-parenting benefits but does not reciprocate by being an allo-parent. Let the two strategies available to males be a Coalition Male and a Non-coalition Male. Non-coalition males forage independently and thus gains fewer resources for paternal care than those in coalitions, but they are able to engage in more matings than males in coalitions. A Coalition Male will form a coalition with another Coalition Male with whom he will not compete for mates. Coalition Males can invest more effort in parental care because (1) they reap an economy of scale and (2) they also expend less effort competing for mates and mating For females, we consider three possible reproductive strategies:  for the evolution of Coalition Males, we look for any synergistic dynamic between allo-mothering and coalition-based monogamous male foragers.Analysis through Monte Carlo simulation shows mixed support for Male Coalitions to evolve. As in a recent work  to EPMs .However, positive assortment of Cooperative Mothers and Coalition Males can generally help favor Coalition Males. Some patterns in This analysis highlights the importance of the parameter ranges which specify the level of extra-pair matings for Coalition and Non-coalition males and whether there exists any assortment within and between the sexes. Given our current hypothesized parameter ranges in Our analysis suggests that female cooperation, rather than bi-parental care, would have provided a more robust system for helping hominid mothers care for infants. Consistent with recent work The monogamy observed in modern humans may have evolved in a context of multi-male, multi-female cooperative breeding group. As groups evolved increasingly complex culture, social recognition of long term relationships between mates may have emerged through a process of gene-cultural coevolution It is argued that females cooperating in the care of young is highly unlikely because of low relatedness between females who disperse on maturity Homo, hominids occupied the drier, less stable and more heterogeneous environments that were expanding in Africa during the early Plio-Pleistocene For cooperative mothering to be a compelling strategy, a division of labor between mothers would have to generate sufficient extra resources to allow a reduction in interbirth interval. The lack of cooperation seen among female apes suggests that it is not sufficiently beneficial in their case. However, if Australopithecine females produced larger babies than extant apes, as By the time members of the hominid line began to exploit drier and more varied habitats about two million years ago, it is likely they lived in cooperative breeding groups. If this is the case, then speculation about the course of human evolution and the adaptations thought to have emerged ought to consider what phenotypes would have been favored in this social system. Cooperative breeding groups would not have provided the selective environment favoring individuals with a capacity for choosing mates, nor individuals with a inclination to form a long-term attachment to a mate, nor males with an inclination to provide certain kinds of parental care e.g. . Rather,Our mathematical analysis suggests that female cooperatives are foundational to our modern life history. Nevertheless, our results should be taken as preliminary as work is needed to consider other potentially important factors and address further the parameter ranges given in Reproductive payoffs to females are described below, with the mathematical details in ad infinitum. Thus the fitness of CMs is divided into interactions with non-cooperative and cooperative female partners with reproductive payoffs discounted by the probability of the interaction taking place.A Cooperative Mother may interact with another CM, Independent Mothers (IM), or an Opportunistic Mother (OM). After CMs interact with an IM or OM once, they will search for another female partner. This continues until the CM meets another CM, then the two engage in cooperative allo-parenting ad infinitum. The survival probability of offspring to the breeding adult stage, without allo-parental and paternal care, is When two CMs interact each has an equal probability of being the allomother first, engages in alloparental care, then enters in reciprocal interactions in the next year with probability When a CM interacts with an OM there is a When OMs interact with an IM or another OM they both act as independent caregivers. An Independent Mother’s reproductive payoff is not contingent on any interactions with other females.Female and male reproductive output are tied together since offspring receive paternal care of amount Males can provision (or not) his offspring by a female with one of the strategies described above. Males who form coalitions and reap an economy of scale will have different fitness consequences due to a likely increased support of their children and less extra-pair reproductive activity. The paternal contribution to infant care, Extra-pair reproductive activity for Coalition Males is Information S1Derives fitness expressions for the female and male strategies described in the main text.(PDF)Click here for additional data file.