Electrochemical anodization of pure aluminum enables the growth of highly ordered nanoporous anodic alumina (NAA) structures. This has made NAA one of the most popular nanomaterials with applications including molecular separation, catalysis, photonics, optoelectronics, sensing, drug delivery, and template synthesis. Over the past decades, the ability to engineer the structure and surface chemistry of NAA and its optical properties has led to the establishment of distinctive photonic structures that can be explored for developing low-cost, portable, rapid-response and highly sensitive sensing devices in combination with surface plasmon resonance (SPR) and reflective interference spectroscopy (RIfS) techniques. This review article highlights the recent advances on fabrication, surface modification and structural engineering of NAA and its application and performance as a platform for SPR- and RIfS-based sensing and biosensing devices. These novel analytical devices based on nanoporous materials are cost-effective, highly sensitive due to the large surface-to-volume ratio, and additionally show excellent selectivity when coupled to biorecognition elements . In partHerein, for the first time, we review the major advances and developments of chemical and biosensing systems based on NAA in combination with surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIfS). First, we present the fundamental aspects of the fabrication and structure of NAA prepared by electrochemical anodization of aluminum. Next, recent advances on the modification of the surface chemistry of NAA using various techniques are reported. Then, we present the most outstanding works in structural engineering of NAA relating aimed at engineering its optical properties. Finally, we present examples of recent advances on SPR and RIfS chemical and biosensing systems based on NAA, providing details of their principles, performance and practical applications.2.et al. in 1953, for the first time, described the structure of anodically grown alumina as a close-packed hexagonally organized duplex structure with a porous and a barrier type oxide layer . Th. Thand cot array ,40.et al. for the first time, reported on the modification of NAA by plasma polymerization .,53.et ali.e., APTES and MPTES) inside NAA pores . Th. Th96]. embranes . Notice embranes –100. Furs of NAA . In thishemistry . The samtemplate ,104. The3 nanotubes, which replicated the length and outer diameter of the NAA templates used in the synthesis process . T. T109]. ved SiOC . This teved SiOC . The remved SiOC ,112. Leeechnique . The preemplates –116. Theet al. prepared carbon-based nanostructures such as carbon fibers and carbon ribbons with a circular mesoporous framework ..i.e., Gri.e., waveguiding layer) on Al  was carefully engineered in terms of porosity, pore density, thickness, and refractive index to tune and optimize the optical WG and enhance the sensitivity of the system. The performance of the optimized NAA-WG was assessed by monitoring the adsorption of BSA molecules onto the nanopores of NAA. The most optimum NAA-WG sensor resulted in an extraordinarily large red shift (>300 nm) of a waveguide mode on adsorption of BSA. This extraordinary red shift was attributed to the large surface area of NAA, which results in a huge adsorption capacity. The theoretical calculations using Fresnel's equation suggested that the sensitivity of the NAA-WG sensor was much higher than that of conventional SPR sensors.For this, the structure of NAA  inside the nanopore surface of a NAA waveguiding film . Auet al. monitored the release of a model drug, indomethacin, in real-time under dynamic flow conditions . This approach provides more accurate information than conventional in vitro drug release techniques performed under static conditions. A comparative study measuring the sensing performance of RIfS and photoluminescence spectroscopy (PLS) using NAA as substrate was recently reported by Santos et al.  for both the analytes and binding conditions than NAA-RIfS platforms. Macias et al. fabricated a bilayered funnel-like NAA-RIfS sensing platform . T. Tet al.s et al.  60]. Fi. Fiet alplatform . This st6.i.e., limited coverage, stability, and terminal groups). In the future we may expect more versatile surface functionalization process to achieve chemistries unimaginable with present techniques and several research groups are already working towards it.We believe that there is still a huge potential for further improvement of basic properties of NAA, which can be utilized for broad and multidisciplinary research fields as materials science, nanotechnology, optics, electrochemistry, cell biology and medicine. Over the last few decades, the different approaches have made it possible to design the structure of NAA, resulting in better control over pore diameter, length, porosity and organization of pores. However, the real understanding of the self-organizing anodization process on aluminum is not complete. Thus, to generate and extend the range of NAA structures available, it is necessary to investigate this phenomenon intensely. For its wider commercial application, the cost is a critical factor for NAA to be used as a consumable, however, the cost of high purity aluminum may be bottom line in this scenario. In terms of surface chemistry, the surface properties of NAA can be finely tuned in limited ways due to inherent properties of the process used to functionalize NAA (This review has summarized and presented the recent progresses in surface functionalization and modification of NAA and the developments for its use in localized plasmon and reflective interference based sensing systems. We have presented a detailed description about structure and surface modifications of NAA. We have also provided detail of the fundamental aspects of LPR and RIfS in combination with NAA platforms along with the most relevant concepts and examples of devices reported based on NAA and these optical techniques. Due to the advanced properties of NAA, it has emerged as a highly attractive alternative for development of broad range stable and functional sensing devices for clinical, industrial and environmental analyses. The ability to modify the surface of NAA with a wide range of functionalities to endow it with desired selectivity and specificity towards target analytes has pushed NAA for applications in several fields. We believe that future developments in structural engineering and chemical modifications of NAA will yield more innovative NAA-based sensing systems. Furthermore, a number of methods to structurally engineer the NAA make it further attractive nanostructures for development of future optical sensing devices. However, this review has demonstrated that there is still plenty of advances and developments to be made in the field of structural engineering, surface modification, and NAA-LPR and NAA-RIfS based sensing platforms.


This special issue of eCAM has brought together several unique investigators interested in invertebrate animal models. Investigators would benefit by examining advances and applications of findings derived from useful terrestrial and marine invertebrate animal models. Results derived from well-defined evidence-based approaches are usually relevant to humans; the advantages are numerous, inexpensive, and noncontroversial and are not subject to rigid, ethical, or moral guidelines.In the historical context, these animals have been crucial in many cultures before the advent of industrial medicines thus providing the only source of aid. Now, there is a move to return to or at least to include these practices lest they disappear since, in certain cultures, they represent the sole source of useful health remedies. However, evidence-based medicine emphasizes the importance of subjecting animal products to rigorous analysis. Results are then standardized and serve to educate populations including those that use exclusively western practices.eCAM stands ready to accept the importance and contributions of bees as valuable sources of beneficial activities (pollination) and products (honey and propolis) that for centuries have enhanced human lives as food and medicine. Others include corals, millipedes, maggots, and earthworms. Corals are marine invertebrates that live in compact colonies. Humans have learned that they are useful in the manufacture of jewelry and chemical compounds that are useful against cancer, AIDS, and pain and serve as skeletons during bone grafting. Millipedes are arthropods  which in some cultures are used during pregnancy and as cures for fever, wounds, earaches, and hemorrhoids and food among the Bobo people of Burkina Faso when crushed; sometimes they are boiled and eaten in tomato sauce.Since the time of Napoleon and even centuries earlier, maggots have been used especially in patients with severe intractable wounds caused by nerve degeneration that results from diabetes. In fact, leeches and maggots are now valuable components of the emerging field of biotherapy, the therapeutic use of living creatures to clean gangrenous tissue often found in ulcers, burns, and postoperative infections. Earthworms are experiencing growing popularity due to their many applications including sources of high protein as food and the production of an anticlotting agent known as lumbrokinase that dissolves blood clots in patients. Aside from observations and usefulness by the layman, certain techniques are valuable as molecular biologists strive to understand the minute structure of molecules in order to improve therapies. Let us not forget the contributions of earthworms as tillers of the soil which enhance agricultural output. Earthworms keep the soil aerated by their constant churning which makes for improved yield of plants regardless of the proposed use.Chih-Yang HuangChih-Yang HuangEdwin L. CooperEdwin L. CooperCatherine Fang-Yeu PohCatherine Fang-Yeu PohWei-Wen KuoWei-Wen KuoTung-Sheng ChenTung-Sheng ChenRonald ShermanRonald Sherman


Mitochondrial dysfunction and defects in oxidative metabolism are a characteristic feature of many chronic illnesses not currently classified as mitochondrial diseases. Examples of such illnesses include bipolar disorder, multiple sclerosis, Parkinson’s disease, schizophrenia, depression, autism, and chronic fatigue syndrome.While the majority of patients with multiple sclerosis appear to have widespread mitochondrial dysfunction and impaired ATP production, the findings in patients diagnosed with Parkinson’s disease, autism, depression, bipolar disorder schizophrenia and chronic fatigue syndrome are less consistent, likely reflecting the fact that these diagnoses do not represent a disease with a unitary pathogenesis and pathophysiology. However, investigations have revealed the presence of chronic oxidative stress to be an almost invariant finding in study cohorts of patients afforded each diagnosis. This state is characterized by elevated reactive oxygen and nitrogen species and/or reduced levels of glutathione, and goes hand in hand with chronic systemic inflammation with elevated levels of pro-inflammatory cytokines.This paper details mechanisms by which elevated levels of reactive oxygen and nitrogen species together with elevated pro-inflammatory cytokines could conspire to pave a major road to the development of mitochondrial dysfunction and impaired oxidative metabolism seen in many patients diagnosed with these disorders. Syndromic or non-syndromic mitochondrial diseases, classified as cytopathies or encephalomyopathies, arise as a result of mutations in mitochondrial or nuclear DNA . HoweverMitochondrial dysfunction in such individuals may well result from the presence of oxidative stress, as there is now ample evidence implicating oxidative stress as one of the major contributing factors in the development of mitochondrial dysfunction and compromised bioenergetic performance -13. In fOxidative stress activates a number of transcription factors, such as NF-kappaB and activated protein 1, leading to the production of pro-inflammatory cytokines (PICs), various chemokine species, and activation and proliferation of lymphocytes. The activation of other immune cells in turn leads to the production of more ROS and RNS, principally in the form of superoxide, nitric oxide (NO), and peroxynitrite -27. The ROS and RNS can also contribute to the development of chronic oxidative stress and inflammation via the oxidative and nitrosative modification of proteins, lipids, and DNA, resulting in modification of DNA bases and tertiary protein structure, lipid peroxidation of cell membranes, and the production of highly reactive aldehydes and ketones. The net result of these processes is the indirect and direct formation of damage-associated molecular patterns capable of activating pathogen sensing receptors on the surface and in the cytoplasm of immune cells ,29-32.The origin of oxidative stress in the brains of people suffering from a range of neuroimmune diseases, such as MS and PD, is still a matter of debate. There is now however strong evidence supporting the hypothesis that the oxidative stress in the brains of such patients stems from the transduction of inflammatory signals to the brain following the establishment of chronic inflammation and oxidative stress in the periphery. There is ample evidence demonstrating that systemic inflammation can lead to the development of chronic neuroinflammation -35. CommFollowing the up-regulation of these receptors, such microglia become exquisitely sensitive to subsequent inflammatory stimuli, leading to an exaggerated production of neurotoxic molecules that may exacerbate the pre-existing pathology and may even accelerate the progression of existing neuroinflammatory or neurodegenerative diseases -41. ActiThe aim of this paper is to outline how excessive levels of PICs, notably TNF-α, ROS, and RNS, can lead to mitochondrial dysfunction and compromised bioenergetics in PD, MS, CFS, depression, bipolar disorder, schizophrenia and autism, by inhibiting the electron transport chain (ETC), the tricarboxylic acid cycle, and fatty acid oxidation, adversely affecting the activity and structure of structural and regulatory proteins and the integrity of essential functional lipid membranes. In short, this paper aims to demonstrate that chronically elevated levels of these pro-inflammatory entities are common denominators in paving the many roads to mitochondrial dysfunction.Chronic activation of the humoral and innate arms of the immune system in both the periphery and the CNS is a characteristic finding in MS patients. Commonly reported abnormalities include elevated levels of activated T helper (Th)2, Th17, and Th1 T cells, abnormal function of regulatory T cells, and activated naive B cells with impaired tolerance together with changes in the overall B cell subpopulation distribution pattern ,29,46. TThere is now ample evidence highlighting the pivotal role of oxidative stress in the pathogenesis of MS -61. SeveAlthough the weight of evidence demonstrates that, while the development of pathology in the early stages of MS is largely driven by inflammation , mitochoA diagnosis of autism similarly in all probability represents a group of illnesses with heterogeneous etiology ,88. EpigThe presence of chronic oxidative stress is commonly reported in CWA -114. Int31P-magnetic resonance spectroscopy have reported decreased production of ATP elevated levels of lactate, reduced levels of carnitine  polymerase 1 ,463, theThe adverse effects of elevated levels of TNF-α on mitochondrial biogenesis have already been discussed; however, excessive levels of TNF-α and other PICs, typifying a state of chronic systemic inflammation, can additionally disable oxidative phosphorylation . This isThis paper has detailed some of the evidence demonstrating the existence of immune dysfunction, oxidative stress, and mitochondrial dysfunction in many patients diagnosed with MS, PD, autism, bipolar disorder, depression, schizophrenia, and CFS. It is proposed that these apparently non-specific findings may contribute to the pathophysiology in each illness. Excessive levels of peroxynitrite, NO, and PICs clearly have the capacity to inhibit the activity of the ETC at several points, alone or synergistically, leading to the depletion of ATP production and promoting a switch to anaerobic glycolysis. Peroxynitrite and TNF-α can also depolarize the mitochondrial membrane via a number of different mechanisms once again having a deleterious effect on the generation of ATP. Peroxynitrite in particular can damage lipids and proteins, altering their conformation and function, causing structural damage to integral mitochondrial proteins and lipid membranes and to proteins regulating the function of the organelle. The capacity of peroxynitrite to inactivate a range of enzymes with an essential function in the generation of energy and the regulation of energy generation, such as p53, can provide other pathways to impaired oxidative metabolism. However, peroxynitrite is not alone in its ability to impair the activity of essential transcription factors as evidenced by the capacity of TNF-α to inhibit the production of PGC-1α and indirectly impair activity-stimulated mitochondrial biogenesis. It must be emphasized, however, that the presence of these inflammatory entities in an environment of oxidative stress is highly unlikely to be the sole cause of the mitochondrial dysfunction and impaired energy production seen in people with these illnesses. Genetic and epigenetic factors are also surely involved. Consequently, it is impossible to calculate the extent of the contribution that these entities make to the phenomenon of bioenergetic impairment seen in these apparently disparate illnesses, but it is likely that they play at least a part.


The neurobiology of suicidal behaviour, which constitutes one of the most serious problems both in psychiatry and general medical practice, still remains to a large degree unclear. As a result, scientists constantly look for new opportunities of explaining the causes underlying suicidality. In order to elucidate the biological changes occurring in the brains of the suicide victims, studies based on post-mortem brain tissue samples are increasingly being used. These studies employ different research methods to provide an insight into abnormalities in brain functioning on various levels, including gene and protein expression, neuroplasticity and neurotransmission, as well as many other areas. The aim of this paper to summarize the available data on the post-mortem studies, to provide an overview of main research directions and the most up-to-date findings, and to indicate the possibilities of further research in this field. Suicide is one of the leading causes of death globally for all ages. Every year, nearly one million people die from suicide according to WHO, 2013. It therefore constitutes a serious medical and social problem, and as such calls for profound and systematic research to enable a better understanding of the underlying pathology, as well as the development of new therapeutic approaches. Suicides and suicide attempts are usually associated with mental illness including affective or psychotic disorders as well as alcohol and/or substance abuse disorders , impulsiPost-mortem brain samples obtained from suicide victims and controls offer many new opportunities to study molecular mechanisms underlying suicidal behaviour. In contrast to earlier studies based on peripheral tissues, research enables a direct insight into the neurobiological abnormalities associated with suicide. However, it is important to be aware that those studies need to meet certain criteria .To begin with, a positive opinion of the local ethical commission is mandatory for every study involving the examination of human brain samples. Next, because the brain tissue degrades very easily, the process of collecting samples for post-mortem studies should be carried out with special care. The collection of the samples should ideally take place no longer than 48 hours after death. In order to establish the quality of collected tissue and determine whether it is adequate for measuring protein levels and gene expression, pH of the samples and the RNA integrity number should be determined. Since methyltransferase- and acetyltransferase-activities measured in brain samples were reported to be relatively preserved and independent of storage duration or post-mortem interval Monoranu et al.  proposedLooking at a possible role of various genetic factors in suicidal behaviour can broaden our knowledge of different neurobiological elements responsible for this pathology. A number of studies have focused on one or several genes at a time, comparing suicide victims with controls. This led to the identification of genetic predisposing factors, such as WFS1 , 23 or pGenetic studies offer a unique possibility of comparing the effects of both long-term substance abuse (in subjects with documented chronic dependence) and acute substance exposure  on gene expression. The same research group performed gene expression analyses in 17 cortical and subcortical brain regions from suicide victims with and without major depression and controls . They obhttp://www.HUPO.org). The field of proteomic studies of post-mortem brain tissue is still relatively unexplored. Schlicht et al. -cAMP binding and PKA activity to be significantly decreased in the PFC of suicide victims , 141. MoProtein kinase C is a regulatory enzyme involved in the modulation of many neuronal and cellular functions, such as neurotransmitter synthesis and release, regulation of receptors and ion channels, neuronal excitability, gene expression, secretion and cell proliferation . The PKCActivation of PKA, as well as PKC, phosphorylates several transcription factors, which then influence gene expression. Cyclic AMP response element binding protein (CREB) is a transcription factor, which has been implicated in the pathophysiology of suicide. Odagaki et al.  quantifiCytokines are small cell-signaling protein molecules which serve as immuno-modulating agents. Changes in the immune system have been suggested to play a role in depression and suicidality, following observations of a negative influence of IFN-alpha on patients' mood , 158, asNeurotrophic factors are a family of proteins that induce the survival, development, and functional differentiation of neurons , which iThe levels of the whole blood glial cell line-derived neurotrophic factor (GDNF) has been shown to be decreased in remitted patients with mood disorders  and one Polyamins, including putrescine, spermidine and spermine, are low molecular weight aliphatic amines involved in cell proliferation, apoptosis, immunity and oxidative stress response –185. ChaTable Abnormalities in HPA axis have been postulated both in mood disorders and suicide. On the one hand, most patients with depression have been shown to present with hypercortisolemia both in plasma and CSF, increased cortisol response to adrenocorticotropic hormone (ACTH) and a deficient feedback mechanism, reflected in an abnormal dexamethasone suppression test  as well Post-mortem studies on brain tissue have a number of limitations. One of them is the relatively short period of time, within which the samples should be collected. For example, the levels of dihydropyrimidinase-related protein-2 were found to decrease within 6 h after death . Thus, tPost-mortem studies of suicidality provide many possibilities for widening our knowledge of the underlying neuropathology and of the influence of medication and substance abuse, as well as for constructing and testing different clinical hypotheses. Several interesting new research directions of post-mortem studies are conceivable for the future, including innovative genetic and epigenetic studies with the use of new data bases and new brain collection programs. Another unique possibility is the comparison of neurobiological factors characteristic for suicide in the samples representing particular psychiatric disorders, as described above. The same goes for differentiating between neurobiological abnormalities related to suicidal behaviour and those associated with completed suicide. Another interesting possibility is the description of the effects of different psychiatric drugs, their actions on genes expression, protein levels and neuro-receptors, and their efficacy in preventing suicide. Last but not least, the impact of chronic or acute substance intoxication on the brain neurobiology and its influence on the suicide risk could also be further researched in post-mortem studies.Studies on post-mortem brain tissue provide numerous possibilities for a deeper insight into the neurobiology of different psychiatric disorders, including suicidal behaviour. With new methods being now introduced to improve the reliability of results, genetic information being openly available for analyses and comparisons and the brain tissue banks being founded and expanded, the post-mortem studies are likely to grow in number and further develop in future.


Stem cells are considered as the next generation drug treatment in patients with cardiovascular disease who are resistant to conventional treatment. Among several stem cells used in the clinical setting, cardiac stem cells (CSCs) which reside in the myocardium and epicardium of the heart have been shown to be an effective option for the source of stem cells. In normal circumstances, CSCs primarily function as a cell store to replace the physiologically depleted cardiovascular cells, while under the diseased condition they have been shown to experimentally regenerate the diseased myocardium. In spite of their major functional role, molecular mechanisms regulating the CSCs proliferation and differentiation are still unknown. MicroRNAs (miRs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the important role of miRs in regulating stem cell proliferation and differentiation, as well as other physiological and pathological processes related to stem cell function. This review summarises the current understanding of the role of miRs in CSCs. A deeper understanding of the mechanisms by which miRs regulate CSCs may lead to advances in the mode of stem cell therapies for the treatment of cardiovascular diseases. Stem cells are gaining interest as the treatment of choice in patients with cardiovascular disease who are resistant to conventional therapies. Stem cells are cell precursors which contribute to the formation of new tissues by a process involving cell differentiation, as well as a series of asymmetric divisions that lead to the production of daughter cells with different cell fates . Recent  in vitro to an appropriate number, so that they can be transplanted back in vivo in order to repair the damaged heart tissue . Fu. Fu65]. Available evidence suggests mixed results for the roles of miR-1 and miR-133 in regulating the functions of adult CSCs and CPCs , 66. IvemiR-1 and miR-499 were demonstrated to reduce proliferation and induce differentiation of human CPCs (72). miR-1 represses histone deacetylase 4 (HDAC4), a transcriptional inhibitor of muscle gene expression, while miR-499 represses sex determining region Y-box 6 (Sox6), a transcription factor involved in muscle differentiation  68]. Mo. Mo68].  Drosophila, wherein miR-1 was shown to inhibit Notch signalling by targeting the translation of the Notch ligand, Delta in vivo  protein . These e of SVPs .Accumulating evidence demonstrates that miRs are also involved and modulated in several pathologies, including cardiovascular pathology. Therefore, a better understanding of the underlying mechanisms through which miRs affect disease processes is vital for the development of stem cell therapies. The known effect of each miR subtype on the different pathologies is summarised in Vascular remodelling is an active process characterised by alterations in the structure and thickness of the luminal walls of blood vessels, which causes a change in systemic vascular resistance and thus blood pressure . A serie in vitro by targeting c-kit, as well as indirectly reducing the expression level of endothelial nitric oxide synthase expression  and therefore improve the survival of CPCs  87]. Mo. Mo87]. hickness .In addition to apoptosis, miRs are also shown to have an effect on the necrotic cell death of CSCs. Liu et al. demonstrated a 4-fold increase in miR-155 expression in CSCs when the cells are exposed to prooxidant hydrogen-peroxide . Exposur in vivo transplantation and in chronic morbidities including diabetes [Apoptosis and necrotic cell death are the major causes for reduction in the number of CSCs followingdiabetes , 10, 90.The potassium channels, KCNE1 and KCNQ1, have been identified in CPCs, where they aid in maintaining cell repolarisation . AbnormaAs indicated in the previous sections, due to their physiological and pathophysiological role in CSCs, miRs have the potential to be modulated to improve CSC function and its efficacy in various cardiovascular diseases. Moreover, the primary role of miRs in promoting the differentiation and proliferation potential of stem cells enables their use in a number of other therapeutic settings.Although limited, emerging evidence strongly supports the major role of miRs in CSC-based therapies. Inhibition of miR-378 was demonstrated to indirectly promote CSC viability by enhancing the expression of connective tissue growth factor (CTGF). CTGF functions as an autocrine growth factor and is known to exert its effect on tissue repair, scarring, fibrosis , cytopromiR-499 has been recently shown to have an important role in influencing the cardiomyocyte differentiation of CSCs. Under normal conditions, miR-499 expression is negligible in CSCs, although cardiomyocytes express high levels of miR-499. However, when required, miR-499 traverses gap junction channels and translocates to structurally coupled CSCs, favouring their differentiation into functionally competent cells through repression of miR-499 target genes, Sox6 and regulator of differentiation 1 (Rod1) . Further In vivo transplantation of miR-133 overexpressing CPCs consistently demonstrated superior recovery of cardiac functions [In a very recent study, Izarra et al. demonstrated the ability of miR-133 in improving the protective ability of CPCs. They demonstrated a marked increase in miR-133 expression in CPCs exposed to differentiation medium. Further, CPCs overexpressed with miR-133 showed a marked reduction in caspase activity through inhibition of proapoptotic Bim.unctions .The application of miRs to CSCs to treat myocardial infarction is a newly emerging treatment strategy and one that has gathered much more evidence for its therapeutic effects in recent years. Despite a lack of documentation for this treatment method, specific miRs such as miR-378, miR-499, and miR-133 have been shown to effectively regenerate myocardial tissue in animal models. However, it is apparent that more research is needed to fully understand whether the capacity of the miRs to regenerate the myocardium can be translated to clinical practice and whether this is as effective in humans as in animal models.The primary function of miRs within CSCs involves regulating the differentiation and proliferation potential of these cells.miRs within CSCs are involved in a number of physiological functions, mostly based on their ability to target the mRNA of transcription factors and other regulatory molecules.miRs within CSCs have implications in pathological settings, as they are responsible for influencing the progression of a number of pathological processes such as apoptosis and necrosis.Modulation of cardiac-specific miRs has the potential to be an effective strategy in CSC therapies for the purposes of treating cardiac-related diseases. The therapeutic potential of miRs has been indicated from an experimental standpoint in animal models. However, whether the same can be replicated in humans and most importantly, whether one or a cocktail of miRs are necessary to produce the desired clinical effect, is yet to be investigated.A number of miR subtypes have been investigated for their role in regulating the differentiation and proliferation of CSCs. However, several questions still remain unanswered such as if the effect of miRs will depend on age, gender, associated diseases, treatments, ethnicity, and environmental factors. Therefore, future studies focusing on answering these important questions will provide novel insights into the pathophysiological role of miRs in CSCs, which eventually result in the development of new therapeutic modalities. Based on the experimental evidence described within this review, a general consensus can be drawn.


The role of extracellular vesicles as an important mediator of cell-to-cell communication has been well established by many studies that have shown their capability for exchanging proteins, bioactive lipids and nucleic acids. Extracellular vesicles have been implicated in several physiological and pathological processes according to the cell of origin. Identification of the innate properties of extracellular vesicles derived from stem cells and from immune cells has led to the possibility of their exploitation in regenerative medicine and immune therapies. As extracellular vesicles are able to cross biological barriers, express surface receptors and contain defined cargoes able to target specific cells/tissues, they may represent a biocompatible and effective tool for drug delivery. Herein, we review and discuss the perspectives related to the therapeutic opportunities of extracellular vesicles. Liposomes, which are synthetic phospholipid vesicles, have been used in the delivery of anticancer agents for the treatment of different solid tumours . AnticanSecreted vesicles are heterogeneous populations of small vesicles released by eukaryotic cells.They have been classified on the basis of the cell of origin, specific function, or of their biogenesis and are known in the literature by different names such as prostatosomes, cardiosomes, tolerosomes, microparticles, ectosomes, microvesicles and exosomes. Moreover, the cell-released vesicles also include apoptotic bodies, generated by blebbing of apoptotic cell membranes. Virtually all cells can secrete vesicles in basal conditions; however, this event is particularly evident for certain cell types and it may increase during cell proliferation and cell activation or after exposure to stress conditions . The twoExosomes have been defined as originating from inward budding of membranes of multivesicular bodies, followed by their fusion with the cell plasma membrane and release into the extra-cellular space , 13. ExoThis distinction, based on biogenesis, size, sedimentation on sucrose gradients, protein and lipid composition, remains confusing because the markers used for defining vesicles are frequently not exclusive, and may vary depending on the cell of origin. In addition, small vesicles have recently been reported to have a broad range and size , 16. ForNaturally-occurring vesicles, given their properties of selectively targeting certain cell types or tissues in order to deliver their cargo, are potential candidates for therapeutic applications.On one hand, the innate therapeutic potential of EV derived from certain cell types can be exploited, for example stem cells; on the other hand, EV may represent a biocompatible and effective tool for drug delivery, as they are able to cross biological barriers .The possibility of exploiting the innate therapeutic potential of EV is based on the observation that, by delivering their bioactive cargo, EV plays a critical role in cell-to-cell cross talk . EV deriRatajczak et al.  demonstrAdult human stem cells, such as bone marrow-derived multipotent stromal cells (MSC) and human liver stem cells (HLSC), secrete EV that contain specific subsets of functional mRNA , 36 and Direct EV-mediated delivery of mRNA into bone marrow cells and induction of transcription was demonstrated by Aliotta et al. . SeveralBased on the properties of EV, Quesenberry et al. , 39 receThe beneficial effects of stem cell-based therapies are not supported by any tangible indication that in vivo stem cells are able to permanently engraft the injured tissues and substitute parenchymal cells, despite their in vitro plasticity. Instead, the current view is that stem cells induce regeneration by paracrine/endocrine mechanisms , 44. ThiIn this context, if EV were able to reproduce the regenerative action of stem cells, they may provide an important new therapeutic perspective. We observed that EV from human bone marrow-derived MSCs were able to promote the recovery of acute kidney injury (AKI) in a manner similar to the cell of origin . MoreoveThe transcription regulators delivered by EV caused modification of gene expression in tubular epithelial cells, with a consequent up-regulation of the anti-apoptotic genes BIRC8, BCL-XL and BCL2 and down-regulation of the pro-apoptotic genes CASP1, CASP8 and LTA . EV wereA renoprotective action has been described for EV derived from EPC in a model of ischemia-reperfusion injury (IRI), characterized by diffuse endothelial and tubular cell damage . EPC-derNon-specific miRNA depletion of EV by Dicer knockdown in EPCs, specific depletion of miR-126 and miR-296 by siRNA transfection in EPC, or inhibition by the use of antagomirRs prevented the renoprotective effect of EV . By meanEV have also been shown to display a therapeutic effect in other organs. For example, in a murine model of myocardial ischemia/reperfusion injury, EV derived from the conditioned medium of MSC were shown to decrease infarction size . MoreoveOur group also showed that EV derived from HLSC were able to stimulate liver repair in 70% hepatectomized rats . In a reA neuro-regenerative potential of MSC-derived EV has also recently been proposed . IschemiIglesias et al.  suggesteAlthough the physiological functions of EV in different tissues are mostly uncharacterized, it is emerging that they not only depend on the EV-carried molecules, but are also influenced by the functional and metabolic state of target cells. Indeed, the same EV may exhibit contrary effects depending on the different states of activation or inhibition of particular metabolic pathways in recipient cells. For instance, EV released from HLSC are able to accelerate liver regeneration  whilst iRaposo et al.  first deBased on the knowledge that EV express surface receptors and contain selected patterns of proteins and exRNA, we can potentially generate EV expressing or containing desired molecules by engineering the cells of origin. The feasibility of this approach has been demonstrated by the Gould group through protein targeting EV by plasma-membrane anchors . MoreoveAnother example of the use of EV in therapy was demonstrated by Ohno et al. , who engTaken together, these experiments provide proof of the concept that EV represent a potential biocompatible vehicle for different therapeutic molecules, enhancing their stability, limiting their potential toxicity and immunogenicity, targeting specific cells/tissues and enabling them to cross biological barriers.Despite the promising results demonstrated in experimental animals and the preliminary clinical trials that have tested exosomes in the field of cancer , several points should be elucidated before envisaging the use of either the innate therapeutic potential or engineered EV in clinics.th passage. We have found that these cells do not require immortalization and the derived EV share several properties with those of MSC.Firstly, there is an urgent requirement to develop large-scale methods for EV preparation. This implies identification of the best suitable cellular sources and culture conditions for GMP production of EV. Culture conditions are critical, as it is known that they may influence the yield and the content of EV and thus their bioactivity. The use of EV for regenerative medicine and for drug delivery needs a source of cells that are expandable in GMP conditions and that generate nonimmunogenic EV. MSC derived from bone marrow, fat or the umbilical cord are potential candidates, as they retain the immune-modulatory properties of MSC. However, their senescence after repeated culture passages may limit their use. To overcome this limitation, Chen et al.  immortalAnother critical point is to develop an easy, reproducible and efficient GMP purification protocol.The gold standard protocol for EV purification against which all other techniques should be evaluated is based on differential ultracentrifugation to remove cell debris and large vesicles, and to collect the small vesicles. As the ultracentrifugation technique is time-consuming with a low yield and poses concerns about the loss of the biological activities of EV, some alternative techniques have been under evaluation, including immunoaffinity , 81 and EV have emerged as an important vehicle of information between cells, as they can transfer bioactive proteins, lipids and nucleic acids. This property can be exploited for therapy in different fields, as EV retain several biological activities of the cell of origin. EV released in physiological conditions have innate therapeutic potential. Stem cell-derived EV mimic the favourable effects of the cell of origin, thus promoting repair and limiting injury in several organs, as they are able to activate regenerative programs and coordinate tissue self-repair. Due to the expression of membrane receptors derived from the stem cell of origin, EV can localize at the site of injury and deliver their cargo to damaged cells. The complex constituent array of EV allow them to influence multiple cellular pathways involved in different pathological conditions. EV derived from immune cells may potentiate the immune response and can therefore be used in cancer therapy. The identification of molecules responsible for the biological effect of EV may provide critical information for engineering EV for therapeutic purposes. The possibility of developing specifically targeted and drug-loaded EV may allow for the development of new therapeutic strategies. Additional investigations into the pathological conditions that may benefit from an EV-based therapy, as well as a definition of suitable, scalable GMP protocols of EV production are needed. Moreover, the biosafety and pharmacokinetics of EV require further studies.


The recent recognition of fungi in the oceanic igneous crust challenges the understanding of this environment as being exclusively prokaryotic and forces reconsiderations of the ecology of the deep biosphere. Anoxic provinces in the igneous crust are abundant and increase with age and depth of the crust. The presence of anaerobic fungi in deep-sea sediments and on the seafloor introduces a type of organism with attributes of geobiological significance not previously accounted for. Anaerobic fungi are best known from the rumen of herbivores where they produce molecular hydrogen, which in turn stimulates the growth of methanogens. The symbiotic cooperation between anaerobic fungi and methanogens in the rumen enhance the metabolic rate and growth of both. Methanogens and other hydrogen-consuming anaerobic archaea are known from subseafloor basalt; however, the abiotic production of hydrogen is questioned to be sufficient to support such communities. Alternatively, biologically produced hydrogen could serve as a continuous source. Here, we propose anaerobic fungi as a source of bioavailable hydrogen in the oceanic crust, and a close interplay between anaerobic fungi and hydrogen-driven prokaryotes. The oceanic igneous crust is one of the few great frontiers of unknown biology on Earth , and des1 to C4), but difficult to quantify at such depths. It is also questionable whether abiotic sources are able to ensure a steady supply of organic compounds over geological time. Ingress of organic matter by seawater could be another source but restricted to shallow parts of the oceanic crust , and cool (<25°C) basaltic crust at North Pond. However, ridge-flanks are areas where the igneous crust is exposed, and where much of the fluid exchange between the ocean and the basement is focused  and blue–gray–greenish clays like saponite represent anoxic zones. The redox gradients can be steep in subseafloor environments and, thus, contacts between the two zones can be sharp. The presence of anoxic environments in the oceanic crust is further supported by anaerobic isolates from the igneous crust (see section below).Alpha-, Beta-, and Gammaproteobacteria lineages , and a cluster of microbes that fall within uncultured sulfate reducers. Furthermore, studies of crustal fluids collected on the ocean ridge flanks of the Juan de Fuca Ridge have indicated the presence of members of the phylum Firmicutes, of which some are only distantly related to any known cultivated microorganism and whose mode of life is unclear   , decompoigure 1) , weatherigure 1)  and zeoligure 1) , and areigure 1) . Thus, f2); radiolysis of water by radioactive uranium, thorium, and potassium isotopes; cataclasis of silicates under stress in the presence of water; and hydrous alteration of ferrous minerals. Hydrous alteration of ultramafic rocks (serpentinization) produces significant quantities of bioavailable molecular hydrogen that theoretically could support a deep biosphere  and molecular hydrogen , a process in which the energy source acts as both the electron acceptor and the electron donor. Instead of mitochondria, anaerobic fungi have hydrogenosomes, organelles capable of coupling the metabolism of glucose to cellular energy production. Hydrogenosomes contain hydrogenase, and in the process of decomposing lignocellulosic plant fiber they produce molecular hydrogen, carbon dioxide, acetate, formate, lactate, and ethanol as metabolic waste products  . Methanogens use carbon dioxide, acetate, or one-carbon substrates as carbon sources and electron acceptors while hydrogen and acetate function as electron donors . Such a consortium could facilitate indigenous populations independent of the surface biosphere (The aptitude of fungi to enter into symbiotic relationships with chemoautotrophic prokaryotes (igure 1)  in deep n donors . Also fon donors . Acetogen donors . An enhan donors . Furthern donors . Thus, tiosphere  and of aiosphere . Some aciosphere .There are also other feedback mechanisms that would promote microbial growth from this system. Methane produced by methanogens would support the growth of methanotrophic communities that in turn serve as biomass accessible for heterotrophic, including fungal, decomposition, and fermentation. Methanotrophs are both anaerobic and aerobic, but methane is highly volatile and can easily migrate from anoxic to oxic areas of the oceanic crust. Thus, the products of an anaerobic fungal/hydrogen-consuming consortium would spill over to other provinces, including oxic ones, and stimulate microbial growth not directly associated with the consortia.We suggest anaerobic fungi as a biological source of molecular hydrogen in anoxic provinces of the igneous oceanic crust. This source might out-compete the abiotic production of hydrogen, at least in basalt hosted systems, where they could represent a steady supply of hydrogen as a base for anaerobic hydrogen-consuming microbial communities such as, i.e., acetogens and methanotrophs. A consortium of anaerobic fungi and prokaryotes could facilitate indigenous populations more or less independent of the surface biosphere , and abiEven though the model is partly hypothetical we wish to stimulate further exploration of the deep biosphere of the igneous oceanic crust including extensive phylogenetic studies of the microbial communities with special emphasis on fungi. Also, large portions of the deep-sea sediments are anoxic, and anaerobic fungi are likely to play an important ecological role there as well.MI designed the study and wrote the paper. AS and SB contributed scientifically to the hypotheses. AN contributed scientifically and designed the illustration.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.


Cluster analysis is widely used in cancer research to discover molecular subgroups that inform subsequent laboratory investigations and define risk classification criteria for subsequent clinical trials. However, for any data set, there are a very large number of candidate cluster analysis methods (CCAMs) due to the many choices for feature selection criteria, number of selected features, number of clusters to define, etc. Frequently, a specific CCAM is chosen without quantifying the validity of its results in terms of reproducibility or distinctiveness of the reported subgroups.Here, we propose the Dunn Index Bootstrap (DIBS) procedure to quantify the reproducibility and distinctiveness of subgroups defined by many CCAMs. DIBS applies each CCAM to the observed data and many bootstrap data sets obtained by subject resampling. The bootstrap results are used to compute metrics of subgroup reproducibility and distinctiveness of the subgroups defined by each CCAM.DIBS was used to characterize the performance of each of 4,032 CCAMs in the analysis of one RNA-seq, two microarray gene expression, and one methylation array data set from three different cancers. In each example, DIBS identified specific CCAMs that defined subgroups of well-established biological and clinical relevance.


Plasmodium falciparum parasites from the GMS. This strategy is more efficient for several reasons. First, a subregional strategy is in line with current commitment to elimination and will build upon the existing national political support for elimination as well as enhancing collaboration among countries. Second, the challenge of human mobility in the GMS is subregional in scope and requires a harmonized elimination strategy. Third, countries will need to improve and intensify malaria operations to reach elimination, and this will be a singular goal across the subregion. Rallying around the goal of P. falciparum elimination will not only utilize existing regional bodies to catalyze political and funding support, but will also leverage the funding already in place to achieve this subregional goal.Artemisinin-based combinations are currently the most effective anti-malarials and, in addition to vector control, have led to significant declines in malaria morbidity and mortality. However, foci of artemisinin drug resistance have been identified in the Greater Mekong subregion (GMS) of the Asia Pacific, threatening the major gains made in malaria control and potentially creating a parasite pool that is more difficult to treat and eliminate. Efforts are underway to halt the spread of artemisinin resistance, including coordination of activities and funding, and identification of areas of suspected artemisinin resistance, now using a newly identified molecular marker. However, targeting resources to the containment of resistant parasites is likely inefficient and monitoring impact is challenging. A more sustainable solution is the rapid elimination of all  The Greater Mekong subregion (GMS) of the Asia Pacific, which includes the countries of Cambodia, China (Yunnan Province), Lao People’s Democratic Republic (PDR), Myanmar, Thailand, and Vietnam, is the epicentre of artemisinin resistance. Foci of resistance have been identified along the Thailand-Myanmar, Thailand-Cambodia, Vietnam-Cambodia, and Vietnam-Laos borders ,2, and rPlasmodium falciparum parasites resistant to chloroquine, then sulphadoxine-pyrimethamine, and finally mefloquine were first detected in this part of the world [Resistance to anti-malarials has historically originated on the Thailand-Cambodia border; he world . Initialhe world , then suhe world . Artemishe world . Given tP. falciparum, guided by the framework of the World Health Organization’s Emergency Response to Artemisinin Resistance in the Greater Mekong Subregion (ERAR)[Efforts are underway to contain the spread of artemisinin-resistant on (ERAR), which oon (ERAR). Even soP. falciparum malaria transmission, regardless of whether resistance has been detected, negates the need to define drug resistance in all sites in real-time, and protects against the spread of resistance to new areas because transmission everywhere is reduced. Thus, there is a strong argument to be made that the only effective and sustainable way to respond to the current resistance situation is the rapid elimination of all P. falciparum parasites from the GMS.In light of these challenges, operationally targeting resources to only those areas with resistant parasites has not been effective. Instead, targeting all areas with P. falciparum-specific elimination goal, and four countries—Cambodia, China, Thailand, and Vietnam—have made major strides toward national elimination of all malaria species [A subregional elimination strategy is in line with the current commitment to malaria elimination in the GMS and will build political support and enhance collaboration across countries. Five countries in the GMS have already declared an intention to eliminate all forms of malaria within their borders Figure . Cambodi species .P. falciparum elimination initiative would bring together national programmes and partners around a single, strategic, time-limited goal, presenting an opportunity to achieve a regional public good. A focused, subregional effort specific to artemisinin resistance would improve coordination and harmonization of activities and funding across the six countries and multitude of partners, through the increase of communication and information sharing, launch of operational collaboration in border areas, and identifying and implementing a joint research agenda.Political commitment for malaria elimination in the GMS is considerable, facilitated by five of the countries’ partnership with, and active participation in, the Asia Pacific Malaria Elimination Network (APMEN), a network of 15 countries across the Asia Pacific region that have declared goals of malaria elimination ,9. LeverP. falciparum control and elimination activities will not be impactful unless the GMS countries have the financial resources to make those plans a reality. Funding is now available for GMS countries to increase their capacity, a major source being the Global Fund grant to the Regional Artemisinin Initiative (RAI) [P. falciparum-free GMS may spark the interest of other funders in a way that containment efforts thus far have not.All of the countries of the GMS have intensified their control and elimination efforts and are, or will soon be, receiving technical and financial support to boost their operational capacities. Many technical partners are working on research and implementation in the region. Some countries, such as China and Thailand, have made major progress in their capacity for response while others, such as Myanmar, have some work to do to bring the malaria program to a level necessary to meet the resistance challenge. However, well-drafted plans for intensified ve (RAI) , but addA subregional effort for elimination meets the challenge of human mobility in the GMS. The populations most at risk for development of resistance are primarily located in relatively underdeveloped and remote border regions. The lack of accessible services leads to inadequate detection of resistant malaria and low coverage of preventive and curative services. National malaria strategies typically focus on at-risk populations within country borders, but this approach fails to address the regional nature of migration patterns. Population movement through remote, forested areas and across porous borders increases the difficulty of accessing these groups by any one country. Thus, a comprehensive subregional strategy that relies upon international coordination is essential.Large-scale regional infrastructure developments are underway that necessitate rapid action to prevent the spread of drug-resistant malaria. In late 2015, the Association of Southeast Asian Nations (ASEAN) will launch the ASEAN Economic Community (AEC). The AEC aims to reduce economic disparities among countries through the development of an integrated regional economy, which will create a freer flow of goods, services, capital, and labour, increasing human mobility in the region. Greater population movement in areas with confirmed resistance can facilitate its spread, and the risk will be compounded by the new transport corridors and infrastructure that will increase access to remote areas.P. falciparum elimination. Such strategies include early diagnosis and treatment, active case detection, case investigation, and effective measures to reduce and prevent transmission. These strategies should be implemented on a national level with robust regional guidance. Execution in a small, foci-based manner will not be as beneficial to the overall health system nor the malaria program. China’s new 1-3-7 malaria surveillance approach provides one operational model of surveillance and response; more countries in the region need to implement surveillance and response measures as an intervention. 1-3-7 defines actions and timelines for effective surveillance activities: case reporting within one day, case confirmation and investigation within three days and appropriate public health response to prevent further transmission within seven days [A GMS elimination goal requires that countries improve and intensify malaria control operations and management. A recent joint assessment of containment efforts found insufficient intensity, coverage and quality in the delivery of malaria control interventions . Regionaven days . NationaP. falciparum elimination goal is the only way to effectively remove the historic epicentre of anti-malarial drug resistance. The newly formed Asia Pacific Leaders Malaria Alliance (APLMA) presents a timely opportunity: one way to signal commitment to a subregional elimination goal is to have members of the Alliance engage with public and private sector development partners in generating support and mobilizing resources. ASEAN is chaired by Myanmar this year, and encouraging ASEAN to formally indicate that improvements in regional economic trade are linked with anti-malarial drug resistance and subregional malaria elimination would accelerate elimination efforts. This is particularly true given the potential impact of the AEC on the spread of resistance.Rallying around an urgent and ambitious subregional P. falciparum elimination throughout the subregion must be aggressively implemented, without limiting their scope to already-identified resistant focal areas or populations. Progress towards this subregional goal is underway, with encouraging steps taken through the establishment of the RAI, funded by the Global Fund, the Regional Malaria and Other Communicable Disease Threats Trust Fund hosted by the Asian Development Bank [Finally, proven malaria strategies with the explicit goal of ent Bank , contribAEC: ASEAN Economic Community; ASEAN: Association of Southeast Asian Nations; APLMA: Asia Pacific Leaders Malaria Alliance; APMEN: Asia Pacific Malaria Elimination Network; ERAR: Emergency response to artemisinin resistance in the Greater Mekong subregion; GMS: Greater Mekong subregion; PDR: Lao People’s Democratic Republic; RAI: Regional Artemisinin Initiative; WHO: World Health Organization.The Global Health Group at UCSF exists in part to support global, regional and country efforts to achieve evidence-based malaria elimination. CSG, RG and MW are part of the APMEN Joint-Secretariat and RGAF is co-Chair of APMEN.CSG, GN and JH conducted research and analysis and wrote the manuscript. AAP, MW, JM, RDG, and RGAF provided comments on drafts and contributed to the final manuscript. All authors read and approved the final manuscript.


Population connectivity, which is essential for the persistence of benthic marine metapopulations, depends on how life history traits and the environment interact to influence larval production, dispersal and survival. Although we have made significant advances in our understanding of the spatial and temporal dynamics of these individual processes, developing an approach that integrates the entire population connectivity process from reproduction, through dispersal, and to the recruitment of individuals has been difficult.We present a population connectivity modelling framework and diagnostic approach for quantifying the impact of i) life histories, ii) demographics, iii) larval dispersal, and iv) the physical seascape, on the structure of connectivity and metapopulation dynamics. We illustrate this approach using the subtidal rocky reef ecosystem of Port Phillip Bay, were we provide a broadly-applicable framework of population connectivity and quantitative methodology for evaluating the relative importance of individual factors in determining local and system outcomes.The spatial characteristics of marine population connectivity are primarily influenced by larval mortality, the duration of the pelagic larval stage, and the settlement competency characteristics, with significant variability imposed by the geographic setting and the timing of larval release. The relative influence and the direction and strength of the main effects were strongly consistent among 10 connectivity-based metrics.These important intrinsic factors  and the spatial and temporal variability represent key research priorities for advancing our understanding of the connectivity process and metapopulation outcomes.The online version of this article (doi:10.1186/s40462-015-0045-6) contains supplementary material, which is available to authorized users. Understanding the causes and consequences of dispersal is a foundational goal in population and community ecology, and evolution. Dispersal, or the exchange of individuals among natal and non-natal sites, is the primary process that ‘connects’ populations, with important impacts on local demography, landscape-wide population dynamics and gene flow. Although there are a diversity of evolutionary drivers of dispersal, such as reducing kin competition and inbreeding , ‘bet heThe importance of population connectivity has also fuelled efforts to integrate this process into conservation and management, particularly in relation to population persistence and viability , reserveper se has elevated the importance of both intrinsic  and extrinsic  drivers [Recent advances in movement ecology, population biology, and land/seascape ecology have improved our understanding of this biological-physical process. The study of the mechanisms of dispersal movements  drivers , 17 and  drivers , 19. Thi drivers , 21, or  drivers . Concurr drivers . The mecStudying dispersal-driven population connectivity in benthic marine species with complex life cycles is particularly challenging due to the strong influence of currents, the age, size, and behavioural complexities of the dispersing individuals, and the spatiotemporal scales (and variability) of the process . DespiteAlthough individual biophysical parameters are important in determining connectivity outcomes, such as larval mortality , larval Here we have taken a system-level perspective and define metapopulation connectivity as the aggregate process integrating natal dispersal, post-settlement survival and reproduction , in both natal and non-natal sites. This process-based conceptualisation includes four-stages of population connectivity Fig.  and provDue to the nearly passive dispersal qualities of the early developmental stages, the strong influence and dynamics of the physical environment , and the potential mobility and sensing capacity of late-stage dispersers, the existing mechanistic models of dispersal are inadequate. As a result, we have developed a framework describing the four stages of population connectivity, incorporating the three stages of dispersal , 20 apprThe first stage in the connectivity process is the initiation of emigration  semi-enclosed temperate marine system in Victoria, Australia, and is ideally suited for exploring the biophysics of marine population connectivity. One of the most prominent and economically important habitat features in the bay are the sub-tidal rocky reefs  is a large ~2,000 km,000 km2 The hydrodynamics of PPB have been well studied and accurately modelled since the mid 1980′s  accompanAn individual-based model of dispersal (modified from ) was user), allocated randomly across patches to represent (unknown) recruitment costs [At the initiation of emigration, the reproductive output (RO) is determined by the fecundity and abundance of the modelled taxon, and the habitat characteristics of the natal site. While individuals move through the environment , a network-based measure characterising the strength and evenness of downstream linkages [Simulations were completed for each of eight selected reef habitat patches to explore the impact of geographic location on connectivity outcomes. In addition, simulations were initiated on three separate dates  was used , 70. Due2) in the recursive partitioning regression analysis for each response variable and the global mean relative influence of each parameter is presented in Table The mean relative influence and 95 % confidence limits were plotted for parameters using the LHS (each with 900 parameter sets) for all eight reefs and three release dates Figs. , 5 and 6Across all response variables, high variability resulted from both the geographic context and timing of initiation. Variation driven by the geographic location of the eight reefs is evident as the horizontal displacement when comparing individual bars (reefs) across the vertical extent of each parameter’s row. Similarly, the variation resulting from different release times is seen as the total spread across colours (release dates) along each horizontal plane (unique reefs). Importance values for all simulations are shown, and those that are not significant are plotted as points along the y-axis and some points and lines may fall on top of each other. Generally, across the majority of response variables, larval mortality rate (A) and the length of the pelagic larval stage (PLD) were the most influential parameters Figs. , Table 3W) was determined primarily by larval mortality (A), followed by the precompetency period (PreP), swim speed (Sp), PLD, and to a lesser extent, reproductive output (RO). The proportion of successful settlers was driven by mortality rate (A), followed by the precompetency period (PreP), with swimming speed (Sp), PLD, and homing distance (HmD) all with modest influences. At the metapopulation scale, larval mortality rate (A), the precompetency period (PreP), and the reproductive output (RO) were the most influential drivers, followed by the PLD and swim speed (Sp).Focusing on the local patch scale, retention of individuals within the natal site Fig. , Table 3Across the majority of population connectivity response variables the following six parameters consistently had low relative influences: larval mortality shape parameter (B), rate of transition to competency (ComR), initial development time (DevP), bio-physical cohesion (K), the target depth (TD), and homing distance (HmD). The strong influence of geographic location or seascape context was clear when comparing values in the response variables across all reefs . Increasing the time spent dispersing effectively increases the likelihood of making some long-distance connections, while at the same time, increases the likelihood of being lost at sea resulting in larval wastage. Quantifying these mixed effects of PLD on connectivity outcomes could help disentangle the equally mixed results present in the empirically-based, albeit indirect, correlations between PLD and genetic distance or gene flow [Despite this spatiotemporal variability, broad patterns in the dominant drivers of connectivity outcomes were apparent and strongly consistent. Across the majority of response variables Table , three pene flow .W) and effectively decreasing metapopulation growth . Although relatively little is known of the development transition phase of many marine species , a trait unique to marine and aquatic taxa, was the third most important parameter. Increasing the precompetency period decreases the potential settlement window, effectively increasing the geographic distance (mdG and mxG) individuals travel while decreasing the proportion that settled locally . These parameters have been highlighted in the past as being relatively important , 40, yetrals see , 81), itrals see , thereforals see , 10.A suite of parameters were identified as having an intermediate level of influence, often with inconsistencies in the strength and direction of effect on connectivity ). This M and λmax, should be used to help identify locations that are important contributors to overall growth rates or capacity to recover from disturbance. Identifying such keystone populations, which are likely to be well-connected populations that are important sources, has been a successful approach for informing conservation efforts in terrestrial ecosystems [Our framework has direct applicability to many marine and aquatic systems, and may assist in gaining a more holistic and integrated view of dispersal-based connectivity to aid in management. Connectivity is a multifaceted process and using a holistic framework to assess the primary drivers of dispersal and population connectivity will lead to greater insight into where best to target management efforts. We suggest that the metapopulation-based metrics, such as λosystems . We suggHere, we have presented clear evidence on the relative importance of the transport and settlement stages to marine population connectivity, the key intrinsic drivers of larval mortality, the length of the pelagic larval phase, and the settlement competency characteristics, and the influence of the extrinsic factors of habitat geography and currents. Gaining a better understanding of these drivers and how they vary across species will greatly enhance our ability to predict contemporary connectivity patterns, will aid in our study of the evolution of larval dispersal strategies , and ma


Epigenetics is currently one of the hottest topics in basic and biomedical research. However, to date, most of the studies have been descriptive in nature, designed to investigate static distribution of various epigenetic modifications in cells. Even though tremendous amount of information has been collected, we are still far from the complete understanding of epigenetic processes, their dynamics or even their direct effects on local chromatin and we still do not comprehend whether these epigenetic states are the cause or the consequence of the transcriptional profile of the cell. In this review, we try to define the concept of synthetic epigenetics and outline the available genome targeting technologies, which are used for locus-specific editing of epigenetic signals. We report early success stories and the lessons we have learned from them, and provide a guide for their application. Finally, we discuss existing limitations of the available technologies and indicate possible areas for further development. The marbles (cells) compete for the grooves on the slope, which define their developmental trajectories, and come to rest at the base of the hill in defined positions. These defined positions demarcate eventual cell fates, meaning the tissue types which the cells adapt [The human body is built from more than 200 different cell types organized in various tissues. It is fascinating that although all these cells stem from a single cell (the zygote) and contain exactly the same DNA sequence (except for the antibody-forming B cells of the immune system ), they ehttp://www.genome.gov/encode/) [http://www.roadmapepigenomics.org/) [http://www.blueprint-epigenome.eu/) [The human genome comprises approximately three billion base pairs, which represent a large repository of information. The fact that different cells contain basically the same DNA but show very distinct phenotypes indicates that regulated access to this information is key to understanding cell identity and, therefore, human development and health. The last 60 years of research in the field of epigenetics was focused mostly on elucidating the molecular mechanisms and the enzymatic machinery responsible for epigenetic control of gene expression, as well as the distribution of various epigenetic marks in different cell types from healthy and diseased tissues and organisms. Progress in the next-generation sequencing and proteomic approaches allowed systematic analysis and identification of novel epigenetic marks and their distribution across the genome and nucleus. Few large-scale collaborative projects, like ENCODE (encode/) ,4, Roadmcs.org/)  and BlueDespite the tremendous progress in understanding the epigenetic signalling pathways, as well as characterization of the epigenetic marks (DNA and histone modifications) and the enzymatic machinery that can write, read and remove these marks, many fundamental questions still remain unresolved, mainly due to technological limitations. For example, are various epigenetic signals the cause or the consequence of the cell transcriptional profile? What is the sequential order of epigenetic transitions between the repressed and activated states? Are epimutations drivers or merely by-products of a diseased state, and finally, what is the contribution of epigenetics to disease (cancer) development ,8?In this review, we try to define the area of synthetic epigenetics and outline the available genome targeting technologies, which are used for targeted editing of epigenetic signals. We report early success stories and the lessons we have learned from them, as well as potential biomedical applications and existing limitations of the available technologies.the design and construction of novel specific artificial epigenetic pathways or the redesign of existing natural biological systems, in order to intentionally change epigenetic information of the cell at desired loci. In this broad definition view, somatic cell nuclear transfer experiments (SCNT) [We define synthetic epigenetics as s (SCNT) ,10, dires (SCNT) ,12, genes (SCNT) ,14 and ts (SCNT)  all conss (SCNT) ,17) as sImportantly, there is a qualitative difference between the nuclear transfer and induced pluripotency experiments when compared to epigenetic editing. In the experiments with somatic cell nuclear transfer and during generation of induced pluripotent stem cells, genome-wide changes in the epigenetic state of the cells are triggered by a mixture of defined oocyte-specific factors or forced expression of a selection of transcription factors, respectively. This is achieved through the restoration of robust pluripotency promoting transcription factor networks with self-enforcing feedback mechanisms, which rewire the transcriptional profile of the cell ,19, and On the other hand, in a bottom-up approach, a local direct change to the epigenome can be introduced by targeting an effector domain to the desired locus. In this epigenetic editing  approachExisting technologies used to modulate transcription of desired genes, like RNAi , gene knThe epigenetic editing tools are perfectly suited to treat and study the molecular mechanisms underlying epigenetic diseases like cancer, chronic diseases or imprinting defects. For example, DNA methylation has been already used to silence overexpressed oncogenes -39 and cNature Methods has distinguished epigenome editing as a “Method to Watch” [The concept of bottom-up synthetic epigenetics (epigenetic editing) relies on the combination of an artificial DNA binding domain, which can directly bind a unique sequence found within the desired locus, with an effector domain able to edit the epigenetic state of that locus Figure . To dateo Watch” . In the For most of the known DNA interacting proteins, no simple DNA recognition code exists that could connect individual amino acid residues with the corresponding DNA base . For thiC2H2 zinc fingers were the first example of predictable DNA interaction domains amenable to rational protein design , andhttp://zifit.partners.org/ZiFiT/ [Currently, two main methods are used to generate engineered zinc finger arrays: context-dependent modular assembly  and bactg/ZiFiT/ ,69).Xanthomonas [Another class of customizable DNA binding domains, the transcription activating-like effectors (TALEs) are important virulence factors initially isolated from the bacterial plant pathogen thomonas . Membersthomonas . Due to thomonas ,72. Becathomonas . HoweverThe newest exciting addition to the genome targeting toolbox repository is the CRISPR/Cas9 system . CRISPR Each of the available programmable genome targeting devices discussed above offers unique advantages and disadvantages  and HaeIII (GGCC) methylated sites in a specific context, by using rounds of selection of phage displayed zinc finger randomized libraries that employed M.HhaI- and M.HaeIII-methylated DNA as baits [Both zinc fingers and TALEs read the sequence in the major groove of DNA ,90. Impoas baits . Such meas baits . The modas baits .Overall, the CRISPR/Cas9 systems offer substantial advantages over zinc finger and TALE arrays, like simplicity of target design, possibility for multiplexing (targeting two or more sites at the same time) and independence of DNA modifications. Moreover, orthologous CRISPR/Cas9 proteins isolated from various bacteria could be employed for simultaneous targeting of different functionalities to the same or different loci . Howeverin vitro to synthetic DNA [Selection of the appropriate effector domain is based on the planned application and intended effect on transcription and epigenetic state. Nature provides numerous possible modification domains, which can be employed for the particular function. Various effector modifiers have been already fused to zinc fingers, TALE arrays and other DNA binding domains to target their activities to endogenous or genome integrated targets, reporter plasmids or viral DNA ,97. Exametic DNA , M.HpaIIetic DNA ,100 and etic DNA ,101), teetic DNA ,103), thetic DNA ), histonetic DNA , G9a [10etic DNA  and Suv3etic DNA ,107 targetic DNA ) and hisetic DNA ), as weletic DNA ), which etic DNA .Many (if not most) of the targeted epigenome modifications studies were focused on manipulating the DNA methylation state , in order to repress active oncogenes or activate silenced tumour suppressor genes, respectively. This might be explained by the fact that in contrast to the not yet fully understood mechanism of maintenance of histone modifications during mitotic division , the mecMethylation of gene promoters around transcription start sites and first exons is strongly correlated with gene repression ,114. OncNo matter which effector domain is selected for targeting, the applicability of that domain should include the evaluation of the extent and stability of the introduced modification and its biological effect, as discussed in the next sections.Despite the dynamic nature of epigenetic information, the overall cell state and global epigenetic states are heritable and maintained remarkably stably during multiple mitotic divisions. This is mainly due to the cooperation and redundancy of the multiple epigenetic signals (DNA and histone modifications), as well as to the transcriptional activity of each particular gene , which cThese studies illustrate that targeted epigenome modification can indeed withstand numerous cell divisions and is superior over effects introduced with transient targeting of repressors. Nonetheless, the introduced epigenetic modification might not be stable in all genomic contexts and will depend on the pre-existence of other activating or repressive epigenetic marks, the location of the locus in the euchromatic or heterochromatic region, as well as on the extent of the introduced modification and the nature of the mark itself. It is likely that small and local changes to the epigenome may not be efficiently maintained, and therefore, the locus would return to the initial state before perturbation happened ,115. TheSince the epigenetic modifier is tethered to the DNA binding domain, which binds tightly to its recognition sequence, the length of the region which can be directly modified is limited. The extent of the linker between the targeting device and the epigenetic modifier is the main determinant of the possible reach distance. In the majority of the studies, the most efficient introduction of modification was observed in the nearest vicinity of the binding site of the targeting domain Due to mechanical constraints of the targeted epigenetic editing machinery, it is unlikely that a single and even very stable binding event would lead to a widespread modification at larger distances. Interestingly, histone modification  and DNA Whereas most of the studies were designed to achieve widespread DNA methylation of the targeted locus, Chaikind and colleagues developed an opposite strategy to selectively methylate a single CpG site in the genome using a split enzyme approach ,129. TwoThe efficiency of introducing epigenetic change and therefore possible biomedical application depend in big part on the vehicle used for delivery of the constructs. So far, only cultured cells and not whole organisms were used for epigenetic editing applications. However, similar delivery methods as used for genome engineering can be applied for this purpose as well. Besides the traditional approaches, like transfections of transient expression plasmids, transduction of adeno-associated viral (AAV) , lenti-,From the time when the field of epigenetics was defined by Waddington until nowadays, we have learned a lot about epigenetic regulation of gene expression and maintenance of cellular identity. Substantial progress was achieved in identification and understanding the role of various epigenetic marks, their distribution in healthy and diseased tissues and the enzymatic machinery responsible for depositing, reading and removing these marks. This progress was accompanied or rather preceded by technological development, like chromatin immunoprecipitation (ChIP), bisulfite sequencing, high-throughput proteomic and whole genome approaches. The bottom-up synthetic epigenetics (targeted epigenome editing), although still in its infancy, constitutes an area of extensive research. New technological developments will likely increase the specificity of targeting devices and the efficiency of effector domains in setting the desired epigenetic marks and will supply engineered systems for spreading the modification across the whole locus, providing efficient and reliable tools for stable modification of the epigenome. The most exciting progress is expected from the CRISPR/Cas9 system, as it allows the biggest flexibility and ease of design of new targets and the possibility to construct target libraries ,138, whiSynthetic epigenetics has the potential to address so far unapproachable areas of basic and clinical research. It provides tools and methods, which allow dissection of the epigenetic signalling cascades and to identify the driver and passenger modifications. It can widen our understanding of epigenetic dynamics and the basis of signal inheritance. Top-down synthetic epigenetic approaches have already made a big contribution in generating disease model cell lines. Epigenetic editing will further foster biomedical research by addressing the epigenetic contribution to complex and simple diseases, through discovery and validation of disease-promoting epimutations and provide means for reverting them. It also offers tools to probe factors responsible for cell identity and allows intelligent control of the cell fate.


Background. Sepsis is among the leading causes of death worldwide and is the focus of a great deal of attention from policymakers and caregivers. However, sepsis poses significant challenges from a clinical point of view regarding its early detection and the best organization of sepsis care. Furthermore, we do not yet have reliable tools for measuring the incidence of sepsis. Methods based on analyses of insurance claims are unreliable, and postmortem diagnosis is still challenging since autopsy findings are often nonspecific. Aim. The objective of this review is to assess the state of our knowledge of the molecular and biohumoral mechanisms of sepsis and to correlate them with our postmortem diagnosis ability. Conclusion. The diagnosis of sepsis-related deaths is an illustrative example of the reciprocal value of autopsy both for clinicians and for pathologists. A complete methodological approach, integrating clinical data by means of autopsy and histological and laboratory findings aiming to identify and demonstrate the host response to infectious insults, is mandatory to illuminate the exact cause of death. This would help clinicians to compare pre- and postmortem findings and to reliably measure the incidence of sepsis. Sepsis can be defined as a syndrome of dysregulated inflammation caused by the failure of infection control and containment mechanisms. It should be considered a major public health problem since it affects millions of people worldwide each year, with an incidence which is dramatically increasing . It accoConsequently, considerable attention is dedicated to sepsis by policymakers and caregivers. However, these efforts are tempered by several limitations. First of all, difficulty in defining sepsis still exists due to the emerging biological insights and reported variation in epidemiology. Recently the Third International Consensus Definitions Task Force defined sepsis as “life-threatening organ dysfunction due to a dysregulated host response to infection” , 8; howeThe objective of this review is to assess the state of our knowledge of the molecular and biohumoral mechanisms of sepsis and to correlate them with our postmortem diagnosis ability.The septic response is an extremely complex chain of events involving inflammatory and anti-inflammatory processes, humoral and cellular reactions, and circulatory changes. When an infectious insult occurs, it initiates a series of events, resulting in the release of inflammation mediators which involves both a local reaction and a systemic response and which, finally, could impact on organs function .Sepsis triggers the production of a diverse array of cytokines that are proinflammatory and anti-inflammatory , 22. CytProinflammatory cytokines trigger a beneficial response, such as increased local coagulation, limited tissue damage, and elimination of the pathogen. Overwhelming production of these proinflammatory cytokines, however, can be very dangerous in that excessive cytokines destroy the normal regulation of the immune response and induce pathological inflammatory disorders, such as capillary leakage, tissue injury, and organ failure. Similarly, the anti-inflammatory cytokines play a critical role in regulating overall immune response, in establishing homeostasis, and in checking the effects of the proinflammatory ones in order to solve inflammation and heal tissue. Therefore, their dysregulation can also trigger pathogenesis , since iκB (NF-κB), followed by the production and secretion of cytokines, chemokines, and nitric oxide [In brief, when an infectious insult occurs, pattern recognition receptors (PRRs), which are expressed on epithelial barriers as well as on resident immune cells such as dendritic cells and macrophages, detect invading microorganisms. A specific family of PRRs named toll-like receptors (TLRs) recognizes conserved macromolecular motifs from microorganisms, called pathogen-associated molecular patterns (PAMPs). The stimulation of TLRs or the nucleotide oligomerization domain NOD-like receptor (NLR) family of intracellular PRRs results in the triggering of downstream signalling cascades. Depending on the particular receptor engaged, this process leads to the activation of a transcriptional response programme that includes nuclear factor ic oxide , 22. Cytic oxide .Therefore, different types of cells, tissues/organs, or protein/other molecules may function as effectors, modulating the immune response through various pro- or anti-inflammatory mediators. Resident macrophages and polymorphonuclear cells (PMCs) initiate the primary host response to the invading microorganisms. They are responsible for the primary phagocytosis and subsequent activation and recruitment of granulocytes and monocytes. Leukostasis of neutrophils  in the liver sinusoids, in the pulmonary vessels, and so forth is the common histological counterpart of this phenomenon . ProlifeEndothelial cells (ECs) activation occurs during sepsis. The interaction between ECs and leukocytes, a hallmark of the inflammatory process, comprises adhesive and migratory molecular events including low-affinity transient and reversible rolling adhesions, integrin-dependent firm adhesive interactions, and migratory events of the leukocytes through the endothelium and, finally, in the interstitial space , 28. A vSepsis is also associated with robust activation of the complement system, as demonstrated by the presence of complement activation products  in the plasma . C3a andOther products derived from activation of the complement system play an important role in sepsis, such as C3b (from C3), which is a key opsonisation factor that reacts with phagocytes receptors to favour internalization of bacteria and their subsequent elimination. The membrane attack complex (C5b-9) causes lysis of Gram-negative bacteria .Finally, sepsis also affects other biological systems, such as the coagulation system and the autonomic nervous system . In the A fundamental step in the comprehension of sepsis pathophysiology is to appreciate that the inflammatory response varies from one compartment to another, and not all compartments behave similarly . ChinnaiκB activation in liver was mediated through TNF and IL-1 receptor-dependent pathways, but, in the lung, LPS induced NF-κB activation was largely independent of these receptors [β in response to TLR4 and TLR3 agonists [Each organ has a distinctive molecular response to systemic inflammation. Several experimental pieces of data confirm this observation. For example, neutrophil sequestration in lung and liver resulted differently regulated by chemokines in a murine experimental model of peritonitis . After ieceptors . Althougeceptors , do not eceptors  and failagonists , thus deThrough the bloodstream a strict cross talk exists between the different organs and compartments. Both pro- and anti-inflammatory mediators are present concomitantly in the bloodstream, evoking different responses in the various organs. The latter, in turn, respond through the local production of different mediators and the activation of different cellular types . Most tiThis concept is of paramount clinical importance as the various organs may respond differently to therapeutic strategies. On the other hand, upon approaching sepsis-related deaths, pathologists must keep in mind that tissue injury can be initiated remotely from an insult in a faraway site and that all organs and compartments may be involved .Different organs and systems are interconnected via humoral and biochemical interactions and are clustered into functional modules sharing many common pathophysiological mechanisms. Diagnostic postmortem strategies based on the measurement of compartmentalized mediators may prove useful as a diagnostic strategy , 48.Sepsis is a disease of microcirculation . NuclearDeleterious effects on the vascular function are mediated by increased synthesis of inflammatory cytokines and chemokines and increased expression of endothelial adhesion molecules –51. Micr complexes I and II/III respiration, MnSOD (manganese superoxide dismutase) activity, and ATP levels. This was associated with increased mitochondrial superoxide levels, impaired renal microcirculation, and impaired renal function [Microvasculature dysfunction in sepsis is almost ubiquitous. Pulmonary MVECs injury and barrier dysfunction result in the leak of protein-rich fluid and circulating neutrophils into the pulmonary interstitium and alveolar spaces –54. Recefunction . Oxidantfunction –62. Cerefunction , and it function . Both enfunction –69.κB, and the expression of inducible nitric oxide synthase and cyclooxygenase-2, with an increase in nitric oxide and vasodilator prostanoids, leading to arterial hyporeactivity [Finally, in recent years a growing body of evidence has been established regarding the role of microvesicles (MVs) in sepsis . These eactivity –74.In recent years, the analysis of circulating-cell-derived MVs has become more defined and clinically more useful, and several groups suggest that it may enter mainstream clinical testing , 76. End in vita diagnosis of sepsis and some have been proposed also as biological markers of the severity of sepsis, but none alone is entirely specific for infection because they can be also detected in the absence of infection [Blood compartment plays a key role in the inflammatory response. Waves of mediators with both anti- and proinflammatory properties are detectable in the plasma of septic patients. Many of these are considered reliable markers fornfection . The futnfection –86. Howenfection .As molecular and humoral mechanisms are thought to play a key part in the pathophysiology of sepsis, postmortem diagnostic strategies based on the concentration of such mediators have been investigated.Many authors propose the detection of procalcitonin (PCT) as a useful and reliable marker of sepsis in routine autopsy investigations –91. PCT,However, due to the detectability of high levels of PCT also in aseptic inflammation  –103, a pAs demonstrated by the studies mentioned above, much attention has been focused on cytokines and other mediators as diagnostic tools in many diseases and they certainly hold promise also for the discovery of reliable postmortem biomarkers of sepsis.Soluble interleukin-2 receptor (sIL-2R) and LBP seem to represent appropriate diagnostic tools for the postmortem diagnosis of sepsis . PostmorOther biomarkers of potential relevance to sepsis/septic shock diagnostics have been proposed more recently.Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes and upregulated in bacterial sepsis. This is associated with a marked plasma elevation in the soluble form of this molecule (sTREM-1). Studies have indicated that sTREM-1 could be a valuable diagnostic biomarker for sepsis . Palmier; it shows promise for diagnostic and prognostic purposes in septic patients [Presepsin (sCD14-ST) is a soluble N-terminal fragment of protein CD14 which is released into circulation during monocyte activation on the recognition of LPS from infectious agents ; it showpatients . It has patients .Endocan , a 50-kDa dermatan sulphate proteoglycan, expressed by endothelial cells in lung and kidney, can be detected at low levels in the serum of healthy subjects. Increased concentrations were described in patients with sepsis, severe sepsis, and septic shock compared to healthy individuals, with serum concentrations related to the severity of illness . PalmierNeopterin , a biochemical product of guanosine triphosphate pathway, has been proposed to aid in the diagnosis of bacterial  and viraConclusively, there is a strong body of evidence that postmortem concentration of serum cytokines and other mediators of inflammation may be an area of great interest with exciting diagnostic possibilities for sepsis/septic shock-related deaths , 119. HoIn a previous review, Pierrakos and Vincent identified nearly 180 distinct molecules that have been proposed as potential biological markers of sepsis . HoweverThe difficulties of the postmortem diagnosis of death due to sepsis are well known. The major limitation is the poor specificity of macroscopic and routine histological findings encountered in such cases. Due to the complex molecular and cellular mechanisms underlying sepsis, proof of the presence of germs alone cannot be of evidential value in establishing a causal relationship between infection and outcome. Thus, the old saying by William Osler 1849 to 1919) “except on few occasions, the patient appears to die from the body's response to infection rather than from the infection” is still true [9 to 1919A complete methodological approach, integrating clinical data by means of autopsy and histological and laboratory findings aiming to identify and demonstrate the host response to infectious insult, is mandatory. Such an approach would be likely to produce an accurate objective surveillance of deaths due to sepsis and improve our knowledge of the clinical-pathological correlation in sepsis, thus contributing to the evaluation of the effectiveness of therapies. Finally, autopsy is a critical tool for protection from false liability claims and settling valid claims quickly and fairly.