Liver stem/progenitor cells (LSPCs) are able to duplicate themselves and differentiate into each type of cells in the liver, including mature hepatocytes and cholangiocytes. Understanding how to accurately control the hepatic differentiation of LSPCs is a challenge in many fields from preclinical to clinical treatments. This review summarizes the recent advances made to control the hepatic differentiation of LSPCs over the last few decades. The hepatic differentiation of LSPCs is a gradual process consisting of three main steps: initiation, progression and accomplishment. The unbalanced distribution of the affecting materials in each step results in the hepatic maturation of LSPCs. As the innovative and creative works for generating hepatocytes with full functions from LSPCs are gradually accumulated, LSPC therapies will soon be a new choice for treating liver diseases. IntroductionThe identification of cells generated from LSPCs–Morphological characteristics–Phenotypic markers–Functional evaluation–In vivo transplantationStrategies for inducing the hepatic differentiation of LSPCs–Modification of the physical parameters in LSPC cultures–The induction of LSPCs by adding feeder cells–Addition of soluble medium factors–The induced differentiation of LSPCs on bio-scaffold–Determination of cell fate via epigenetic modificationDetailed mechanisms of hepatic differentiation from LSPCs–The initiation of hepatic differentiation–The progression of hepatic differentiation–The accomplishment of hepatic differentiationConclusions and perspectivesi) LSPCs have great potential for generating numerous functional cells in the liver such as mature hepatocytes and cholangiocytes; (ii) LSPCs are present in the liver regardless of donor age#x2014;they are present in the ductal plates in foetal and neonatal livers and in the Canals of Hering in paediatric and adult livers iii) LSPCs with liver fate specification have been isolated and identified, including foetal liver stem/progenitor cells  and adult liver stem/progenitor cells ; (iv) LSPCs can be genetically manipulated, implanted without major surgery and cryopreserved for future use; (v) the requirements for immunosuppression are not strict in most LSPC transplantation cases.Liver transplantation is currently the most effective treatment for liver failure, but its use is limited by the scarcity of organs for transplantation, the high cost, and lifelong immunosuppression necessary after transplantation. Cell-based therapy is a promising strategy to solve these problems. Studies on liver stem/progenitor cells (LSPCs) have shown especially promising results for overcoming the above-mentioned limitations In addition to the promising therapeutic effects for liver failure, LSPCs also play a critical role in liver regeneration after severe liver damage In vitro culture systems as well as in vivo studies have elucidated detailed molecular mechanisms, including intercellular signalling networks and intracellular transcriptional regulatory webs, that co-ordinately regulate the hepatic differentiation of LSPCs. Understanding the cellular and molecular bases of hepatic differentiation from LSPCs will be invaluable in producing fully functional hepatocytes that can be applied for cell therapy and pharmaceutical screening in the future Despite the fact that LSPCs are promising for cell therapy and are essential for liver regeneration, the fundamental problem in clinical applications lies in generating mature functional cells from LSPCs in vivo microenvironment via (i) reconstitution of the cell matrix, (ii) cell–cell interactions (feeder cells), (iii) addition of soluble medium factors, (iv) chromatin modulation, (v) overexpression of liver-enriched transcription factors (LETFs), and (vi) treatment with biomaterials. No matter how the differentiation of LSPCs is triggered, we hypothesize that the unbalanced distribution of materials such as inductive molecules, facilitative signalling pathways and the accompanying LETFs, is crucial in deciding the cell fate of LSPCs  typical morphological characteristics; (ii) expression of lineage-specific markers; (iii) essential functional achievements; (iv) in vivo transplantation for cell therapy. We must identify the cell fate of LSPCs according to the features of the primitive resident cells comprising the liver.This section discusses how to identify undifferentiated LSPCs and induced hepatocytes. The following criteria are discussed  and tightly packed colony formation When LSPCs differentiate into hepatocytes, the cells grow in size to >18#x00A0;#x03BC;m and display a cordlike colony morphology. These induced cells can form hepatic plate-like structures consisting of long spindle or polygonal epithelia-like cells with dual-nuclei or multi-nuclei when viewed under a light microscope. In ultrastructural studies, these cells acquired typical hepatocyte features such as large numbers of mitochondria, rough endoplasmic reticulum (ER) and Golgi complexes.The differentiation of LSPCs into mature cells is a steady process, and stem cells, progenitor cells and mature cells differ in their molecular phenotypes i) LSPCs themselves show a wide range of phenotypic heterogeneity; (ii) many of the currently used markers remain controversial among different research groups. Thus, in this review, we present the markers that are the most effective for analysis.Although great progress has been made in the isolation and identification of LSPCs, there are still no widely accepted markers that are specific for LSPCs. The reason for this is twofold:  133  et#x00A0;al. demonstrated the pure enrichment of ALSPCs using combinations of several positive markers, including CD13, Dlk1 and CD133 Compared to FLSPCs, older and more numerous research studies have identified markers of ALPSCs. Until now, several monoclonal antibodies have long been used as the #x2018;golden standards#x2019; to identify OCs (representative ALSPCs) including oval cell 1 (OV-1) and OV-6 in rats et#x00A0;al. have successfully isolated ALSPCs by SP from injured rat liver Adult liver stem/progenitor cells show a wide range of phenotypic heterogeneity, and more and more new molecules have been reported to be ALSPC markers, including c-kit i) select the most widely accepted markers; (ii) combine more than two markers; (iii) pair stem cell markers with early liver lineage markers; and (iv) consider the species of LSPC origination.In summary, when judging whether LSPCs differentiate or not, keep the following in mind: , mid/late markers  and late markers  #x03B1; and cytochrome P450 #x005B;CYPs#x005D;) in vitro, cells sequentially converted from expression of early markers  to mid/late markers  to late markers  Hepatogenesis in vitro culture and after induction should be able to generate ALB-positive hepatocytes and CK-7-positive cholangiocytes. In addition, as mentioned above, LSPCs should be able to functionally reconstitute the liver parenchyma efficiently after injury.The main characteristic of stem cells is self-renewal and multipotent differentiation  expression analysis of genes found in mature hepatocytes, such as LETFs and CYPs; (ii) metabolism of xenobiotics and endogenous substances ; (iii) synthesis and secretion of ALB, clotting factors, transporter proteins, bile, lipids and lipoproteins; and (iv) storage of glucose (glycogen), folate, vitamin B12, copper, iron and the fat-soluble vitamins A, D, E and K. Detailed information on the essential aspects can be found in Figure#x00A0;We first need to establish a list of mature hepatic functions that can be easily measured. In other words, we need fast and easy tests that provide relevant and robust information on the hepatic capabilities of the LSPC-derived hepatocytes. From a functional point of view, any candidate hepatocyte-like cell type should represent a minimal set of hepatic functions of a true hepatocyte in vivo identification of differentiated cells is essential, and in some sense it is the #x2018;golden standard#x2019; of certifying the cell fates of differentiated LSPCs in vivo experiment to confirm the identity of LSPC-derived hepatocytes is to restore damaged hepatocytes and recover liver function in animal models. To monitor the implanted cells and their contributions, the transplanted cells should be labelled with either fluorescent materials As we have mentioned in our previous articles, the in vivo microenvironment of LSPCs in vitro strategies for inducing hepatic differentiation of LSPCs come from monitoring the in vivo microenvironment. In other words, when it is needed to promote transplanted LSPCs or resident LSPCs to differentiate into hepatocytes, we only have to rebuild the required microenvironment for hepatic differentiation, including addition of some necessary materials. The co-ordinated signalling between stem cells, non-stem niche cells and the scaffold and the integration of stem cell-autonomous characteristics, including a dynamic interplay between transcription, epigenetic control and post-transcriptional regulators, represent an interactive system organized to facilitate cell fate decisions in a spatiotemporal manner Hepatocytes obtained from LSPCs and other stem/progenitor cells have not yet matured to the stage at which they can efficiently repopulate the liver of an adult. In other words, to use LSPCs in regenerative medicine, an effective procedure to accomplish hepatic maturation from LSPCs must be developed. The methods for controlling the cell fate of LSPCs can be determined from the In recent years, simulated microgravity, a physical force, has been shown to differentially regulate the proliferation and differentiation of stem cells. Microgravity, which is simulated using indigenously fabricated three-dimensional 3D) clinostats, can induce the differentiation of stem cells within 2–3#x00A0;days. Furthermore, microgravity interplays with signalling pathways (such as Wnt/Notch) in stem cells, thus better inducing the differentiation of LSPCs into hepatocytes D clinostIn vivo, it is believed that the surrounding cells can play important roles in controlling the cell fate of LSPCs. For example, within the niche, macrophages use paracrine signalling to control cell fate of LSPCs via TWEAK (tumour-necrosis-factor-like weak inducer of apoptosis) and the Wnt signalling pathway. After hepatocellular injury, macrophages ingest hepatocyte debris and release Wnt, which promotes LSPC differentiation into hepatocytes In vitro, LSPCs can be induced to differentiate by co-culture with feeder cells such as fibroblasts, hepatocytes and mesenchymal cells i) angioblasts, (ii) myofibroblasts, (iii) hepatic stellate cell precursors, (iv) mature stellate cells (pericytes) and (v) mature endothelial cells. In one word, if the feeder cells are stem-like cells, they will probably induce LSPC proliferation; however, if they are mature cells, they are more likely to induce LSPC differentiation  and components of the ECM lead to the differentiation of LSPCs In addition, biomaterials can potentially influence stem/progenitor cell proliferation and differentiation in both a positive and a negative way , HNF4#x03B1; and C/EBP#x03B1; induces a mature hepatocyte phenotype in an expandable LSPC cell line in vitro and in vivo researches. We could use these mechanisms to correct the abnormal liver development and rescue the insufficient liver regeneration. Meanwhile, the knowledge of molecular mechanisms of hepatic differentiation could also guide LSPCs transplantation-based treatment of severe hepatocytes loss.Although LSPCs are thought to differentiate into hepatocytes, the details of the differentiation process are not well understood. In the above sections, we described the conditions needed for LSPCs to differentiate into fully mature hepatocytes. In the present section, we will clarify the related mechanisms for inducing the hepatic differentiation of LSPCs. In other words, both positive factors and negative factors collectively regulate the process of hepatic differentiation from LSPCs. However, at present, most of the researches are concentrating on the positive factors, and there is lack of knowledge of negative factors responsible for hepatic differentiation. In this review, we mainly pay attention to the positive factors. Generally speaking, different positive factors are unequally assigned to progeny cells, resulting in distinct directions of LSPCs differentiation. The differentiation of LSPCs is a gradual process that can be divided into three main stages: initiation, commitment and maturation  The precise characterization of undifferentiated LSPCs and induced hepatocytes is of utmost importance for the future exploitation of stem cell technology. Phenotyping based on surface markers has thus far been insufficient. Instead, characterization should be performed at the morphological, molecular and functional levels ii) Liver stem/progenitor cells react differently to cytokines/GFs at successive developmental stages iii) Finally, the molecular mechanism surrounding hepatic differentiation from LSPCs is essential for cell-based therapies. The essential factors responsible for the initiation, progression and accomplishment of hepatic differentiation should be specially considered. In detail, a more scrupulous understanding of the instructive signals emanating from the LSPC niche, together with a deeper analysis of the cell-intrinsic mechanisms governing replication- versus differentiation-inducing signals, is needed to reliably expand and differentiate LSPCs.From the discussion of the hepatic differentiation of LSPCs, composed of current In general, an unlimited supply of stem cell-derived hepatocytes would be an invaluable resource for the development of novel cell-based therapies. Studies of the molecular mechanisms regulating the hepatic differentiation of LSPCs will facilitate the reality of such cell-based therapies for treating liver diseases. By clarifying the mechanisms of hepatic differentiation regulation, LSPC-based therapy will have a bright future.


AbstractColeoptera: Carabidae) involve many indirect ecological processes, likely reflecting the function of numerous forest ecosystem components. Describing and quantifying these associations at the landscape scale is basic to the development of a surrogate-based framework for biodiversity monitoring and conservation. In this study, we used a systematic sampling grid covering 84 km2 of boreal mixedwood forest to characterize the ground-beetle assemblage associated with each tree species occurring on this landscape. Projecting the distribution of relative basal area of each tree species on the beetle ordination diagram suggests that the carabid community is structured by the same environmental factors that affects the distribution of trees, or perhaps even by trees per se. Interestingly beetle species are associated with tree species of the same rank order of abundance on this landscape, suggesting that conservation of less abundant trees will concomitantly foster conservation of less abundant beetle species. Landscape patterns of association described here are based on characteristics that can be directly linked to provincial forest inventories, providing a basis that is already available for use of tree species as biodiversity surrogates in boreal forest land management.Spatial associations between species of trees and ground-beetles ( Although some ground-beetle species are recognized as forest habitat specialists , the potCarabidae are a group of choice for forest health assessment at local scales  e.g.,  and thus56°46'13"N, 118°22'28"W, ~90 km northwest of Peace River, (see Picea glauca (Moench), Populus tremuloides Michx., Populus balsamifera L., combined with Picea mariana (Mill.) BSP and Larix laricina (Du Roi) K. Kock in wetter sites, with occasional Abies balsamea (L.) Mill., Betula papyrifera Marsh and Pinus contorta Loudon, representing the boreal-montane transitional nature of the lower foothill ecoregion. Viburnum edule (Michx.) Raf., Rosa acicularis Lindl., Sherpherdia canadensis (L.) Nutt., Alnus crispa (Ait.) Purch, Alnus tenufolia Nutt., and Ledum groenlandicum Oeder are common forest understory plants, and open meadows, fens, and bogs sometimes dominated by willow  or alder (Alnus spp.) shrubs are interspersed on this typical boreal landscape.The study was conducted at the EMEND  research site in the boreal mixedwood forest of northwestern Alberta, Canada. The approximate project centre is at 2 , we installed three pitfall traps in each of the same 200 sites . Traps were located at 0º (North), 120°, and 240° on a circle 15 m in diameter centred on the previously established site. Pitfall traps were a plastic cup (11 cm diameter by 13 cm depth), containing a plastic inner collecting cup and covered by a 14 cmails see . Traps wails see . All beeroth 1960. NomenclAs a first step in linking within-stand canopy heterogeneity to ground-beetle assemblages, we calculated the relative basal area for each tree species in every site. Basal area is directly related to canopy cover  and, thuIn order to visualize how the assemblage of ground-beetles was arrayed on the EMEND landscape, we performed a non metric multidimentional scaling (NMS) ordination of 194 sites based on the standardized abundance of the beetle species. We used the Bray-Curtis dissimilarity index to build the distance matrix and chose the highest number of dimensions providing a reduction of five in the stress . The NMSBeetle assemblage response to forest canopy was more clearly illustrated by plotting the relative basal area of tree species for every site on the beetle ordination diagram. This procedure was undertaken because interesting ecological trends were obscured by the sole use of centroids or vectors. Centroid calculation, vector projection, and ordination were performed using the vegan package  within tStereocerus haematopus (Dejean), Calathus advena (LeConte) and Pterostichus adstrictus Eschscholtz accounted for over 70% of catches, and together with the next seven most abundant species , accounted for 95% of the beetle catch. Nine beetle species , Elaphrus clairvillei Kirby, Notiophilus semistriatus Say, Poecilus lucublandus (Say) and Trichocellus mannerheimii (R.F. Sahlberg)) were trapped only in the 4 disturbed sites.We collected and identified 9,845 individual ground-beetles representing 48 species . StereocPicea glauca was found in the highest number of sites followed by Populus tremuloides, Picea mariana, Populus balsamifera, Abies balsamea, and Larix laricina  and Platynus mannerheimii (Dejean). The centroid for Trechus apicalis Motschulsky was also placed in quadrant III, although sites with Trechus apicalis were also widely distributed in the second and fourth quadrantPageBreak.A two dimensional NMS solution arrayed the carabid assemblages collected from 193 sites in four quadrants  with a sPicea glauca  are characteristic of open habitats  in Populus balsamifera occupies wetter sites than Populus tremuloides  were associated with Picea glauca, the tree species similarly recorded from the highest number of sites. This is also true for the association between the carabids Pterostichus adstrictus and Calathus ingratus and the tree species, P .tremuloides (second highest number of sites). Pterostichus punctatissimus, the sixth most common carabid, was associated with Picea mariana (noted at the third highest number of sites). Likewise Agonum retractumPageBreak, Trechus chalybeus, and Patrobus foveocollis were associated with Populus balsamifera (fourth highest number of sites), and Pterostichus brevicornis and Carabus chamissonis were predominately collected at sites with Abies balsamea (fifth highest number of sites). As outlined above, Agonum retractum and Platynus mannerheimii were associated with Larix laricina (recorded at the sixth highest number of sites). We suggest that each tree species indicates its own set of edaphic and perhaps even broader environmental conditions. If so, beetle species requiring conditions related to the most frequent tree encountered on a landscape will also be the most commonly encountered in systematic sampling efforts to the extent that beetle population sizes follow that of tree species. If a habitat is less frequent on a landscape, the beetle requiring this habitat should also be less frequently collected, and this is what we observed. Thus, including all tree species in stand-level forest inventories can have real practical value in developing regional conservation strategies.In general, associations between carabid and tree species, as described previously, match species of the same rank order of abundance on the EMEND landscape  and 2. FPageBreakDespite the indirect nature of potential links between distributions of tree and ground-beetle species, the ecological features associated with their distributions appear to be similar, allowing us to discern surprisingly clear patterns of association. Variation in carabid assemblages over this section of the boreal forest reflects the specific presence of all tree species present on this landscape. It is unknown at present the extent to which these associations simply reflect a response to common features or if, perhaps, the trees themselves contribute to conditions  that promote success of particular invertebrate species. Furthermore, although associations between ground-beetle and tree species are strong and interpretable, the potential implication for predicting distribution of other invertebrate taxa remains to be investigated.Nevertheless, these observations are of interest for regional conservation purposes. Because the boreal forest covers vast areas, it is impossible in cost-effective practice to assess biodiversity reliably, and thus surrogates are needed . Our res


Pain remains a poorly understood and managed symptom. A limited mechanistic understanding of interindividual differences in pain and analgesia response shapes current approaches to assessment and treatment. Opportunities exist to improve pain care through increased understanding of how dynamic epigenomic remodeling shapes injury, illness, pain, and treatment response. Tightly regulated alterations of the DNA-histone chromatin complex enable cells to control transcription, replication, gene expression, and protein production. Pathological alterations to chromatin shape the ability of the cell to respond to physiologic and environmental cues leading to disease and reduced treatment effectiveness. This review provides an overview of critical epigenetic processes shaping pathology and pain, highlights current research support for the role of epigenomic modification in the development of chronic pain, and summarizes the therapeutic potential to alter epigenetic processes to improve health outcomes. Pain is the number one reason patients consult a health care provider in the United States, with one in every three emergency room patients and more than 60% of all primary care patients listing pain as their chief complaint . ChronicPain genetics is a broad term that describes both classic Mendelian techniques used to identify inherited variation in pain sensitivity and analgesic response as well as newer gene-level DNA and RNA sequence measurement sciences. While these techniques and approaches are not novel, their application to understanding pain and pain management has provided new mechanistic insights and treatment possibilities. Genome-wide association studies (GWAS) and focused candidate gene association studies (CGAS) have identified more than 350 genes that are relevant in both clinical and experimental pain, with identification of many hundreds more pain and analgesia regulating genes . PolymorHowever, not all attempts to isolate the effects of DNA variance have been successful. Many complex pain conditions including rheumatoid and osteoarthritis as well as fibromyalgia and neuropathic pain do not link to heritable factors. Genetic approaches also have not been generally useful in identifying factors that shape the trajectory of pain symptoms distinct from the pathophysiology of disease –18. AlthThe human genome contains more than 6 billion individual base pairs of amino acids packaged onto 23 paired chromosomes, supporting more than 30,000 regionally localized genes. These transcription and regulatory sequences of nucleic acid molecules are responsible for coding the instructions that make proteins and other critical cell products. Compacting the nearly 2 meters of chromosomal DNA into the relatively small nucleus inside each cell, while continuing to support transcription, replication, and ultimately gene expression, requires a series of highly coordinated packaging processes that serve to temporally and functionally control access to DNA throughout the cell cycle –21. HistCells have evolved considerable diversity for altering the way DNA compacts around histone proteins, providing an almost unlimited ability to control and shape DNA readout. Chromatin remodeling processes include those that specifically target the genomic DNA within the chromatin structure, specifically and principally DNA methylation. Other processes target the histone proteins, including processes that add or remove methyl, acetyl, and phosphate groups. Still other processes shape the actions of small regulatory noncoding and gene-silencing RNAs. DNA methylation involves the addition of a methyl group to a cytosine in a DNA dinucleotide; this modifies the covalent bonding between DNA and histones providing a stabilizing effect on gene expression , 29. EntThe linear structure of histone proteins includes sequences of amino acids that have been translated from messenger RNA and then folded and held together by both weak and strong covalent bonds. These amino acid sequences or residues can undergo a wide variety of posttranslational enzymatic modifications that also influence how DNA compacts around the histone core. Similar to methylation in the DNA dinucleotide, the addition of a methyl group to a histone protein generally inhibits gene expression, while the addition of an acetyl group into a histone protein generally loosens the interaction of DNA and histones favoring gene transcription , 24, 25.Chromatin modifications and remodeling are most pronounced when cells experience rapid environmental changes and chemical stress –35. Manyα  as well as T-cell and B-cell regulating interleukins [Several critical “first responder” transcription factors, including NF-kB (nuclear factor k-light-chain-enhancer of activated B cells), c-Jun, c-Fos, and several hormone activated receptor proteins, serve as drivers for wide-ranging epigenetic responses to cellular stress. Present but inactive in many vascular, nerve, and immune cells, these transcription factors become activated in response to cell insult and are able to rapidly access chromosomal DNA as demethylation unspools chromatin structure to initiate production of a reparative cascade of inflammatory cytokines including TNF-rleukins –44. The rleukins . Epigenerleukins –49. Two of the most therapeutically intriguing insights arising from epigenetics research are suggestions that epigenetic mechanisms play a critical role in the transition from acute to chronic pain, and that a wide range of environmental factors across the lifespan serve as epigenetic primers for individual pain and analgesic response , 26, 38.µ-opioid receptors and increased pain with continued opioid use [Variable production of stress-induced glucocorticoids, variable response to exogenously administered steroidal anti-inflammatory agents, and even glucocorticoid resistance are all identified as the likely mechanisms responsible for autoimmune illness and pathologic chronic pain, with each linked to underlying epigenetic processes. Diverse and high individual methylation patterns are associated with alternative activation of promoter sites producing different sensitivities to glucocorticoids. These diverse methylation patterns have been shown to be associated with a number of environmental factors including diet, maternal care, and early life stressors. This provides compelling mechanistic evidence in support of long-observed linkages between early sexual or physical abuse, neonatal pain, previous injuries, and chronic pain later in life , 46. Epiioid use . The critical role of deacetylation of histone proteins in shaping pain pathophysiology and analgesic response is highlighted in a series of experiments, where deacetylation has been pharmacologically inhibited –57. CurrWhile pharmacologic blockade or inhibition of acetyl group removal from histones is showing early promise, current approaches to therapeutically manage the methylation status of DNA present a more mixed picture of success. Glucosamine and L-methionine, for example, are endogenously produced molecules utilized by the body for biosynthesis of cartilage-repairing glycoproteins and glycosaminoglycans. Pharmaceutical grade versions of these molecules are both prescribed and taken over the counter as nutritional supplements for joint health and pain management for patients with osteoarthritis. Evidence suggests that these molecules also disrupt or alter methylation status of chromatin. To date, however, no well-designed studies report improved pain relief using these supplements over placebo controls –62. MoreOne exciting growth area for symptom research, including pain and pain management, is evidence suggesting that the epigenetic state of chromatin interacts with and is critically shaped by context and environment. A new emphasis on how epigenetics may shape and be shaped by best practice and even moment-to-moment care decisions is likely to have a profound impact on the practice of both medicine and nursing, especially as it relates to critical event management. Neonatologists now recognize that micro- and macronutrition during pregnancy have an impact far beyond the early months of life, with nutritional support strategies epigenetically linked to later development of a wide range of adult illnesses including asthma, hypertension, colitis, and malignancies –65. Crit


Physiological adaptations that allow air-breathing vertebrates to remain underwater for long periods mainly involve modifications of the respiratory system, essentially through increased oxygen reserves. Physiological constraints on dive duration tend to be less critical for ectotherms than for endotherms because the former have lower mass-specific metabolic rates. Moreover, comparative studies between marine and terrestrial ectotherms have yet to show overall distinct physiological differences specifically associated with oxygen reserves.Acrochordus granulatus.We used phylogenetically informed statistical models to test if habitat affects hematocrit (an indicator of blood oxygen stores) in snakes, a lineage that varies widely in habitat use. Our results indicate that both phylogenetic position (clade) and especially habitat are significant predictors of hematocrit. Our analysis also confirms the peculiar respiratory physiology of the marine Contrary to previous findings, marine snakes have significantly–albeit slightly–elevated hematocrit, which should facilitate increased aerobic dive times. Longer dives could have consequences for foraging, mate searching, and predation risks. Alternatively, but not exclusively, increased Hct in marine species might also help to fuel other oxygen-demanding physiological adaptations, such as those involved in osmoregulation. Evolutionary habitat transitions often involve substantial modifications in morphology, physiology, and behaviour, particularly if the new habitat imposes novel physical challenges. For example, transition to an aquatic life leads to selection on such attributes as the ability to move through water, to remain underwater for long periods, and to dive to considerable depths Physiological adaptations that allow air-breathing vertebrates to remain underwater for long periods primarily involve modifications of the respiratory system, essentially through increased oxygen reserves  have an exceptionally large oxygen store (combining both high blood volume and high blood oxygen carrying capacities Dermochelys coriacea) have high myoglobin concentration in their muscles Most of the previous comparisons of terrestrial and diving, air-breathing, ectothermic “reptiles” have failed to find common physiological differences specifically associated with respiration and metabolism Snakes are well suited to identify possible evolutionary adaptations to aquatic life. First, almost every family has undergone independent transitions from terrestrial toward aquatic or marine habits, thus allowing examination of possible parallel or convergent physiological adaptations among lineages Here, we analyze hematocrit in relation to the use of aquatic environments in snakes by use of both phylogenetic and non-phylogenetic statistical models Information on hematocrit  was collected from the literature and from unpublished studies , which require developed diving capacities, amphibious Laticaudinae were included in this group.Notechis scutatusSome populations of semi-aquatic species can be observed in xeric environments  multiple regression (Ordinary-Least-Squares - OLS - regressions) with dummy variables that code for Clade and Habitat . We repeated the analysis using Phylogenetic Generalized Least Squares (PGLS) models and also with phylogenetic models that use a branch-length transformation based on the Ornstein-Uhlenbeck (OU) model of evolution for residual Hct variation . Thus, we found no statistically significant phylogenetic signal in the residuals of our dependent variable after accounting for variation related to the Habitat and Clade independent variables Based on partial F tests, both the OLS and RegOU models indicate that with effects of Habitat controlled, Acrochordidae have significantly elevated levels of Hct relative to the Colubrinae clade . AdditioBoth Clade and especially Habitat accounted for a substantial amount of the variation in Hct , 2. A siAcrochordus granulatus has the highest Hct in our dataset . Blood-oxygen carrying capacities of Acrochordus granulatus exceed those of other reptiles and approach levels characteristic of endothermic mammals and birds A. granulatus versus other ecological specificities , a lineage known to be very atypical in terms of ecology, morphology, and physiology compared to other Caenophidia Although our results also suggest that aquatic species have lower Hct than their terrestrial counterparts , we recoper se and other oxygen-demanding habits on increased Hct in marine snakes . For instance, marine reptiles display specific physiological adaptations related to osmoregulation . However, this example does not allow robust conclusions about marine snakes in general, most notably because of the atypical ecology, morphology, and physiology of this lineage. Future exploration should focus on a promising “model lineage” of highly aquatic snakes - Homalopsidae, especially the widespread Cerberus rynchops - in which different populations of the same species use a continuum of contrasting aquatic habitats . Overall, exploring the effects of increased blood viscosity on cardiovascular and respiratory systems  in snakes remains a largely open field of investigation.Alternatively, increased Hct is also likely to impose costs, especially in terms of increased blood viscosity. Such issues have been investigated in the case of Additionally, we acknowledge that our analysis does not take into account the role of the right lung , which is obviously a major component of respiratory physiology Lastly, we note some limitations of our analysis. The use of Hct as a proxy for blood oxygen stores is a simplification, and other blood-related traits are likely to play a role in oxygen storage and distribution, and thus in the overall respiratory physiology of marine snakes. Most notably, blood volume is a likely candidate to examine in order to refine estimates of oxygen stores in snakes Although our results show that marine snakes have increased Hct as compared with terrestrial species, this difference seems modest in comparison to what is found in marine endotherms Appendix S1Online_Appendix_S1.xls. Data file in Excel file format with species code (PDICode) in column 1. Column 2 is the left to right order of taxa in our phylogenetic tree  and S3. (XLS)Click here for additional data file.Appendix S2Details on the phylogeny construction.(DOC)Click here for additional data file.Appendix S3http://mesquiteproject.org). It is in the Newick Standard File Format.File of Phylogenetic tree. This file (Hct67P_2.phy) of the phylogenetic tree (described in Online (PHY)Click here for additional data file.


History drives community assembly through differences both in density (density effects) and in the sequence in which species arrive (sequence effects). Density effects arise from predictable population dynamics, which are free of history, but sequence effects are due to a density-free mechanism, arising solely from the order and timing of immigration events. Few studies have determined how components of immigration history  alter local dynamics to determine community assembly, beyond addressing when immigration history produces historically contingent assembly.Colpoda steinii, mediated alternative community states; early arrival or high introduction density allowed this species to outcompete or suppress the other competitors . Multivariate analysis showed that density effects caused greater variation in community states, whereas sequence effects altered the mean community composition.We varied density and sequence effects independently in a two-way factorial design to follow community assembly in a three-species aquatic protozoan community. A superior competitor, A significant interaction between density and sequence effects suggests that we should refine our understanding of priority effects. These results highlight a practical need to understand not only the “ingredients” (species) in ecological communities but their “recipes” as well. Community assembly has been a prominent concept in ecology; a variety of sometimes divergent views have reflected different assumptions and a confusing array of terminology. At one extreme, communities have been viewed as the product of random dispersal events, after which deterministic species sorting overrides immigration history. For example, Diamond Empirical efforts to understand historical forces driving community assembly have included observational comparisons of natural communities at different localities at various disturbance levels , Colpoda steinii (species B), and Eimeriidae gen. sp. (species C) are protozoans commonly found in water-filled leaves of the purple pitcher plant, Sarracenia purpurea, in the Apalachicola National Forest, Florida, USA. The first is a suspension feeder; the latter two use both suspension feeding and grazing We assembled three-species protozoan communities in 10-mL microcosms, created as described below, by adding 0.50-mL aliquots from appropriate stock monocultures sequentially in a two-way factorial design (3 density levels×6 sequence levels), replicated in three temporal blocks, each of which contained 18 microcosms. Successive blocks were started at 1-hour intervals . Sequence treatment included six categorical levels, corresponding to all possible orders for introduction of the three species . For eacMicrocosms initially contained 10 mL  of sterile water that was inoculated with bacteria by addition of 1.6 mg of Tetramin fish food  and exposure to open air for 24 hours before the introduction of protozoan species. The first, second, and third species were added from stock monocultures after 0, 12, and 24 hours, respectively. Differences in introduction density were created by dilution of high-density stock cultures with medium similarly inoculated with bacteria; We used permutational MANOVA (PERMANOVA) to test for density and sequence effects on the final community structure of the three protozoan species in microcosms on day 3 (72 hours after microcosm establishment). PERMANOVA uses an additive partitioning of a pairwise distance metric  according to a multifactorial ANOVA design, with significance testing by permutation to accommodate the frequent violation of the assumptions of MANOVA in community data Although PERMANOVA can test for differences in the centroids of multivariate species composition between levels of a factor, we were also interested in differences in the among-plot variability in species composition , as did sequence treatments , and notably, these two effects interacted to determine the protozoan community structure . Strong density effects also caused greater dispersion in the protozoan community structure , density and sequence effects interacted to determine the final community structure . Only thIn summary, whichever species arrived first had an advantage and more strongly influenced later-colonizing competitors, regardless of its initial density. A superior competitor, species B, mediated alternative community states; when arriving first, it outcompeted or suppressed the other two species, whereas it was vulnerable to extinction under the simultaneous disadvantages of late arrival and low density. In contrast, species A and C were able to overcome any disadvantage due to low initial density.Our experiment and analyses demonstrated that density and sequence effects were distinct ecological mechanisms that differed qualitatively in their impacts on assembly of a three-species protozoan community and, most importantly, that density and sequence effects on assembly interacted. Density effects caused greater dispersion in the protozoan community structure without substantially changing the average community states, whereas sequence effects often altered the community states, possibly through changing the locations of the community attractors themselves . HistoriAlthough most of our experimental protozoan communities appeared to have stabilized after 72 hours (approximately 9 generations), we cannot be certain that they do not actually represent transient community states. We suggest that the durations of our experiments were generally sufficient, on the basis of the criteria presented by Grover and Lawton In prior studies, priority effects have often been invoked as a post hoc explanation for the observed community changes, as the investigator looked back to initial conditions to interpret current conditions [see, e.g., Robinson and Dickerson Sequence effects may be characteristic of particular types of systems—they may lead us forcefully to dissect purely historical processes into trait-based mechanisms  in island restoration, biocontrol management, and biological invasion continues to be important for understanding a large-scale imprint of assembly-history dynamics. Although our study was of a competitive community, further mechanistic lines of inquiry into assembly-history dynamics for predator-prey interactions, mutualisms, and multitrophic food webs will enrich our understanding not only of the ingredients (the species) but also of the recipes  for ecological communities in an invasion-driven world.


Pulmonary embolectomy is a treatment option in selected patients with high-risk pulmonary embolism (PE). Efficiency of thrombus degradation in PE largely depends on the architecture of its fibrin network, however little is known about its determinants. We present the case of a 56-year-old woman with high-risk PE and proximal deep-vein thrombosis, whose thrombotic material removed during embolectomy from the right atrium and pulmonary  arteries has been studied using scanning electron microscopy (SEM). SEM images showed that distally located thrombi are richer in densely-packed fibrin fibers and contain more white cells and less erythrocytes than the proximal ones and the atrial thrombus. Fibrin fibers alignment along the flow vector was observed in the thrombi removed from high-velocity flow pulmonary arteries, and not in the atrial thrombus. The content of denser fibrin network and platelet aggregates was increased in segmental thromboemboli. Our findings describe the relation between thrombus architecture and location, and might help to elucidate thrombus resistance to anticoagulant therapy in some PE patients. Pulmonary embolism (PE) is associated with a substantial morbidity and mortality . Right hFibrin network, that represents a major component of venous thrombi, is lysed at a rate dependent on its architecture, with impaired lysis in case of dense clots . Fibrin Recently, we have shown that the structure of the pulmonary thrombus obtained during embolectomy is organized, fibrin-rich, and not layered . To our 2), a former smoker, with a history of chronic venous insufficiency who suffered from recent-onset schizophrenia was admitted to our institution for PE after a 48 h treatment with unfractionated heparin in a regional hospital. Two months prior to admission she was hospitalized in a psychiatric ward and received no thromboprophylaxis. On admission, the patient presented with progressing dyspnea, cyanosis and chest discomfort with elevated D-dimer . Contrast-enhanced computed tomography revealed large emboli at the right pulmonary artery (PA) trifurcation and in the left PA , but not the vessel caliber, reported in acute myocardial infarction . MoreoveMechanisms underlying such fibrin phenotype in pulmonary thromboemboli obtained from our PE patient remain to be elucidated. Most likely, transient factors, including those linked to hemodynamic abnormalities and massive thrombosis, determine thrombus properties to a greater extent than genetic factors in our case . We cann


The long-term impact on bone health of lifelong HIV infection and prolonged ART in growing and developing children is not yet known. Measures of bone health in youth must be interpreted in the context of expected developmental and physiologic changes in bone mass, size, density and strength that occur from fetal through adult life. Low bone mineral density (BMD) appears to be common in perinatally HIV-infected youth, especially outside of high-income settings, but data are limited and interpretation complicated by the need for better pediatric norms. The potential negative effects of tenofovir on BMD and bone mass accrual are of particular concern as this drug may be used more widely in younger children. Emphasizing good nutrition, calcium and vitamin D sufficiency, weight-bearing exercise and avoidance of alcohol and smoking are effective and available approaches to maintain and improve bone health in all settings. More data are needed to inform therapies and monitoring for HIV-infected youth with proven bone fragility. While very limited data suggest lack of marked increase in fracture risk for youth with perinatal HIV infection, the looming concern for these children is that they may fail to attain their expected peak bone mass in early adulthood which could increase their risk for fractures and osteoporosis later in adulthood. Worldwide, more than 2 million children are infected with HIV. In most of the cases, HIV infection was acquired during pregnancy or intrapartum or through breastfeeding. Effective antiretroviral therapy (ART) has dramatically reduced morbidity and mortality for children with perinatal HIV infection, many of whom are now adolescents or even young adults. Even as the prevention of AIDS-defining illnesses and of progressive immunosuppression is appropriately celebrated, the long-term impact of lifelong HIV infection and prolonged ART in growing and developing children is not yet known. An area of particular concern is the potential effect of HIV infection and ART on bone, which undergoes profound changes in size, mass and strength from foetal life through to young adulthood. This article will focus on available data and remaining questions related to bone outcomes in perinatal HIV infection in the context of normal bone development, non-HIV factors that impact bone, and composition of ART as well as an approach to detection, prevention and management of bone problems in this group.Bone is composed of organic (bone matrix) and mineral components. Bone mass refers to the weight of bone. Bone mineral density (BMD) refers to the bone mass divided by its volume. In practice, BMD is not usually directly ascertainable (would require bone biopsy), and it is estimated by radiologic or other methods. Bone mineralization describes the incorporation of calcium and other minerals into organic bone matrix . Low BMD2). The main limitation of the aBMD for children is that relatively smaller bones (e.g. in a child with short stature) can lead to lower aBMD and thus underestimates of true BMD [Dual-energy X-ray absorptiometry (DXA) is the most commonly used modality for estimating bone mineral content (BMC) and BMD. The technique measures how much radiation (two beams emitted at different energy levels) gets absorbed while passing through bone or other body tissue to estimate the density of that region . Denser true BMD . These mtrue BMD . A DXA Btrue BMD .Other modalities used to estimate BMD include quantitative computed tomography (qCT) and quantitative ultrasonography (qUS)  4]. qCT. qCT4]. The potential impact of HIV infection and treatment on the bone health of youth with perinatal HIV infection must be evaluated in the context of normal, physiologic bone growth and development . The effPerinatally infected youth are potentially first exposed to HIV infection and antiretroviral drugs (ARVs) during intrauterine life. The foetal period is a critical period for the development of bone mass. The foetus is entirely dependent upon its mother for calcium and other minerals necessary for normal bone development. Foetal bones accumulate about 30 g of calcium from transplacental transfer, with 80% of calcium accretion taking place in the third trimester .Infancy represents an equally critical period in bone growth, development and mineral accretion. In contrast to the foetus, infant bone health depends on maternal (breast milk) and non-maternal sources of calcium and other bone components and is affected by environmental factors, including nutrition, infections, drug and toxin exposures and activity level. During infancy, the increase in bone diameter by 50%, despite a concomitant increase in BMC by at least 50%, results in a decrease in bone density of about 30%. In this complex process, however, actual bone strength increases threefold because of accompanying changes in bone architecture . There aThroughout childhood, bone size, BMC and aBMD continue to increase . NormatiPBM is attained by the end of the second or early in the third decade of life , generalOutside the context of HIV infection, there are numerous demographic, genetic, nutritional and lifestyle factors, as well as medical conditions and treatments, that are well known to impact bone health and BMD . The relIntrauterine factors may have both short- and long-term impacts on bone health. Maternal macronutrient and micronutrient intake during pregnancy has been linked to effects on bone mass in progeny through 6–16 years old . Some haPreterm birth, low birth weight and poor growth early in life all may have a lasting negative impact on bone health ,19. WhilThrough childhood and adolescence, major influences on bone mass accrual and bone health include genetic determinants, physical activity (or lack thereof), nutritional status and hormonal changes during puberty. Up to 80% of the variability in PBM may be explained by genetic factors . Weight-http://www.nof.org/articles/6).Many illnesses and medications have been associated with low BMD and/or increased fracture risk ,26. OsteCross-sectional studies of BMD among HIV-infected youth  suggest In one longitudinal study, BMD increased over one year in 32 HIV-infected Italian children with a mean age of 12.4 years, but it remained significantly lower than HIV-uninfected controls .Many –37 but nStudies of BMD and fracture risk in HIV-infected youth in low-income settings have not been published.Many factors contribute to low PBM in children with HIV infection, including delayed growth and puberty, low lean body mass, altered levels of hormones and inflammatory cytokines, vitamin D deficiency, malabsorption and physical inactivity. HIV-specific factors, which are important contributing factors to bone loss in children, include advanced HIV disease, uncontrolled viremia and ART initiation and type . A largep=0.003) [p=0.1) [Not all antiretroviral drugs have the same effect on BMD. Protease inhibitors and tenofovir are most often associated with low BMD. Lopinavir/ritonavir , indinavp=0.003) . On the p=0.003) . Anotherp=0.003) . The lac [p=0.1) . There a [p=0.1) .The pathogenesis of low bone mass among HIV-infected individuals is multifactorial, including traditional risk factors such as smoking, physical inactivity and vitamin D deficiency and also HIV-related factors including HIV infection itself, chronic immune activation and the direct effects of antiretroviral therapy. In vitro studies have shown that HIV viral proteins gp120  and Vpr Careful review of occurrence and circumstances of fracture can help identify children and youth with increased bone fragility. Fractures that occur after minimal trauma, vertebral compression fractures, a single instance of traumatic fracture of a lower extremity long bone, and two or more fractures of upper extremity long bones should raise suspicion of bone fragility . In loweThere is no consensus that all HIV-infected youth (or adults) should undergo routine DXA screening. If DXA is available, it should be considered for HIV-infected youth with a suspicious fracture history and/or multiple risk factors for poor bone health. As for children in general, lumbar spine and total body less head are the sites recommended for assessments. The hip is not a reliable site for measurement in growing children due to significant variability in skeletal development and lack of reproducibility . A BMD ZThe normative databases used as a reference should be based on large samples of healthy children that are similar in gender, age and race/ethnicity . For exaDelay in growth and puberty is quite common among HIV-infected children. BMD interpretation should be adjusted for absolute height or height age in children with linear growth or maturational delay . SubstitIn children who have a baseline BMD assessment by DXA, there is no clear recommendation for how often DXA scans should be repeated as part of monitoring bone health. Intervals shorter than six months are unlikely to yield significant changes in BMD; intervals of one to two years may be reasonable for children with low BMD at baseline or ongoing risk for skeletal losses .Many of the approaches to maintaining good bone health in youth with perinatal HIV infection are similar to those used for youth in general . AdolescEven though continuous ART resulted in lower BMD than intermittent BMD in adults in the SMART study , untreatFor youth with perinatal HIV infection who have very low BMD (Z-score<−2.0) and/or fractures that are suspicious for bone fragility, clinicians should implement a multi-pronged approach . CalciumAlternative ARV agents can be considered. In particular, replacing tenofovir and/or boosted PIs with agents that have not been associated with negative bone effects  may be beneficial but evidence is lacking. Such regimen changes should only be undertaken if the new regimen is expected to be sustainable, maintain virologic suppression and be well tolerated.Drugs like bisphosphonates that inhibit bone resorption have been widely used to treat osteoporosis, especially in post-menopausal women. Alendronate, one of the most widely used oral bisphosphonates, improved BMD in HIV-infected adults with low BMD in a randomized, placebo-controlled trial . This drMeasures of bone health in youth must be interpreted in the context of expected developmental and physiologic changes in bone mass, size, density and strength that occur from foetal through adult life. The potential effects of HIV infection, ARV drugs and other factors on the bones of perinatally HIV-infected youth begin in utero and persist through the critical bone growth and development periods during childhood and adolescence and into young adulthood when PBM is attained. Low BMD appears to be more common in perinatally HIV-infected youth in lower resource settings, likely due to differences in genetic/ethnic and environmental factors, but data are limited and are complicated by the lack of well-characterized paediatric DXA BMD norms for each setting. The potential negative effects of tenofovir on BMD and bone mass accrual are of particular concern as this drug may be used more widely in younger children. Emphasizing good nutrition, calcium and vitamin D sufficiency, weight-bearing exercise and avoidance of drugs  are effective and available approaches to maintain and improve bone health in all settings. More data are needed to inform therapies and monitoring for HIV-infected youth with proven bone fragility. While very limited data (with no data from low-resource settings) suggest lack of a marked increase in fracture risk for youth with perinatal HIV infection, the looming concern for these children is that they may fail to attain their expected PBM in early adulthood which could increase their risk for fractures and osteoporosis later in adulthood.


Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. In 2010, individuals aged 65 years and older constituted approximately 12.9% and 8% of the population in the United States and worldwide, respectively [Aging-associated diseases include degenerative conditions affecting tissue and organ function. For example, neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) are conditions marked by the progressive deterioration of structure and function leading to neuronal death. A retinal disorder, age-related macular degeneration, is caused by the gradual degeneration of cells in the macula of the retina and is the leading cause of vision loss in adults over age 55. Conditions such as osteoarthritis and osteoporosis, which are marked by the degeneration of cartilage and bone, respectively, cause the majority of knee, joint, hip, and spine injuries in older individuals.Aging-associated diseases may also arise from cell dysfunction. Such conditions may include cancer, heart disease, chronic obstructive pulmonary disease (COPD), and diabetes. Cancer is caused by metabolic changes in cells that lead to DNA damage that can fuel the uncontrollable and inappropriate proliferation of cells. The risk of cancer increases significantly with age. Heart disease is typically caused by prolonged exposure of the heart to hypertension, hypercholesterolemia,diabetes, and other cardiovascular risk factors, as well as an age-dependent increase in the prevalence of left ventricular hypertrophy, diastolic dysfunction, and atrial fibrillation . COPD isThe prospect of repairing or replacing damaged, dysfunctional or missing cells with new functional cells has shifted the therapeutic paradigm toward restoring tissue function in individuals affected with aging-associated diseases. The primary candidate for the development of these therapies is stem cells, particularly human embryonic stem cells (hESC), which has the capacity to self-renew indefinitely and differentiate into all tissue-specific cell types Figure . In thisHuman embryonic stem cells (hESCs) are pluripotent stem cells derived from various stages of embryonic development. hESCs are uniquely capable of proliferating indefinitely and differentiating into all tissue cell types. The unrestricted potential of hESCs has made these cells especially attractive for therapeutic applications. In particular, the regenerative capacity of hESCs could be the key to successful treatment of aging-associated diseases which, as discussed in the previous section, are characteristically marked by progressive dysfunction and/or loss of somatic cells.in vitro and retain their pluripotency indefinitely. hESCs have also been derived and established from single blastomeres of the 4- or 8- cell embryo [hESCs are typically derived through the microsurgical removal of the inner cells mass (ICM) of the blastocyst-stage pre-implantation embryo Figure . Cells pl embryo -8, 16-cel embryo , 10, or l embryo -14, havel embryo , hundredPluripotent hESCs maintain specific morphological and molecular properties that are shared by the majority of hESC lines. Morphologically, single hESCs have an enlarged nucleus and distinct nucleoli. In culture, proliferating hESCs form compact cell colonies of spherical cells Figure . Under dA panel of molecular markers has been identified in 59 independently derived pluripotent hESC lines by the International Stem Cell Initiative, a consortium of stem cell researchers from more than 15 countries . These min vivo and in vitro techniques that are routinely used to determine pluripotency of a hESC line. The most commonly used in vivo method involves the induction of teratoma formation after transplantation of undifferentiated hESCs into immunodeficient mice [in vitro through the formation of embryoid bodies (EBs). EBs are spherical colonies of non-adherent, differentiating hESCs that contain cell populations representative of all three embryonic germ layers. Under suitable conditions, EBs can differentiate into specific cell types. As will be discussed below, EB formation is typically used as an intermediate step when directing hESCs into tissue-specific cell populations.hESCs are capable of differentiating into cells that constitute the three germ layers. This can be tested using established ent mice -28. TeraThe most promising feature of hESCs is the ability to derive lineage-restricted progenitors that are capable of differentiating into specialized post-mitotic cell types that can be used in cell-based therapies. Furthermore, hESCs provide a virtually inexhaustible source of specific cell populations, due to their ability to divide indefinitely. Current research studies are focused on identifying and refining ways for directing the differentiation of hESCs to enrich for pure, homogenous populations of specific cell types that can be used to either replace damaged cells or coax neighboring cells to function properly. In the following sections, we will provide some examples of how differentiation of hESCs is directed towards tissue-specific cells, particularly those with potential to treat aging-associated diseases.Endodermal derivatives include cells that populate the lung, liver, pancreas, urinary bladder, pharynx, thyroid, parathyroid, and digestive system. The initial step in generating endodermal cells is the formation of definitive endoderm. D'Amour et al. showed thThe induction of definitive endoderm from hESCs can lead to the generation of specific progenitor populations such as pancreatic islet β cells, hepatocytes, or alveolar epithelial cells. These are being developed with the intent of treating diseases such as diabetes, liver disease, or lung disease, respectively. Among the most successful examples to date is the generation of pancreatic islet progenitors devised by Kroon et al.  through In a similar manner, hepatocytes are obtained after differentiation of hESCs into definitive endoderm , 36. A hThe use of hESCs to treat lung injury has also been an area of active investigation. A significant step towards directed differentiation of lung-specific cells was reported by Wang et al. , 39, in in vitro and in vivo, have also been derived from hESCs co-cultured with stromal cells [Directing the differentiation of hESCs into mesoderm requires the activation of the TGFβ signaling pathway and can be accomplished through the stepwise and dosage-dependent addition of Activin A, BMP4, and growth factors, such as vascular endothelial growth factor (VEGF) and basic FGF (bFGF) . Mesoderal cells . Thus, tal cells .in vivo [Cardiomyocytes, which represent another therapeutically important derivative of mesoderm, have been successfully generated from hESCs using several methods . Throughin vivo . Anotherin vivo . Successin vivo , 53 and in vivo .in vitro differentiated tendon structures in injured immunosuppressed mice restored ankle joint movements that rely on an intact Achilles tendon [hESCs can readily form connective tissue, such as bone or cartilage, as can be appreciated from teratoma formation assays Figure . Thus, hs tendon . Furthers tendon .The dominant differentiation pathway in hESC cultures leads to the formation of ectoderm, which gives rise to cells of the nervous system and the epidermis. hESC-derived neural progenitor cells are characterized by rosette-like neural structures that form in the presence of FGF2 or EGF through either spontaneous differentiation from an overgrowth of hESCs, or after hEBs are plated onto adherent substrate , 60. TheThe withdrawal of FGF2 and EGF, and addition of other factors can lead to the differentiation of neural rosettes into specific neural subtypes. Neural crest stem cells derived from neural rosettes can differentiate into peripheral sympathetic and sensory neurons through the addition of BDNF, GDNF, NGF and dbcAMP, or into Schwann cells in the presence of CNTF, neuregulin 1β and dbcAMP . NeuroglThe ability to differentiate hESCs into neuronal and non-neuronal subtypes has generated much interest due to their potential use in drug testing or cell replacement therapies for a number of neurodegenerative diseases. In particular, the successful derivation of dopaminergic neurons, particularly those of the midbrain subtype, could potentially be used to treat Parkinson's disease, which is marked by the progressive loss and dysfunction of these neurons. In Alzheimer's disease, where the degeneration of basal forebrain cholinergic neurons causes debilitating cognitive dysfunction, hESC-derived cholinergic neurons may also be useful for therapy. However, hESCs may also be helpful without requiring cell replacement. As observed in a clinical trial in which autologous fibroblasts programmed to express human NGF were implanted in the forebrain of individuals with mild Alzheimer's disease, a marked improvement in the rate of cognitive decline was observed . One canSimilarly, hESC-derived motor neurons might be used in the treatment of ALS, which is characterized by the progressive loss of motor neurons in the cortex, brain stem, and the spinal cord. Studies of ALS disease models have also suggested that non-neuronal cells, such as oligodendrocytes and Schwann cells, may be involved in the pathogenesis of this disease , 71. ThuThe efficacy of transplanting hESC-derived oligodendrocytes to treat acute spinal cord injury is now being tested in the first clinical trial with hESCs to be approved by the United States Food and Drug Administration (FDA) . OligodeRetinal pigment epithelium (RPE) cells are another specific cell type derived from neuroectoderm. RPE cells support the neural retina by phagocytosing and renewing the photoreceptor outer segments of rhodopsin. Recent reports have shown that RPE can be induced from hESCs in the presence of nicotinamide and Activin A under serum-free conditions . hESC-deCellular therapies involving hESCs are in development and have begun to enter clinical trials. The International Stem Cell Banking Initiative (ISCBI) has been created by the International Stem Cell Forum, a group of national and international stem cell research funding bodies, to develop a set of best practices and principles for banking, testing, and distributing hESCs for therapy . In the in vitro. Under these conditions, hESCs could possess animal viruses and other unknown substances capable of eliciting a detrimental immune response in transplanted hosts. Currently, hESC lines under development for clinical use undergo extensive microbiological testing as strictly recommended by ISCBI. In the United States, the FDA legally requires documentation of the source, the potential genetically modified components, and pathogenic agents in any hESC-derived cell intended for therapeutic use. Thus, avoiding exposure to xenobiotics is an ongoing effort. Recently, replacement media have been developed that would allow maintenance of hESCs in xenobiotic-free conditions. These include xenobiotic-free serum replacements such as Knockout Serum Replacer (Invitrogen) or xenobiotic-free culture media such as HESGRO (Millipore) or TeSR (STEMCELL).Many of the hESC lines currently in use have been exposed to animal products during isolation and propagation of hESCs Feeder-free culture systems are now being developed to reduce the risk of contamination with foreign agents when hESCs are cultured on feeder cell layers. Feeder-free and xenobiotic-free defined culture media that consist of a combination of recombinant growth factors known to inhibit differentiation and maintain hESCs in the pluripotent state are now commercially available. However, some reports have associated feeder-free culture conditions with greater chromosomal instability and an increased risk of propagating genetically altered hESCs . For thihESC lines derived using human feeder cells have also been reported. For example, hESC lines have been successfully derived on human fibroblasts generated from neonatal foreskin , 78 and The best characterized hESC lines to date are among the earliest lines derived. However, they may not be the best lines for therapeutic application as many of these lines were derived using animal products. Chromosomal and genomic instability has been detected in several hESC lines, with acquisition of loss of heterozygosity or copy-number variation in cancer-related genes , 82. Manin vivo transplantation of undifferentiated hESCs in mouse models results in teratoma formation. Evidence of tumor formation has also been observed in differentiated hESC derivatives transplanted in vivo [A primary safety concern when using pluripotent hESCs is their potential to form germ layer tumors. As discussed above,  in vivo , 85. Thu in vivo . Thus, din vitro and in vivo [As discussed earlier, enrichment of specific cell types can be achieved using molecules introduced at critical time points during culture. However, many of these methods yield only moderate enrichment that is not yet scalable for clinical application. It may be desirable to enrich first for partially differentiated, proliferative hESC intermediates with specific cell fates. These could then be expanded before further differentiation into cells for therapy. For example, the expression of the cell surface antigen, CD133, on proliferating hESCs identifies cells predestined toward a neuroectodermal fate . CD133-p in vivo .In the absence of specific cell surface antigens like CD133 to identify tissue-specific precursors, molecular beacons have been used to select for specific subpopulations of hESCs. King et al.  first deTransplanted hESCs encounter immune rejection  because Ideally, having genetically identical donor and patient cells is the best way to circumvent immune rejection. Thus, there is expressed interest in developing and using somatic cell nuclear transfer to generate patient-specific hESC lines. Using this technique, the DNA obtained from either a patient's skin or muscle cell would be transferred into an unfertilized egg that has had its DNA removed. Subsequently, the egg is artificially fertilized and allowed to develop until it reaches the blastocyst stage to derive hESCs. The resultant hESC line would have an immunologic profile matching the patient and could be used for cell therapy. This technique has been conducted successfully in animals using species-specific ESCs, but derivation of hESCs through somatic cell nuclear transfer has not yet been achieved.Other strategies to generate hESC lines with the closest match to potential transplant patients include engineering “universal donor hESCs,” a blood antigen O cell in which the expression of HLA is suppressed, or chimeric hematopoietic cells derived from hESCs capable of inhibiting the immune response when co-transplanted with the desired hESC-derived cells . AlternaWhile not the focus of this review, other sources of human-derived stem cells are also being explored for use in clinical settings. Among these are adult stem cells and induced pluripotent stem cells (iPSCs). Unlike hESCs, these stem cells can be obtained directly from the individual to be treated. Thus, as a source of cells for therapy, they are able to circumvent the immunocompatibility issues that hamper many non-autologous transplantation therapies. Furthermore, the utilization of these stem cells in both clinical and basic research studies does not face ethical and political issues that otherwise surround the use of embryonic stem cells. However, adult stem cells and iPSCs have significant limitations as well that are potentially overcome by hESCs at this time.Adult stem cells are derived from non-embryonic tissues and typically reside in their tissue of origin. Similar to hESCs, adult stem cells are capable of self-renewal. However, unlike hESCs, they have restricted potential and are able to differentiate only into cells from the tissue of origin. In some cases, adult stem cells are not able to generate all cell types of the tissue of origin, nor can they sustain growth over time. The latter problem has been encountered specifically in stem cells obtained from aging individuals , 96.iPSCs are generated by reprogramming differentiated somatic cells into a pluripotent state. This can be achieved by inducing the expression of three core reprogramming factors Oct4, Sox2, and Nanog. Several methods have been employed to express these factors and induce pluripotency. Among these are retroviral, lentiviral, and adenoviral transduction, which carry the risk of permanent and harmful genomic integration. Indeed, some established iPSC lines are genetically unstable, exhibiting large-scale genomic rearrangements, copy number variations, and abnormal karyotype even in early passage stages , 98. To In addition to genomic changes, recent studies have also revealed that iPSCs contain epigenetic features that indicate either incomplete or aberrant reprogramming. In particular, iPSC DNA methylation patterns are frequently reminiscent of the somatic cell of origin , suggestAs cell replacement therapies are envisioned and realized, their use in the treatment of aging-associated diseases becomes a compelling prospect. hESCs provide much promise as a potential tool in designing such therapies, as well as in drug discovery. It is clear that there are still major scientific challenges as well as ethical and legislative issues that must be addressed. However, it is encouraging to see that clinical trials involving the use of hESCs have begun, and that extensive efforts are underway to efficiently, successfully, and safely differentiate hESCs into specific cell types. These studies will pave the way toward leveraging the therapeutic benefit of hESCs for regenerative medicine, particularly in aging-associated diseases.


Oncolytic viruses are replication competent, tumor selective and lyse cancer cells. Their potential for anti-cancer therapy is based upon the concept that selective intratumoral replication will produce a potent anti-tumor effect and possibly bystander or remote cell killing, whilst minimizing normal tissue toxicity. Viruses may be naturally oncolytic or be engineered for oncolytic activity, and possess a host of different mechanisms to provide tumor selectivity. Clinical use of live replicating viruses is associated with a unique set of safety issues. Clinical experience has so far provided evidence of limited efficacy and a favourable toxicity profile. The interaction with the host immune system is complex. An anti-viral immune response may limit efficacy by rapidly clearing the virus. However, virally-induced cell lysis releases tumor associated antigens in a ‘dangerous’ context, and limited evidence suggests that this can lead to the generation of a specific anti-tumor immune response. Combination therapy with chemotherapy or radiotherapy represents a promising avenue for ongoing translation of oncolytic viruses into clinical practice. Obstacles to therapy include highly effective non-specific host mechanisms to clear virus following systemic delivery, immune-mediated clearance, and intratumoral barriers limiting virus spread. A number of novel strategies are now under investigation to overcome these barriers. This review provides an overview of the potential role of oncolytic viruses, highlighting recent progress towards developing effective therapy and asks if they are a realistic therapeutic option at this stage. The anti-tumor activity of viruses has been observed throughout the 20th century. Viral infections were noted to precede apparently inexplicable cancer remissions in multiple case reports of spontaneous remissions . VirusesOncolytic viruses are self-replicating, tumor selective and lyse cancer cells following viral infection. Non-oncolytic non-replicating viruses, in contrast, may also be used in cancer therapy, but as vectors to deliver gene therapy. Oncolytic viruses are dependant upon the host for replication, and differences in the cellular processes of normal host cells and tumor cells provide the potential for tumor-selective replication. Characteristics of cell transformation, including increased cell cycling, oncogene activation, altered receptor expression and defective signalling pathways, have been shown to enhance the ability of some viruses to replicate in neoplastic cells . OncolytSignificant obstacles need to be overcome in the design of effective oncolytic virus treatment schedules. Locally administered virus does not generally spread to other tumor sites , and sysThis review aims to provide an introduction to the potential of oncolytic viral therapy, highlighting progress in understanding critical interactions with the immune system and the recent development of strategies designed to overcome current obstacles to successful therapy.A group of naturally occurring or attenuated replication competent oncolytic viruses have been identified, which are non-pathogenic or induce only mild symptoms in humans, and possess intrinsic tumor selectivity . This grViruses use a range of strategies to regulate host cellular processes and promote efficient replication within host cells. Genetic modification of viruses can be used to interfere with these viral strategies, preventing replication within normal host cells, whilst permitting replication in tumor cells. Replacement or modification of viral promoters can provide tumor selectivity . Human DThe process of carcinogenesis involves genetic instability, with one estimate of over 11,000 genomic alterations occurring in a cancer cell . NeoplasAnti-viral defence mechanisms are inactived in many tumors. The double stranded RNA (dsRNA) dependant protein kinase (PKR) and type I interferon (IFN) pathways are important, overlapping, anti-viral mechanisms in normal cells.Reovirus is an example of a naturally occurring oncolytic virus, whose tumor selectivity involves a defective PKR anti-viral response. Reovirus has been found to replicate more rapidly in Ras-activated cells . In normThe type I interferons, IFNα and IFNβ, have anti-proliferative properties, and defects in these pathways are commonly found in tumors, promoting cancer growth . SeveralViruses can achieve tumor specificity if they obtain entry to cells via receptors that are overexpressed on tumor cells or via mutated receptors that are unique to tumor cells. For example, the Edmonton strain of measles virus can obtain entry to cells via CD46, a receptor which is overexpressed in some tumors . VirusesViruses can be genetically modified to provide tumor selectivity by placing regulatory viral genes under control of a tumor or tissue specific promoter. Promoters for tumor antigens such as prostate specific antigen and alphafetoprotein have been incorporated into genetically modified adenoviruses , within the limits of viral manufacture, by all routes. Flu-like symptoms were the most common toxicities and were increased in patients receiving vascular administration. There was no clear correlation between the development of flu-like symptoms and viral dose or dose administration regimen. Neutralizing antibodies were generated following all routes of administration and viral doses. Interestingly, following intra-peritoneal delivery to patients with ovarian cancer, toxicity including viraemic symptoms and abdominal pain appeared more commonly in patients with bulky disease . It was  disease . As the  disease . In thes disease . These r disease . No obje disease .HSV1716 is an engineered oncolytic virus which lacks copies of the gene encoding the virulence factor ICP34.5  such thaDespite the clinical experience of a favourable toxicity profile with high viral doses, compared with the toxicity of conventional cytotoxic therapies, safety concerns remain concerning the use of replicating oncolytic viral therapy. In contrast to gene therapy with non-replicating adenovirus or retrovirus vectors, the interactions of replicating virus with both host and environment are far more difficult to predict. Live replicating virus can be shed from patients following treatment, raising the possibility of person-to-person transmission. For example NDV has been isolated from urine up to three weeks after treatment . The mutDespite intensive investigation of the direct cytotoxic ability of oncolytic viruses, little attention has been given to the potential to induce anti-tumor immunity. Matzinger has proposed that the key role of the immune system is not primarily the identification of self and non-self, but the recognition of ‘danger’ signals . Virus-iThe inflammatory context created by virus-induced cell death is more likely to produce a stimulatory immune response as opposed to tolerance . Howeverin vitro human systems and clinical trials.There is some limited data regarding the ability of oncolytic viruses to generate an anti-tumor immune response, drawn from animal models, Different modified strains of HSV have been shown to induce systemic anti-tumor immune response in murine models . In one in vitro allogeneic human system to provide ‘proof of principle’ that oncolytic virus-induced cell death can lead to cross presentation of TAAs. Tumor cell lysates induced by parvovirus H-1 stimulated DC maturation and cross presentation of melanoma antigens to CTL clones, in contrast to tumor cell lysates induced by ultraviolet light or freeze-thaw cycles. Greiner et al. , basement membrane and necrotic areas, to access tumor cells . Tumor vStrategies designed to enhance the delivery of drugs to tumors  can be aOncolytic viruses are tumor selective potent anti-cancer agents. Currently in the early stages of their emergence as useful therapy, their promise is yet to be fulfilled. Clincial studies so far have provided evidence of limited activity both following local and systemic administration. Human tumors are very diverse, and the response to virotherapy is variable. Significant challenges remain to be overcome before oncolytic viruses can emerge as broadly useful therapy. Efforts are being made to enhance the anti-tumor potency of oncolytic viruses, whilst maintaining tumor selectivity. The anti-viral immune response is a major obstacle to effective virotherapy, although the release of TAAs and provision of a ‘danger’ signal offer the tantalising possibility of generating anti-tumor immunity. Rapid clearance of virus from the bloodstream has limited systemic application. A host of different solutions, including cellular carriers and modification of virus particles, have recently emerged to enhance systemic delivery. Oncolytic viruses have a favourable toxicity record, and safety concerns regarding the use of live replicating viruses continue to be addressed. A lack of cross-resistance, non-overlapping toxicity, and evidence of synergistic interactions with radiotherapy and chemotherapy lays the basis for future combination virotherapy treatment. The considerable concerns by institutions worldwide as to the toxicity of these agents to patients, staff, patients’ immediate relatives and shedding into the environment have resulted in more defined regulatory guidelines and appropriate risk assessments. This process combined with the considerable costs when compared to standard cancer chemotherapeutics has led to questioning of the viability of virus based approaches. Despite this scrutiny, it is clear that oncolytic viruses are moving closer to fulfilling their exciting clinical potential and may become the standard of care for certain cancer scenarios in the future.