Teeth exhibit limited repair in response to damage, and dental pulp stem cells probably provide a source of cells to replace those damaged and to facilitate repair. Stem cells in other parts of the tooth, such as the periodontal ligament and growing roots, play more dynamic roles in tooth function and development. Dental stem cells can be obtained with ease, making them an attractive source of autologous stem cells for use in restoring vital pulp tissue removed because of infection, in regeneration of periodontal ligament lost in periodontal disease, and for generation of complete or partial tooth structures to form biological implants. As dental stem cells share properties with mesenchymal stem cells, there is also considerable interest in their wider potential to treat disorders involving mesenchymal  cell derivatives, such as in Parkinson's disease. Teeth are complex organs containing two separate specialized hard tissues, dentine and enamel, which form an integrated attachment complex with bone via a specialized  ligament. Embryologically, teeth are ectodermal organs that form from sequential reciprocal interactions between oral epithelial cells (ectoderm) and cranial neural crest derived mesenchymal cells. The epithelial cells give rise to enamel forming ameloblasts, and the mesenchymal cells form all other differentiated cells  . Teeth cRepair, restoration and replacement of teeth is unique among clinical treatments because of the huge numbers of patients involved. Paradoxically, although teeth are nonessential for life and thus not considered a major target for regenerative medicine research, in comparison with neural or cardiac diseases, for example, this very fact makes teeth ideal for testing new cell-based treatments. Because the patients are not usually ill, if anything goes wrong it is far less life threatening, and the accessibility of teeth means that treatment does not require major surgery. Added to this is the existence of highly proliferative stem cell populations in teeth, which can be easily obtained from naturally lost or surgically removed teeth. These stem cells can be used for tooth repair, restoration and regeneration and, significantly, non-dental uses, such as developing stem cell-based therapies for major life-threatening diseases. An important but often overlooked advantage of teeth as a source of stem cells is that postnatal root formation  is a developmental process, and thus cells involved in root formation are more embryonic-like than other sources of dental stem cells. The humble tooth clearly has a very important role to play in future developments in regenerative medicine.In this review, we outline the important biological properties of dental stem cells and illustrate examples of research showing the rapid progress being made in using these cells for tooth repair. We also highlight the major obstacles that need to be overcome before any form of usable, cell-based tooth replacement becomes available to practising dentists.in vitro and, to some extent, in vivo. The different cell populations do, however, differ in certain aspects of their growth rate in culture, marker gene expression and cell differentiation, although the extent to which these differences can be attributed to tissue of origin, function or culture conditions remains unclear.Several populations of cells with stem cell properties have been isolated from different parts of the tooth. These include cells from the pulp of both exfoliated (children's) and adult teeth, from the periodontal ligament that links the tooth root with the bone, from the tips of developing roots and from the tissue  that surrounds the unerupted tooth. All these cells probably share a common lineage of being derived from neural crest cells and all have generic mesenchymal stem cell-like properties, including expression of marker genes and differentiation into mesenchymal cell lineages  The possibility that tooth pulp might contain mesenchymal stem cells was first suggested by the observation that severe tooth damage that penetrates both enamel and dentine and into the pulp stimulates a limited natural repair process, by which new odontoblasts are formed, which produce new dentine to repair the lesion in vivo transplantation into immunocompromised mice demonstrated the ability of DPSCs to generate functional dental tissue in the form of dentine/pulp-like complexes in vitro such as odontoblasts, adipoctyes, chondrocytes and osteoblasts The first stem cells isolated from adult human dental pulp were termed dental pulp stem cells (DPSCs) in vitroin vivo and an ability to form sphere-like clusters Stem cells isolated from the pulp of human exfoliated deciduous (children's milk) teeth (SHED) have the capacity to induce bone formation, generate dentine and differentiate into other non-dental mesenchymal cell derivatives in vivo, where pulp removed because of infection is replaced with stem cells, have revealed that the tissue formed has architecture and cellularity closely resembling that of dental pulp, a tissue important for tooth vitality Studies using SHED as a tool in dental pulp tissue engineering SHED and other dental stem cells are derived from cranial neural crest ectomesenchyme, and so developmentally and functionally would appear identical, but studies have shown that they do differ and have different gene expression profiles. SHED have significantly higher proliferation rates compared with DPSC and bone marrow-derived mesenchymal stem cells DPSC are highly proliferative and retain their stem cell characteristics after prolonged culture in vitro and in vivoThe periodontal ligament (PDL) is a fibrous connective tissue that contains specialized cells located between the bone-like cementum and the inner wall of the alveolar bone socket that acts as a ‘shock absorber’ during mastication . The PDLThe periodontal ligament is under constant strain from the forces of mastication, and thus PDLSC are likely to play an endogenous role in maintaining PDL cell numbers. This might explain why they are better than other dental stem cell populations at forming PDL-like structures in vitro coincident with alkaline phosphatase upregulation. SCAP cells exhibit higher rates of proliferation in vitro than do DPSC A unique population of dental stem cells known as stem cells from the root apical papilla (SCAP) is located at the tips of growing tooth roots . The apiThe dental follicle is a loose ectomesenchyme-derived connective tissue sac surrounding the enamel organ and the dental papilla of the developing tooth germ before eruption in vitroDental follicle progenitor cells isolated from human third molars are characterized by their rapid attachment in culture, expression of the putative stem cell markers Nestin and Notch-1, and ability to form compact calcified nodules There are several areas of research for which dental stem cells are currently considered to offer potential for tissue regeneration. These include the obvious uses of cells to repair damaged tooth tissues such as dentine, periodontal ligament and dental pulp The periodontium is a set of specialized tissues that surround and support the teeth to maintain them in the jaw. Periodontitis is an inflammatory disease that affects the periodontium and results in irreversible loss of connective tissue attachment and the supporting alveolar bone. The challenge for cell-based replacement of a functional periodontium is therefore to form new ligament and bone, and to ensure that the appropriate connections are made between these tissues, as well as between the bone and tooth root. This is not a trivial undertaking, as these are very different tissues that form in an ordered manner  during tooth development. One aim of current research is to use different populations of dental stem cells to replicate the key events in periodontal development both temporally and spatially, so that healing can occur in a sequential manner to regenerate the periodontium N-isopropylacryl-amide) (PIPAAm)-grafted dishes that induce spontaneous detachment of the cells as viable cell sheets upon low temperature treatment. HPDL cells sheets were implanted into athymic rats that had the periodontium and cementum removed from their first molars. Fibril anchoring resembling native periodontal ligament fibres, together with an acellular cementum-like layer, was observed, indicating that this technique could be applicable to future periodontal regeneration. Although promising, this approach does not take into account any replacement of bone that might be required.A conceptually simpler approach to periodontal regeneration methods involves engineered cell sheets to facilitate human periodontal ligament (HPDL) cell transplantation The outstanding issue with these approaches is the extent to which any reconstituted periodontium can maintain integrity and function during mastication over long periods of time. Current treatments for severe periodontitis are poor, however, and thus, despite their flaws, any new dental stem cell-based treatments are likely to be the subject of intensive clinical research in the near future.de novo regeneration of dental pulp in emptied root canal space using dental stem cells Dental pulp needs to be removed when it becomes infected, and this is particularly problematic for root pulp that requires endodontic  treatment. The restoration of tooth pulp is thus a much sought after goal in dentistry because the current practice of replacing infected pulp with inorganic materials (cements) results in a devitalized (dead) tooth. A recent study demonstrated The current state of the art in tooth replacement is a dental implant that involves screwing a threaded metal rod into a predrilled hole in the bone, which is then capped with a plastic or ceramic crown. Implant use requires a minimum amount of bone to be present. Because these implants attach directly to the bone without the PDL ‘shock absorber’, the forces of mastication are transmitted directly to the bone, which is one reason implants can fail. In cases where there is insufficient bone for implants, such as tooth loss as a consequence of the bone loss that occurs in postmenopausal osteoporosis, implants have to be preceded by bone grafts. The ultimate goal in dentistry is to have a method to biologically replace lost teeth; in essence, a cell-based implant rather than a metal one. The minimum requirement for a biological replacement is to form the essential components required for a functional tooth, including roots, periodontal ligament, and nerve and blood supplies. Paradoxically, the visible part of the tooth, the crown, is less important because, although essential for function, synthetic tooth crowns function well, and can be perfectly matched for size, shape and colour. The challenge, therefore, for biological tooth replacement is ultimately one of forming a biological root.Currently, the major challenges in whole tooth regeneration are to identify non-embryonic sources of cells with the same properties as tooth germ cells and to develop culture systems that can expand cells that retain tooth forming potential . This isThe induction of odontogenic potential lies in the dental epithelium No sources of epithelial cells capable of inducing odontogenesis have been identified to date, other than the endogenous dental epithelium of early stage embryos. The main limitation for identifying sources of epithelial cells that can be grown in culture and form teeth after association with inducing mesenchymal cells is that these epithelial cells retain an immature state.in vitro on feeder layers, they can be induced to form enamel-like tissues following recombination with primary (uncultured) dental pulp cells The epithelial rests of Malassez (ERM) are a group of cells that remain during root formation; thus, these cells are present in adult teeth and can be isolated and cultured Oral mucosa epithelial cells from embryos and adults have been used in recombination experiments and shown to give rise to complex dental structures, but not whole functional teeth, when combined with embryonic dental mesenchyme in vivo, is daunting. Identification of key intracellular factors  is likely to be a more fruitful direction because these are more easily manipulated.There are sources of epithelial cells that can contribute to tooth formation following culture, suggesting that exogenously adding factors to these cells could make them inducible. Such factors, include signalling proteins of the fibroblast growth factor bone morphogenetic protein and Wnt families, but the issue of reproducing the temporal, spatial and quantitative delivery of these, as seen in vitro expansion was demonstrated when it was shown that expanded adult bone marrow stromal cells would form teeth in vitro when combined with inductive embryonic oral epithelium in vitro illustrated in the above section suggest that the alternative approach of identifying mesenchymal cells with inductive capacity might be more fruitful. The cells that have this capacity in vivo are the early embryonic neural crest-derived ectomesenchyme cells that have already received the first inductive signals from the dental epithelium . Similarly, equivalent cells from human embryos have been isolated and shown to form teeth in re-association experiments .If ectomesenchyme cells have odontogenic-inducing capacity, can this be maintained Adult dental pulp mesenchymal stem cells are an obvious source of cells to replace embryonic ectomesenchyme because they are derived from cranial neural crest and are ‘dental’ cells. Indeed, these cells retain expression of many genes expressed in neural crest, in addition to a number of stem cell ‘marker’ genes. However, it has yet to be shown that adult dental pulp mesenchymal stem cells retain any odontogenic inductive or responsive capacity. One interesting direction is to identify the factors expressed by ectomesenchyme cells  that render them capable of forming teeth that are not expressed by adult dental stem cells. Approaches similar to those developed for producing induced pluripotent stem cells can be used to convert adult dental stem cells into ectomesenchyme cells that can form teeth.4 cells dissociated from multiple tooth germs are required to generate a single new tooth germ in vitro expansion of epithelial and mesenchymal cells that will retain their odontogenic properties.Functional teeth can be experimentally bioengineered in mice by re-association of dissociated tooth cells Despite some progress, there remain major obstacles to formulating safe, simple and reproducible cell-based approaches for tooth repair and regeneration that could be used on patients. It is clear that there is both a clinical need for such treatments and a vast patient resource. Dental stem cells have many advantages, and results to date suggest that teeth are a viable source of adult mesenchymal stem cells for a wide range of clinical applications. Ultimately, the use of these dental stem cells over other sources of mesenchymal stem cells for therapeutic use will not only depend on ease of use and accessibility, but also on the efficiency and quality of repair in relation to cost. Dental pulp cells grow well in culture and, unusually, the proportion of cells with stem cell properties appears to increase with passage. The molecular basis of this phenomenon needs to be investigated because it might provide a paradigm for increasing stem cell numbers in cultures of other cell types.For whole tooth regeneration, there remain many major issues that will take considerable time to resolve. Most immediate is the identification of epithelial and mesenchymal cell populations that can be maintained and expanded in culture to provide the large numbers needed to make a tooth. Related to this is the issue of whether the cells will need to be autologous  or allogeneic . Finally, an additional fundamental issue that needs to be considered is that human tooth development is a much slower process than in mice. Human tooth embryogenesis is approximately eight times slower, and postnatal development lasts several years. Thus, whereas growth, implantation and eruption of bioengineered mouse teeth might take a few weeks, the equivalent time to create a functional human tooth might be many months or even years. Research thus needs to be done to investigate ways of possibly accelerating human tooth development.


Brain function and behavior undergo significant plasticity and refinement, particularly during specific critical and sensitive periods. In autistic and intellectual disability (ID) neurodevelopmental disorders (NDDs) and their corresponding genetic mouse models, impairments in many neuronal and behavioral phenotypes are temporally regulated and in some cases, transient. However, the links between neurobiological mechanisms governing typically normal brain and behavioral development  and timing of NDD impairments are not fully investigated. This perspective highlights temporal patterns of synaptic and neuronal impairment, with a restricted focus on autism and ID types of NDDs. Given the varying known genetic and environmental causes for NDDs, this perspective proposes two strategies for investigation: (1) a focus on neurobiological mechanisms underlying known critical periods in the  normal-developing brain; (2) investigation of spatio-temporal expression profiles of genes implicated in monogenic syndromes throughout affected brain regions. This approach may help explain why many NDDs with differing genetic causes can result in overlapping phenotypes at similar developmental stages and better predict vulnerable periods within these disorders, with implications for both therapeutic rescue and ultimately, prevention. Cognitive disorders, including intellectual disability (ID) and autism spectrum disorders (ASD) are genetically and phenotypically highly heterogeneous. To date, more than 450 candidate genes are associated with ID and many hundreds with ASD—numbers predicted to rise with the routine usage of high throughput sequencing technology  are documented well for the mouse visual cortex and occur during a restricted postnatal period. In Fmr1-KO mice, a short MD period induced a significantly smaller reduction in response in the deprived cortex and an enhanced potentiation of input from the open eye compared to wildtype (WT) mice  in the knock-out model rescues certain aspects of synaptic morphology only during a 2 day time-window, but not during earlier development or later in the adult . For human NDD syndromes, this could point towards prenatal and early neonatal changes in brain formation and function at stages not currently tested in the clinic. The implications of abnormalities in brain activity at such early developmental stages would be significant initially for detection and screening for NDDs in the fetus or newborn baby and raise possibilities for therapeutic interventions, technological challenges notwithstanding. It may also challenge the notion at which point a child is considered to be presymptomatic, if changes in brain activity are found at increasingly younger developmental stages.During development of sensory systems in the neurotypical brain, critical periods occur in a sequential pattern from brainstem, to thalamus to cortical regions as synapses form, refine and mature. Given that critical periods at thalamocortical and cortico-cortical synaptic pathways are affected in NDDs, we propose that dysregulation of synaptic pathways occurs at the subcortical level in NDDs at earlier stages than are currently known, during “presymptomatic” stages (Hypothesis 2). Thus, dysregulation and potential developmental delay for one known critical period would have a knock-on effect for synaptic circuits regulated at later timepoints at downstream synaptic pathways. Little is known regarding subcortical brain regions in NDD mouse models. However, alteration of GABAergic transmission and reduction of GABA-A receptor subunits is reported at postnatal day 7 in ventrolateral brainstem of MECP2 KO mice . Thus, a gene with limited postnatal expression in the brain would not give rise to aberrant prenatal synaptic phenotypes since the gene is not normally activated in cells prior to birth. One upshot of this idea is that discovery of prenatal expression patterns of a gene implicated in NDDs may not only lead to detection of prenatal synaptic phenotypes but highlight additional previously unknown functions of a gene during early developmental stages of the nervous system.Observations in mouse models of genetic NDD syndromes, demonstrate that alterations in synaptic networks occur during early brain development. Taking the Fmr1-KO mouse model, for example, reported thalamocortical and cortico-cortical synaptic impairments correlate with FMRP expression that occurs in the normally developing brain (Harlow et al., Alterations in activity levels during early neuronal network development lead to remodelling and compensatory changes in synaptic strength, a phenomenon known as homeostatic plasticity (Turrigiano and Nelson, The transience of synaptic impairments observed during sensitive time-windows (Meredith et al., Compensatory mechanisms may also operate during developmental stages of NDDs at the level of systems processing and behavior Johnson, . In an iIn conclusion, establishing the mechanisms that underlie early time windows for aberrations in synaptic circuits and impaired behavioral development in NDDs has the potential to reveal new approaches for pharmacotherapeutic correction of brain activity during early development or even new neurobiological gene targets (Levenga 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.


Recently, a wide range of nanotechnologies has been approached for material modification by realizing the fact that the extracellular matrix (ECM) consists of nanoscale components and exhibits nanoscale architectures. Moreover, cell-cell and cell- ECM interactions actively occur on the nanoscale and ultimately play large roles in determining cell fate in tissue engineering. Nanomaterials have provided the potential to preferentially control the behavior and differentiation of cells. The present paper reviews the need for nanotechnology in regenerative medicine and the role of nanotechnology in repairing, restoring, and regenerating damaged body parts, such as blood vessels, lungs, and the heart. Regenerative medicine holds great promise for restoring the normal, healthy functions of human tissues after damage. Its potential to treat a broad range of degenerative and ischemic diseases in tissues or organs has been improved with significant progress in understanding biological mechanisms. Based on current technologies, growing tissues and organs in the laboratory becomes a reality in regenerative medicine . Tissue Currently, engineered biomaterial scaffolds with biological functionalization through cell seeding have been widely used to regenerate healthy tissues for replacement. Instead of simply introducing healthy cells into a diseased region, cells are actually seeded onto biomaterial scaffolds before transplantation . These bNanomaterials have provided the potential to preferentially control the behavior and differentiation of cells by controlling nanoscale properties . With thNanoscale materials and therapeutics have been shown to play significant roles in tissue engineering applications since cells respond to nanoscale stimuli in spatial parameters , 4, 11. It is truly challenging to engineer ECM proteins and/or nanomaterial surfaces to function similar to the natural ECM proteins in tissues and organs. However, there have been enormous endeavors towards this goal through nanotexturing, nanopatterning and nanomaterials, although these techniques still need to be further developed , 13–15.ECM structure is also important to mimic because it generates the level of integrity required for structural support to resident cells, as well as regulating a deposition pattern of growth factors . For exaCells also encounter and interact with many topographical features which can range from folded proteins to banded collagen . This imNanoparticles are also an exciting subject in nanotechnology that has been investigated in the field of regenerative medicine. These particles are usually loaded with therapeutics and tagged with appropriate antibodies. These antibodies are specific to ligands in the diseased tissue so the nanoparticles circulate in the body for a long period and bind to the desired tissue. A current method of treatment is the intravenous injection of therapeutic agents –27. The The ECM of each organ in the body differs in their composition and spatial organizations . The ECMet. al. observed the behavior of smooth muscle cells (SMCs) on a biodegradable poly(L-lactide-co-ɛ-caprolactone) (75:25) scaffold fabricated to aligned nanofibrous structures by electrospinning [in vivo-like cell orientation and migration along the aligned nanofibers [Currently, there are a few conventional strategies used to enhance vascularization in tissue-engineered constructs. A favorable way to vascularize engineered tissues is to activate the natural angiogenic potential of the body ; howeverspinning . When conofibers . Tissue nofibers . TNs wernofibers .One of the largest causes of morbidity and mortality worldwide is ischemic tissue diseases. Therapeutic angiogenesis has emerged as a potential treatment plan because it enhances microvascular perfusion in ischemic tissue by delivering pro-angiogenic molecules. This treatment can be optimized through a better understanding of the nanoscale regulation of the process that involves both delivering growth factors and exerting complex signaling cascades. One of the most potent angiogenic factors that have been recognized is vascular endothelial growth factor (VEGF). Therefore, many pro-angiogenic treatments have attempted to utilize VEGF to enhance microvasculature in ischemic tissue. A primary obstacle of the treatments with VEGF is inadequate retention of the protein at the desired site of action. Webber et al., have developed a supramolecular nanostructure that mimics VEGF by reacting with VEGF receptors on the cell membrane . This naet al. have created a wide array of poly(lactic acid-co-glycolic acid) (PLGA) nanorough surfaces by using 190, 300, 400 and 530 nm polystyrene nanobeads through PDMS template molds  are another nanotechnology with potential for interventional lung cancer treatment. NP research is currently one of the most intensive scientific interests due to a wide range of potential applications in the field of regenerative medicine. NPs can effectively carry drugs or small molecules in order to modulate functions at the cell- and tissue-level, exemplifying how nanotechnology can impact physiological function on a much larger scale. One recent study investigated the effects of magnetic NPs loaded with doxorubicin on lung cancer cell function . The autNanoscale architecture plays a large role in manipulating protein adsorption, cell attachment, and cell functions . When coet. al. incorporated gold nanowires into alginate scaffolds which bridged the electrically resistant pore walls of alginate and improved electrical coupling between neighboring cadiomyocytes [When heart tissue is damaged due to a heart attack, cardiac patches can be treated for site-specific regeneration. These patches are three dimensional porous biomaterial scaffolds seeded with healthy cardiomyocytes , 47, 48.myocytes . These nAs previously stated, the effectiveness of nanoparticles has been investigated in inhibiting growth of lung carcinoma cells . NanoparThe field of nanotechnology has witnessed an explosion in research efforts over the past decade. Specific advances have been seen in nanotechnology applications to biomedicine that have revolutionized the way we view disease diagnosis and treatment, and tissue regeneration and repair. Scientists and engineers working together within the framework of nanotechnology have worked diligently to not only develop and apply their devices and methods within their own laboratories but have also sought far-reaching interdisciplinary collaborations to identify novel applications for these technologies. Continuous advances in nanotechnology will undoubtedly be observed as science and technology progress. Maintaining an open dialogue between researchers, clinicians, and industry partners is crucial for translatability of these revolutionary technologies. Without question, it is a very exciting time to work in the field of nanotechnology toward regenerative medicine as the number of researchers expands; the findings and technologies advance exponentially; and new avenues for research and application are discovered. Altogether, nanotechnology has provided a new set of tools that can help researchers solve current problems involving both the pulmonary and cardiovascular systems. However, nanotechnology in regenerative medicine is still at an infant level and is a complicated interdisciplinary field which needs the collective collaboration of physicists, chemists, biologists, engineers, and clinicians. To help mature the exciting field of nanotechnology, researchers must unravel the mechanisms of cell-biomaterial interactions at the nanoscale and develop unique nanotechnology applications in regenerative medicine.


Pathophysiology of Parkinson's disease (PD) is characterized by increased firing rates of cells in the basal ganglia, a tendency toward bursting and abnormal synchronization in cells of subthalamic nucleus (STN) and globus pallidus pars externa (GPe) . In advaWe use a STN-GPe network model consisting of 8-20 STN and GPe cells connected to each other via a sparse structured architecture . The dynDepending on both cell and synaptic parameters, the network model without STDP displays a variety of activity patterns, including synchronous, correlated dynamics and irregular firing. In the model with STDP, the nearly clustered activity patterns disappears and we observe only irregular firing or synchronized activity. Application of DBS replaces the clustered activity by high frequency firing. Switching DBS off, the network without STDP returns to a pathological activity pattern. In contrast, with STDP the temporary application of DBS changes the connectivity such that the network does not fall back to the clustered activity.Synaptic plasticity is the nervous system’s way to learn and adapt to sensory inputs, and this adaptation can strengthen both desired and pathological states. STDP pushes network dynamics to irregular  or synchronized  state, depending on parameter values. Moreover, our results suggest that DBS can use STDP to teach the network to fire in a less pathological manner. This teaching ability of DBS can be exploited to develop more efficient stimulation protocols.


Changes in epigenetic marks such as DNA methylation and histone acetylation are associated with a broad range of disease traits, including cancer, asthma, metabolic disorders, and various reproductive conditions. It seems plausible that changes in epigenetic state may be induced by environmental exposures such as malnutrition, tobacco smoke, air pollutants, metals, organic chemicals, other sources of oxidative stress, and the microbiome, particularly if the exposure occurs during key periods of development. Thus, epigenetic changes could represent an important pathway by which environmental factors influence disease risks, both within individuals and across generations. We discuss some of the challenges in studying epigenetic mediation of pathogenesis and describe some unique opportunities for exploring these phenomena. The field of epigenetics grew from attempts, beginning over 70 years ago, to understand mechanisms whereby multiple cellular phenotypes arise from a single genotype during the complex process of developmental morphogenesis termed epigenesis. The term “epigenetics” was initially reserved for mechanisms by which phenotypic state, as determined by differential gene expression, could be stably retained through cell division by non-genetic factors. Various mechanisms have been proposed to have the potential to encode this phenotypic information; these include enzymatic methylation of cytosine bases (DNA methylation), post-translational modification of tail domains of histone proteins (histone modifications) and associated nucleosome positioning or chromatin remodeling, non-coding RNAs, and transcription factor regulatory networks Ptashne . Epigeneepigenetic processes those that stably affect gene expression through mechanisms not involving the primary nucleotide sequence, and epigenetic state as the configuration of chromatin and DNA marks utilized by these processes. By contrast, genetic state is widely understood to refer to the primary nucleotide sequence itself, while genetic processes maintain or change nucleotide sequence.The term epigenetics has more recently been used in the scientific literature to describe various unspecified non-genetic mechanisms influencing phenotype. This broader usage emerged from mouse studies addressing transgenerational nutritional effects on phenotype, as well as human studies of phenotypic differences between monozygotic twins. In the popular press “epigenetics” has become almost synonymous with nutritional and environmental influences on gene expression. Thus, while “epigenetics” initially referred to largely self-contained developmental processes, it has come to describe environmental influences on phenotypic readout of genotypes. This semantic evolution has caused confusion and controversy regarding the meaning of “epigenetics” at a time of intensified interest in the possible role of epigenetic mechanisms in disease. In this review we define as Epidemiologic research addressing epigenetic mechanisms as mediators of environmental exposures on disease risk is constrained by important ethical considerations. These often preclude both experimental exposure to candidate environmental causes, and invasive collection of cell types of greatest developmental and functional relevance to disease processes. Inquiry has therefore progressed largely by integrating information about biological mechanisms obtained in model systems with observational data provided by humans. To address the current state and future promise of this research, we undertook this review with two goals: to illustrate the potential of epigenetic processes to mediate exposure–phenotype relationships and to discuss study design and statistical analysis methods needed to investigate such mechanisms in relation to origins of human disease. We begin by discussing genetic, developmental, and environmental determinants of epigenetic state in human and model systems, then describe some of the diverse data implicating epigenetic mechanisms in various human diseases, both within individuals and across generations. We conclude by discussing technical challenges, suggesting promising opportunities for epidemiologic research in environmental epigenetics, and offering some thoughts about translational significance and future directions of this field.epigenome, varies by cell type. Considering the tremendous diversity of epigenetic marks, which include dozens of different post-translational histone modifications and more than 50 million sites of potential DNA methylation in a diploid human genome (and thus >250M possible epigenotypes!), it seems that no two human cells would have identical epigenomes. Indeed, within each individual there are many epigenomes, and these change over time as a consequence of both normal developmental and pathological processes, as well as environmental exposures and random drift. Despite this potential for considerable variability of epigenetic patterns within and between individuals, there can also be remarkable consistency. In a study of 11 tissues in 6 autopsies, DNA methylation patterns in a highly selected set of loci were found to be highly conserved, with intraclass correlations of 0.85 across tissues within individuals and 0.83 across individuals within tissues  density  DNA, followed by more general loss of methyl marks in the zygote and embryo during cleavage divisions, while sparing parent-of-origin specific imprints. By the blastocyst stage, de novo DNA methylation distinguishes inner cell mass cells  from relatively hypomethylated trophectoderm cells  Fig. .Fig. 1ReGerm cell lineage specification begins in cells of the proximal epiblast, and involves a second extensive erasure of DNA methylation that removes parental imprint marks Fig. . ThereafDevelopmental reprogramming can result in dramatic epigenetic differences between the two alleles. The association between mono-allelic gene expression and DNA methylation has long been recognized, both in the context of X-inactivation in females  and PPARα gene promoter regions in rat liver  are very short non-coding RNA molecules that can downregulate protein-coding genes by destabilizing mRNAs or blocking translation. The possibility that exogenous microRNA consumed in food may epigenetically regulate gene expression has emerged from recent studies demonstrating the presence of plant-derived miRNAs in sera of humans and other mammals  is associated with reduced methylation of several repeated sequences, including Sat2  are involved in numerous cellular processes including cellular redox alterations, immune response, signaling pathways, chromatin remodeling and gene expression  of maternal (egg-derived) and one set of paternal (sperm-derived) chromosomes could develop normally. However, abnormal development and early demise occurred in all conceptuses receiving either two maternal sets or two paternal sets of chromosomes  are more likely to produce agouti offspring, while dams of the same strain without the Avy epiallele (who have yellow coats) are more likely to produce yellow offspring. A continuous gradient in coat color is related to the penetrance of the Avy epiallele  in agouti mice. Dams carrying the meta-stable AIn a separate model, experimental exposure of pregnant rats to endocrine-disrupting agents  produced changes in male infertility, body weight, and cancer risk in up to four subsequent generations  from chronic exposures may lend useful specificity for addressing some research questions. Persistent biomarkers , when available, are promising if the specific period of exposure is not crucial. Exposure assessed prospectively and tightly linked to proposed periods of vulnerability of the epigenome  would be ideal. However, this rarely occurs in epidemiologic settings with sufficient power to detect most disease associations. However, estimating exposure–epigenetic state associations may be more feasible.Exposures of limited duration corresponding to disasters , availability of regulated or withdrawn compounds , or therapeutic indications of limited duration  can sometimes be retrospectively assessed with reasonable accuracy. Cohorts based on such exposures, although rare and often small, may be especially valuable in efforts to elucidate transgenerational processes.Since assessment of covariates may be equally challenging, there is considerable potential for confounding of exposure–epigenetic state or exposure–disease associations by factors associated with exposure, except in rare circumstances when exposure can be randomly assigned to study participants.Traditional molecular biological methods of analyzing gene loci often involve amplification through cloning, whole-genome amplification (WGA), or polymerase chain reaction (PCR). All of these techniques erase any existing marks in extracted DNA or chromatin. Therefore, methods have been developed to alter the DNA using methods influenced by the epigenetic state prior to amplification. The three main methods employed are affinity enrichment, restriction digestion, and chemical conversion by sodium bisulfite Laird . AffinitThe requirement of relatively large amounts of intact chromatin for ChIP-chip or ChIP-seq has limited the study of histone modifications and other protein-associated epigenetic marks to biospecimens with large amounts of material with intact cells. The vast majority of studies on ChIP have used cultured cell lines. More recently, techniques to analyze small quantities of fresh tissue have been explored , which is only partially observed at particular time points tk by flawed assays Mik. Using standard measurement error modeling approaches, one might then treat the latent process as being determined by one’s constitutional genotype Gi and exposure history Ei(t), through a linear longitudinal random-effects model, with the latent process in turn being a risk factor for disease state Yi(t), through a standard survival analysis model  are transmitted to the parents via transmitted epigenotypes (Gmm* etc.), which are modified by the parents’ environments and in turn transmitted to the offspring generation to determine their phenotypes, in combination with their own exposures at the relevant developmental stage.To address transgenerational phenomena, we propose an extension of this approach illustrated in Fig. Much of the early work on the role of epigenetics in disease has focused either on global methylation, differentially methylated regions associated with several imprinted loci, or promoter regions of selected candidate genes. Now, facilitated by the advent of high-density methylation arrays like the Illumina Infinium Beadarray that can interrogate up to 480K CpG sites, the kinds of agnostic genome-wide scans that have become popular for investigating germline genetic variation [genome-wide association scans (GWAS)] are likely to become widely used for epigenetics as well. There are two forms such studies could take. The first is a genome-wide scan for epigenetic variation in relation to disease risk—what has recently been called an epigenome-wide association scan (EWAS)  and trans-associations (associations with epigenetic state of genes located at some distance from the SNP or on separate chromosomes). Although the vast majority of associations in HapMap cell lines studied by Bell et al. were of the cis type, they found a trans association of a SNP in the DIP2B gene (thought to be involved in DNA methylation) with the first principal component of genome-wide methylation.The second approach to agnostic genome-wide epigenetic research uses methylation quantitative trait loci (mQTLs). These seek genome-wide SNP variation associated with genome-wide epigenetic state , as summarized by Jaffe et al. . Not onlDespite the wealth of research on the role of epigenetics in disease, there has been much less activity on the environmental determinants of epigenetic changes and their role of epigenetic state in mediating exposure–response relationships, either within individuals or across generations. Rakyan et al.  provide Studies of the genetic determinants of disease, whether based on candidate site hypotheses, pathway-based hypotheses, or GWAS technology, have utilized cross-sectional studies in what is conceptually a case–control approach, since the “exposures” in question were genotypes presumed to be immutable. Analogous studies of disease outcomes in light of epigenetic alterations, however, are fundamentally flawed, not only because earlier causal events could always be responsible for observed epigenetic change, but also because any stage in the pathophysiology of the disease and/or any treatment prior to sample collection could itself generate epigenetic change. De novo cohort studies of the genetic determinants of disease require biosamples at the outset, are slow to yield results, and often prohibitively expensive, because the rare outcomes of greatest interest can only be assessed if the genomic subsets are very large. Cohort studies of the causation of endpoint phenotypes or even intermediary risk factors by epigenetic characteristics are likely to be just as difficult. Moreover, observational studies of human epigenetic events as usually conducted are confounded by age, genomic characteristics, and the earlier environmental milieu, including the prepartum component.One can, however, study epigenetic alterations as outcomes, and by so doing, come to understand their role as intermediary agents between disease outcomes and the toxic agents known to cause them. Cross-sectional studies of cells from exposed and unexposed individuals are interpretable under the assumption that the epigenetic alterations are unlikely to have produced the behavior that led to the toxic exposure. Over the long term, comparison between the epigenetic experiences of exposed and unexposed cohorts will require repeated epigenetic characterization of persons with particular toxic exposures or particular characteristics, such as jobs that stand as surrogate measures of such exposure.In contrast to observational studies of singletons, twin studies can provide several advantages for studies of epigenetics . Moreover, mRNA and protein encoded by HERVWE1 was more highly expressed in placental tissue of smaller co-twins  member 1 gene  specific exposures induced measurable changes in epigenetic state, (2) new epigenetic state was shown to persist after cessation of exposure, and (3) measureable phenotype was associated with exposure-related changes in epigenetic state. A fourth element, exposure-induced alteration of epigenetic marks of germ cell lineage DNA, seems necessary for transgenerational transmission of environmentally induced epigenetic changes. On this background, we interpret observational human data illustrating associations of exposures to epigenetic state and epigenetic state to disease to indicate that epigenetic mechanisms may plausibly mediate exposure effects on a wide range of human diseases.unlike genetic mutations, epigenetic profiles are potentially reversible. Therefore, epigenetic approaches for prevention and treatment, such as nutritional supplementation and/or pharmaceutical therapies may be developed to counteract negative epigenomic profiles.Perhaps the greatest significance of this line of research is the potential for designing novel interventions articulated by Dolinoy and Jirtle :unlike gAs an example, Huang et al.  have devWe need to better understand basic epigenetic mechanisms that operate during selected periods when the epigenome may be particularly vulnerable to environmental exposures  as well as mechanisms that routinely maintain proper epigenetic states. We also need to understand mechanisms relevant to cumulative effects of exposure and associations with aging, since duration of exposure and aging are co-linear. A better understanding of basic mechanisms at different time points in life would guide genetic epidemiology studies in selecting environmental exposures that are more likely to affect epigenetic processes, identify the most relevant periods of exposure, identify biomarkers of exposure, and suggest biological intermediates to study.It may be helpful to more deliberately deconstruct the steps whereby an environmental exposure may affect risk of disease via epigenetic mechanisms. For example, studies of the association of environmental exposures with selected epigenetic events as endpoints (rather than intermediates in a complex pathway) may be fruitful and will likely help us better conceive of studies to elucidate an entire pathway, from an exposure acting at least in part through epigenetic mechanisms to disease risk.As we proceed, it will be important to consider seriously the environmental exposures to study, since we will want to select the most promising candidate exposures (those most likely to affect epigenetic processes), measuring these exposures at relevant time periods and with reasonable accuracy. Also, we cannot ignore genetic influences on epigenetic processes. We see above that studies of the epigenetic state of specific candidate genes are yielding interesting results and more such studies are warranted. Given limited knowledge of this genetic–epigenetic interface, to complement the candidate gene approach, integrative genomics approaches should be contemplated in order to combine GWAS data with epigenomic data. Clearly, statistical and bioinformatics approaches will be required to enable the efficient conduct of these analyses, especially as we expand to genome-wide scale. Similarly, research on feasible, efficient study designs, akin to current research on optimum designs for sequencing-based studies, will be needed. Finally, as with other emerging fields , standardization of terminology and testing protocols will facilitate future communication of hypotheses, scientific approaches, and results.


In some cases, the adaptations may persist after the initial oncogenic stimulus is gone in a type of “hit-and-run” oncogenesis. These pathophysiological changes may interfere with cancer prevention therapies targeted solely to the initial oncogenic insult, perhaps contributing to resistance development. Characterization of these accommodating adaptations could provide insight for the development of cancer preventive regimens that might more effectively biomodulate preneoplastic cells toward a more normal state.In a medical sense, biomodulation could be considered a biochemical or cellular response to a disease or therapeutic stimulus. In cancer pathophysiology, the initial oncogenic stimulus leads to cellular and biochemical changes that allow cells, tissue, and organism to accommodate and accept the oncogenic insult. In epithelial cell cancer development, the process of carcinogenesis is frequently characterized by sequential cellular and biochemical adaptations as cells transition through hyperplasia, dysplasia, atypical dysplasia, carcinoma  For example, it has been applied to specific therapies for kidney disease, that is, the impact of freeze-dried Lactobacillus acidophilus on the bacterial overgrowth syndrome occurring in end-stage kidney disease.17To some extent, all therapeutic interventions could be considered biomodulation, as they are all directed toward a change in the individual's pathophysiology at both cellular and systemic levels. For the purposes of discussion here, in situ or invasive stages. The value of considering cancer prevention in the context of biomodulation would be to conceptually encompass the therapeutic approach as both an assault on the initiating event as targeted therapy, as well as an attack on the secondary adaptations that a preneoplastic cell, and surrounding local stromal and systemic environments, may have made to survive or adapt to that initiating stimulus (BRCA1), breast cancer 2, early onset (BRCA2), phosphatase and tensin homolog (PTEN), and tumor protein p53 (TP53). It is possible that specific biomodulatory events accompany epithelial cancer progression in these cases. Identification of these events may lead to more targeted chemopreventive approaches for individuals carrying these mutations. Some types of sporadic cancer development are also characterized by genetic changes, sometimes in the same genes found with familial germ-line predisposition. It will be important to determine whether the types of biomodulatory changes that occur when cancer develops secondary to a germ-line risk factor are the same as those that may occur when the mutation occurs in somatic cells as part of sporadic cancer development. It is quite possible that genetic background, both germ-line and somatic, will influence the response to specific biomodulatory approaches.Evolution of cancer in epithelial tissues frequently develops as a sequence of coordinated changes in both the epithelial and stromal compartments . Biomodustimulus . The proThis short review concentrates on what type of molecular changes might occur in an epithelial cell as secondary cellular adaptations that occur over time following a carcinogenic stimulus, to determine if they are disease drivers, and to test if they can be recognized before therapy targeted to the initial stimulus or whether they only become apparent after the initiating stimulus has been removed. It is possible that cancer prevention approaches that target both disease-initiating stimuli and secondary adaptations would more efficiently effect disease reversal.in vivo phenomenon can be modeled in some tissue culture cells in vitro. For example, long-term exposure to estrogen can induce persistent changes in gene expression in the estrogen receptor alpha (ERα)-positive MCF7 cell line.50Cancer development is not a unitary process but instead is executed by different processes. A relatively direct mechanism of cancer development and maintenance is “oncogene addiction.” In this situation the growth and survival of the cancer cell is dependent upon maintenance of a cancer causing stimulus, such as a defined oncogene,+ T cells+ T cells playing a role in regression following loss of Breakpoint cluster region-Abelson (BCR-ABL) as well.myc oncogene.37Experimentally, a conditional system, such as the tetracycline responsive gene expression systemS-methyltransferase (MGMT),Conditional experimental systems also enable examination of cellular and biochemical changes that occur after the initiating oncogenic stimulus has been downregulated. In the case of TAg expression in striated duct cells in the submandibular salivary gland, dysplasia reverses when TAg is downregulated at four months of age but persists when TAg is downregulated at seven months of age.In the case of TAg-induced age-dependent reversible and irreversible hyperplasia and dysplasia, there are multiple molecular dissimilarities.37Similar to the salivary gland experimental system, loss of cell cycle checkpoints also occurs during breast cancer progression. Some endocrine-resistant breast cancers in women are reported to exhibit higher levels of pRb, consistent with loss of cell cycle control.The message from experimental studies of cancer progression and reversal is that when oncogene addiction is not the case, secondary pathophysiological adaptations induced by oncogene exposure may need to be identified and targeted for successful therapy . Studies+ and ERα− disease is found in these cases.in situ (DCIS)), it does not absolutely prevent it.Development of such strategies might advance breast cancer prevention approaches. For example, tamoxifen is the only FDA-approved drug for chemoprevention of invasive breast cancer in premenopausal high-risk women; however, for every ten women who take tamoxifen, one to two will still develop invasive breast cancer. Both ERαThinking of cancer prevention as biomodulation with attention not only to the initiating stimulus but also encompassing consequent alterations in cellular and biochemical pathways may provide a more robust approach to developing effective chemoprevention. State-of-the-art approaches to accomplish this may include the application of RNAseq, proteomics, and metabolomics to identify the multiplexed changes that occur as part of the process of biomodulation in epithelial tissue that has become preneoplastic. Validation of specific targets within what may be a multitude of changes will be challenging and difficult to accomplish utilizing only previous clinical trial designs. More personalized and intensive comprehensive studies that incorporate serial testing of the response to candidate agents and include the use of novel approaches for primary cell culture, both epithelial and stromal, as well as evaluation of systemic factors such as immune response, may be required. The use of genetically engineered mouse models are likely to facilitate development of this comprehensive approach as they provide a renewable source of material for testing the positive and negative predictive value and sensitivity of specific algorithms used for the development of new chemopreventive approaches targeting both the original lesion and the secondary changes that occur in the course of cancer development.


The findings of this study call for a holistic approach to assessing the risks posed to reef fish aggregations by fishing, grounded in the principals of fisheries science and emerging social-ecological thinking.Reef fishes that exhibit predictable aggregating behaviour are often considered vulnerable to overexploitation. However, fisher knowledge of this behaviour is often heterogeneous and, coupled with socioeconomic factors that constrain demand for or access to aggregated fish, will influence susceptibility to fishing. At two case study locations in Papua New Guinea, Ahus and Karkar islands, we conducted interview-based surveys to examine how local context influenced heterogeneity in knowledge of fish aggregations. We then explored the role of fisher knowledge in conferring susceptibility to fishing relative to socioeconomic drivers of fishing effort. Local heterogeneity in knowledge of aggregating behaviour differed between our case studies. At Ahus, variable access rights among fishers and genders to the main habitats were sources of heterogeneity in knowledge. By contrast, knowledge was more homogenous at Karkar and the sole source of variation was gear type. Differences between locations in the susceptibility of aggregations to fishing depended primarily on socioeconomic drivers of fishing effort rather than catchability. While Ahus fishers were knowledgeable of fish aggregations and used more selective gears, Karkar fishers were less constrained by tenure in their access to aggregation habitat. However, fishing effort was greater at Ahus and likely related to high dependency on fishing, greater access to provincial capital markets than Karkar and a weakening of customary management. Moreover, highly efficient fishing techniques have emerged at Ahus to exploit the non-reproductive aggregating behaviour of target species. Understanding how knowledge is structured within fishing communities and its relation to socioeconomic drivers of fishing effort is important if customary practices for conservation, such as  The depletion of reef fish biomass is often attributed to overfishing driven by socioeconomic drivers such as local human population density and distance from reefs to markets The exploitation of reef fish spawning aggregations is an obvious example of fishers utilising knowledge on fish behaviour to target populations when their density has increased. A large number of important food fishes on coral reefs aggregate periodically at high density to spawn Regardless of their biological predictability, fisher knowledge of aggregations is heterogeneous and will influence the extent to which aggregations are perceived as predictable and exploited by fishers. Fisher knowledge maybe stratified by factors such as gender, age, location and cultural background Fisher knowledge of fish aggregating behaviour will be influenced by cognitive processes  and the formation of heuristic models Assessing the status of aggregating reef fish populations is problematic due to the data-poor context of their fisheries Studies documenting fisher knowledge of reef fish aggregations have primarily gathered information in order to identify research, conservation and management priorities The study was approved by the Human Ethics Research Committee of James Cook University . A permit was obtained for research in Papua New Guinea (Permit number 10350012505). Due to low levels of literacy at our study locations, the Human Ethics Research Committee approved for fishers to provide verbal consent to participate in the study, according to the following procedures. An information sheet detailing the aims of the research and how data were to be used was translated verbally to participants, which also specified their rights to withdraw their information at any time and guaranteed their anonymity as participants. This was followed by verbally translating a consent form to participants. Upon consent, the consent form was signed by the lead author (JR) and translator, copies of which are stored with the unprocessed data at James Cook University. For follow-on contact with the lead author, copies of the information sheet were left with clan leaders at each location.We studied reef fisheries at two locations in Papua New Guinea, representing two extremes of fishing pressure and comprising gear and fishing practices common to the region tambu) to restrict fishing in certain areas in an effort to influence catchability (i.e. make fish less wary of spear fishers) or rebuild biomass for feasts Fishers at the study sites use a combination of gear types including line, net and spears. Fishing effort at Ahus primarily comprises use of lines and spearguns , whereas effort at Karkar comprises both of these gears (72%) in combination with hand spearing (24%). By comparison, a small proportion (<4%) of fishing effort at both study sites involves use of nets Epinephelus fuscoguttatus, Plectropomus areolatus), two emperors  and two snappers . All species are high tropic level predators (trophic levels 3.6–4.5) and were selected to contrast forms and functions of aggregating behaviour (Interviews were conducted with fishers at Ahus (n = 16) and Karkar  in October 2012 to quantify fisher knowledge on aggregating behaviour and to develop an index of knowledge pertaining to fisher perceptions of aggregation predictability. At Karkar, interviews were conducted with all fishers for whom fishing was a regular livelihood activity. In Ahus, where the proportion of residents engaged in fishing was high by comparison, a sample of fishers, representative of fisher gender, clan membership and gear use, was taken. After pilot studies (n = 4) with Karkar fishers, a semi-structured questionnaire was designed to investigate fisher knowledge of the form, function and predictability of aggregating behaviour for six species of reef fish common to fisheries in both locations. The six species comprised two groupers (ehaviour .−1) that they typically obtain for each species when using their primary gear. Thirdly, fishers were questioned on their knowledge of aggregating behaviour, initially focusing on whether they observe a species to display solitary, shoaling (groups of three or more fish displaying unsynchronised swimming) and schooling (groups of three or more fish displaying synchronised swimming) behaviour Firstly, species recognition by fishers was established using a combination of pictures (landed specimens) and local names specific to location. Secondly, we asked fishers whether they caught each species frequently, infrequently or not at all and, if they encountered the species, to estimate the ‘poor’, ‘normal’ and ‘good’ catch rates (fish.tripsee below), an index of fisher knowledge of aggregating behaviour was calculated for each fisher and species by summing the scores for the seven aggregation attributes  increased with spawning.Seven attributes of the spatial, temporal and physical manifestations of aggregating behaviour were employed to quantify fisher knowledge of aggregations and their perceived predictability . AggregaTo investigate sources of variation in fisher knowledge within case study locations, data relating to socioeconomic indicators were collected during interviews. Indicators were selected based on literature pertaining to sources of variation in knowledge among coral reef fishers and included gender, dependency on fishing as a livelihood, and access rights to major reef habitats and gear types . Gender A productivity-susceptibility analysis (PSA) using the bivariate framework of Hobday et al. (2011) −1). The mean ‘good’ catch rate across fisher responses was taken on each gear used for a species, from which gear selectivity was ranked by order of catch rate. Fourthly, we replaced post-capture mortality The four susceptibility attributes of Hobday et al. (2011) as detailed above) and markets that they access, respectively. Management strategy was a location-level attribute adopted from Patrick et al. (2010) The attributes of Hobday et al. (2011) t-test, assuming unequal variances. For each species, associations between fisher knowledge on aggregation form at Ahus and Karkar were analysed by constructing contingency tables of the frequency of observation for each of the eight categories . Cramér's V contingency coefficient was used as the measure of association; the coefficient ranges between 0 (no association) and 1 (perfect association). Since expected frequencies were less than five for a high proportion of cells in the contingency table, p-values were calculated using Monte Carlo simulation .The mean ‘normal’ catch rate estimated by fishers for each species was compared between locations using a p-values reported rather than asymptotic values due to small sample sizes (n≤17). Owing to the risk of type 1 errors arising from multiple comparisons, p-values were adjusted with a false discovery rate (FDR) correction for multiple testing using the Benjamini-Hochberg method Several methods were employed to examine how fisher knowledge of aggregations varied by location and species. Firstly, for each species and aggregation attribute, fisher respondent scores were averaged in each location and the difference between the averages (Ahus minus Karkar) plotted. Secondly, Mann-Whitney U tests were used to compare fisher responses in each location, again for each species and attribute, with exact significance (2-sided) Redundancy analysis (RDA), which combines concepts of ordination and regression p-values are reported due to the small number of paired-samples (n = 6).Estimates of productivity attributes were averaged to give a single productivity score per species. To assess the relative importance of catchability and socioeconomic drivers of fishing pressure for the six species, each susceptibility attribute was first scored for each fisher respondent individually and then averaged across fishers to give a single attribute score for each location. Within location, the susceptibility attributes were combined by averaging across two sets of attributes; (1) the full set of 10 susceptibility attributes, and (2) the four catchability attributes . ConsequL. gibbus and L. harak were encountered frequently and L. fulviflamma and L. lentjan infrequently, again based on median fisher response. Reported catch rates of snappers tended to be greater than those of emperors and groupers . The groupers were primarily perceived as solitary by fishers from Karkar while a larger proportion of Ahus fishers recognised that they are generally solitary but also form spawning aggregations . Emperor aggregating behaviour was also considered more complex than that of groupers, encompassing reports of schooling by two fishers, but also tended towards solitary and shoaling behaviour .Fishers from Ahus and Karkar had different perceptions on the forms of aggregating behaviour exhibited by the two groupers  aggregating behaviour was largely shared among fishers, with the species orientated on the second canonical axis that only accounted for 6.1% of the variation. Therefore, with the exception of snappers, knowledge of species aggregating behaviour at Ahus was heterogeneous and explained by fisher gender and right of access to the major reef habitats  aggregating behaviour again clustered on the first canonical axis but was restricted to only two fishers primarily using spearguns. Use of this gear constituted the only factor that significantly explained variation in knowledge  were equal in their productivity scores    . However= 0.156) . This oc= 0.156) . With th= 0.156) . The scotambu areas, in regulating fishing effort.Supportive of previous research findings in Papua New Guinea Fisher knowledge of aggregating behaviour was particularly heterogeneous at Ahus. Since variation at Ahus related to gender and access to reef habitats, it appears to stem from the decentralised tenure system that specifies ownership rights to space, species, gear and the techniques for using gears among kinship groups Variation in fisher knowledge of aggregating behaviour differed among the three families of reef fish. Compared to emperors and groupers, knowledge of aggregations of the two snappers was greater and relatively homogenous. Snapper aggregation locations were perceived by fishers as spatially predictable and were associated with particular features of the reef, corresponding with empirical evidence for these species Heterogeneity in fisher knowledge relating to grouper spawning aggregations was structured by access rights or gear type, depending on location. Grouper spawning aggregations mainly form on outer reef slopes and channels L. harak, a generally solitary species common to that habitat L. lentjan spawning in aggregations were also limited to Ahus fishers, and are consistent with anecdotal reports of this behaviour from other countries L. lentjan primarily feed in deeper water As with groupers, heterogeneity in fisher knowledge of emperor behaviour also stemmed from variable rights of access among fishers. Thus, lagoon fishers at Ahus developed specialist knowledge of the behaviour of Our study was a first step in quantitatively exploring the factors that influence local ecological knowledge of reef fish aggregating behaviour within communities, but was limited to a small number of socioeconomic indicators that reflect the contemporary context of the two communities. Consequently, we did not quantify important historical aspects of these communities and the role of oral histories in transferring knowledge, which may have influenced the patterns observed in our data. For example, though we pooled respondents from the two study villages of Karkar Island in our analyses, knowledge of grouper spawning aggregations was higher  among Muluk fishers than neighbouring fishers from Wadau. Since fishers exhibit similar dependency on fishing, this finding may reflect the differing historical context of two communities, which may have settled on the coast at different times Comparing several locations in Melanesia, Hamilton et al. (2004) This study demonstrated the utility of PSA in examining how attributes relating to catchability and socioeconomic drivers of fishing effort influence the susceptibility of reef fish populations to aggregation fishing. Given that experts with access to scientific information have scored susceptibility indictors in previous applications of PSA E. fuscoguttatus, L. gibbus and P. areolatusL. gibbusKnowledge of fish aggregating behaviour is commonly utilized by fishers to improve catchability and returns from a fishery tambu at Ahus will have been lost with the recent breakdown in this governance system An advantage of PSA is that the susceptibility attributes included can be adapted to local contexts or issues of importance. For example, Patrick et al. (2010) PSA and other semi-quantitative approaches are, however, sensitive to the assumptions made in developing indicators An additional caveat in the use of indicator-based approaches is that they can be overly reductionist in attempting to simplify complex socio-ecological systems. Consequently, PSA can be combined with more detailed social, economic or ecological research to better understand the management implications of more complex interactions that indicators fail to capture. It is ideally applied as a participatory risk assessment tool for supporting communication, promoting understanding, building consensus and prioritizing actions as part of community-based management planning. In our application of this tool, five populations were identified to be at medium risk from aggregation fishing. However, a participatory application of PSA in the two communities may have yielded different results. For example, resource users could develop their own indicators or choose to weight indicators according to their own priorities tambu areas, are to be supported by working with relevant kinship groups. While knowledge alone does not imply that fishers will maximize extraction from a fishery, shifts in socioeconomic drivers may serve to increase fishing pressure. For example, a breakdown in ownership rights and resulting spread of knowledge among kinship groups has been identified as a cause for concern in relation to pressure on spawning aggregations in PNG tambu areas, would be exacerbated if the system of ownership rights also weakened. However, aggregations forming for purposes other than reproduction are also predictable and may be highly susceptible to fishing if efficient gears are used To conclude, heterogeneity in fisher knowledge relating to reef fish aggregating behaviour will be influenced by social, economic and cultural factors that are specific to the local context. Understanding how knowledge is structured within a community will be important if customary practices for conservation, such as


These discoveries suggest that sociality, a key ungulate strategy to reduce predation-related mortality, selects for inherently longer-lived organisms, and that strong sexual selection, which is known to compromise survival rates in the wild, can constrain also intrinsic lifespan. The contrasting results for males and females indicate that selection on longevity in the two sexes is partly uncoupled.Selection on intrinsic lifespan depends on both external factors affecting mortality and inherent tradeoffs in resource allocation between viability traits and other fitness-related traits. Longevity is therefore likely to vary between species in a sex-specific manner due to interspecific and intersexual differences in behavioural ecology. Here I focus on the bovid family to test two central hypotheses on longevity selection using the comparative method: firstly, that a reduction of extrinsic mortality in social species strengthens selection on intrinsic lifespan, and secondly, that mortality costs associated with intense sexual selection lead to shorter intrinsic lifespan. The results show that longevity ( The extent to which intrinsic lifespan is visible to selection depends on extrinsic mortality rates due to environmental conditions Intrinsic lifespan may also depend crucially on the intensity of sexual selection I here adopt the comparative method with phylogenetic control to investigate the impact of sociality and sexual selection on the evolution of intrinsic lifespan, using the family Bovidae as a model taxon. Bovids were chosen as the study system not only because they provide a classical example of a taxon where sociality has evolved due to anti-predator benefits First, I test the two hypotheses on the association between sociality and longevity. If antipredator benefits of group-living lead to positive selection on intrinsic lifespan in social species due to reduced extrinsic mortality, I predict that longevity will increase with group size. However, if sociality evolves primarily under intense predation and remains associated with high extrinsic mortality in spite of antipredator benefits from group-living, I predict that longevity will decrease with group size. Next, I investigate the hypothesis that sexual selection has a negative impact on intrinsic lifespan. As a proxy measure of the intensity of sexual selection, I use sexual body size dimorphism (SSD), a key sexually selected trait in bovids, whose evolution is linked to the advantage that large body size confers in contests over mates 10-transformed to make them suitable for regression analysis. The data on maximum longevity was obtained primarily from Focusing on all extant bovid species, data was collected from the literature on the maximum recorded lifespans of males and females. These records invariably came from captive populations and were assumed to broadly reflect intrinsic longevity, i.e. independent of ecologically induced mortality due to predation, food limitation, mate competition and disease transmission The comparative method with phylogenetic control was used to identify predictors of longevity Contrasts in both male and female longevity followed a normal distribution, and I performed separate multivariate regression analysis on the two sexes in PASW Statistics 17.0.2. I tested the predictive power of the following independent variables: sex-specific body mass, group size, and SSD. Because antipredator strategy in ungulates depends on body size (see Introduction), the interaction between body mass and group size was also included as an independent variable in order to assess whether any effect of sociality on longevity was size-dependent. All regressions were through the origin. Non-significant variables were removed by backward elimination. Due to correlations between the independent variables, I assessed the potential effect of collinearity on the results of the multivariate analyses by calculating the variance inflation factors. In all cases, the variance inflation factors were <1.5 and thus well below the commonly accepted critical thresholds for significant collinearity of 5–10 t = 4.26, df 98, P<0.001; Longevity in both males and females was positively correlated with group size across the bovid species ; howeverThis study reveals both sociality and sexual selection (as measured by SSD) as independent predictors of interspecific variation in longevity in bovids. Although the intensity of sexual selection is partly dependent on sociality The negative relationship between male longevity and SSD suggests that in species where the fitness of males is strongly linked to their success in mate competition, viability costs associated with the development of sexually selected traits constrain selection on male lifespan. This interpretation is consistent with studies of wild populations, which show male survival rates to be compromised by intense mate competition The positive link between longevity and group size supports the hypothesis that selection for long intrinsic lifespan is stronger in social species due to lower extrinsic mortality from predation as a result of safety in numbers i) the use of a more representative and comprehensive data set in the present study , and (ii) correction of errors in group sizes in the data set used in the former analysis  In summary, the positive link between longevity and sociality, a key antipredator strategy in ungulates, points to lower extrinsic mortality in social species as a factor selecting for inherently longer lifespan. At the same time, the negative link between male longevity and SSD, a key sexually selected trait, suggests that investment in reproductive fitness components may not only compromise survival in the wild but also constrain selection on intrinsic longevity. Now further studies are needed to establish the critical conditions under which these selective forces have had a significant impact on longevity selection.Table S1The data set.(DOCX)Click here for additional data file.


Noteworthy changes in HGF concentration in seminal plasma have been reported in different andrological diseases. All together these data indicate that HGF has a role in the control of spermatogenesis and sperm quality either directly, acting on male germ cells, or indirectly acting on tubular and interstitial somatic cells of the testis.In the last decades, a growing body of evidence has been reported concerning the expression and functional role of hepatocyte growth factor (HGF) on different aspects of testicular physiology. This review has the aim to summarize what is currently known regarding this topic. From early embryonic development to adult age, HGF and its receptor c-Met appeared to be clearly detectable in the testis. These molecules acquire different distribution patterns and roles depending on the developmental stage or the post-natal age considered. HGF acts as a paracrine modulator of testicular functions promoting the epithelium–mesenchyme cross-talk as described even in other organs. Interestingly, it has been reported that testicular HGF acts even as an autocrine factor and that its receptor might be modulated by endocrine signals that change at puberty: HGF receptor expressed by Sertoli cells, in fact, is up-regulated by FSH administration. HGF is in turn able to modify endocrine state of the organism being able to increase testosterone secretion of both fetal and adult Leydig cells. Moreover, c-Met is expressed in mitotic and meiotic male germ cells as well as in spermatozoa. The distribution pattern of c-Met on sperm cell membrane changes in the caput and cauda epididymal sperms and HGF is able to maintain epididymal sperm motility  The hepatocyte growth factor (HGF) is a pleiotropic cytokine originally purified as a potent mitogen for hepatocytes , 2 and sc-Met is the unique HGF receptor and it is normally expressed by cells of epithelial origin whilst HGF expression has been mainly found restricted to cells of mesenchymal origin. c-Met receptor presents tyrosine-kinase activity and, upon HGF stimulation, this receptor triggers several transduction pathways responsible for its multiple biological responses including proliferation, motility, migration, morphogenesis, tubulogenesis, differentiation, and angiogenesis –10. In pvia blood vessels to injured organs allowing their repair and homeostasis  and continues through the entire period of pre-natal development. Actually, at 11.5 dpc only HGF is present in the celomic epithelium underlying male urogenital ridges whereas, at the same pre-natal age, c-Met is not already expressed in the gonads even if it is clearly detectable in the mesonephric mesenchyme , 33. Oneex vivo organ culture condition  c-Met distribution pattern drastically changes, being down-regulated in the testicular cords and up-regulated in the interstitial fetal Leydig cells . HGF is This phenomenon may be relevant for the onset of the first cross-talk among gonad, hypothalamus, and pituitary gland, and potentially involved in the acquisition of secondary sex-specific features and brain testosterone imprinting Figure .Even if the studies on pre-natal morphogenetic functions of HGF have been established using mouse models, the post-natal roles of HGF on testicular physiology have been determined mainly using rats since testicular cell isolation is better characterized for this animal model. Intriguingly, many parallel mechanisms exist comparing ovarian and testicular function and some of them, with particular attention to endocrine function, will be highlighted in this part of the review.Correct Sertoli cell differentiation is crucial in order to maintain the local microenvironment necessary to sustain spermatogenetic process , 57. Sevin vivo Sertoli cell 17β-estradiol production in a similar way of what observed in granulosa cells. Consistent with these data, FSH has been demonstrated able to up-regulate c-Met expression in Sertoli cell cultures  is expressed exclusively in primary spermatocytes (It has been reported by several groups that c-Met is expressed in both human and rodent male germ cells: this observation is interesting since it means the expression of this receptor is conserved at least among mammals –68. In ratocytes  stronglyGerm cell apoptosis is finely controlled in the testis in order to guarantee the best selection of differentiating male gametes and HGF has been demonstrated to be involved even in the control of this biological process. This growth factor, in fact, has been demonstrated to act also as a survival factor for male germ cells since it is capable to significantly decrease germ cell apoptosis . The lowin vivo and in vitro (It is well known that germ cell proliferation and survival are controlled by endocrine signals: for instance FSH stimulates spermatogonial proliferation both in vitro –74. In ain vitro , 76 probin vitro , 78. By in vitro , 79–83 tin vitro maintenance of epididymal sperm motility even if, actually, the factor was not able to significantly increase the percentage of motile cells (There has not been a report of any effect of HGF on meiotic germ cells, even if they express c-Met receptor on their surface . Howeverle cells . Interesle cells . Howeverle cells  and the le cells . It was le cells –89. It iThe described morphogenic and motogenic effect on cultured pre-natal and post-natal Sertoli cells allowed to hypothesize that HGF may modulate junctional capability of this cell type. It is well known, in fact, that HGF is a “Scatter Factor” able to modulate junctional behavior of target cells. In adult mammalian testes, Sertoli cells form junctional complexes with neighboring Sertoli cells that have been described from a long time . These jMyoid cell lineage was the first isolated testicular cell type in which HGF/c-Met system has been discovered . Even ifex vivo organ culture. Intriguingly, the steroid modulator activity is not confined in male gonad but was also described in theca cells, which are the Leydig cell ovarian counterparts. In rat theca cells, in fact, HGF suppressed LH-dependent androsterone secretion, while stimulated basal and LH-induced progesterone production (As well as mouse fetal Leydig cells, also Leydig cells isolated from pubertal rats expressed c-Met receptor . Physioloduction . It is fBesides its effect on testosterone secretion, it is worth mentioning that HGF exerts a broader effect on Leydig cell secretory activities: HGF administration to isolated rat Leydig cells was able to increase the secretion of active form of several proteases such as MMP2 and uPA . InteresDifferent types of molecules can be substrates of MMPs including growth factors, tyrosine-kinase receptors, extracellular matrix proteins, and cell adhesion molecules. HGF administration on isolated Leydig cells significantly reduces the amount of fibronectin indicating that this growth factor might modify interstitial extracellular matrix components and in turn changes significantly adhesive microenvironment and cytokine availability Figure . It is fin vivo by this growth factor could be a useful prerequisite in order to address idiopathic andrological diseases.Hepatocyte growth factor has been well established as a key regulator of the development and homeostasis of many organs. An increasing amount of evidences is demonstrating its important role in several aspects of pre-natal and post-natal testicular physiology. A huge job has been done but we must deal with a major one to figure out what might be the implications of this factor in the reproductive health of human beings. A better understanding of the molecular mechanisms carried out 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. The Guest Associate Editor Gilda Cobellis declares that, despite being affiliated to the same department as author Giulia Ricci and having a collaboration in another journal, the review process was handled objectively and no conflict of interest exists.