Autophagy is known for bulk clearance of cytoplasmic aggregated proteins, whereas the specificity of protein degradation by UPS comes from E3 ubiquitin ligases. Few E3 ubiquitin ligases, like C-terminus of Hsc70-interacting protein (CHIP) not only take part in protein quality control pathways, but also plays a key regulatory role in other cellular processes like signaling, development, DNA damage repair, immunity and aging. CHIP targets misfolded proteins for their degradation through proteasome, as well as autophagy; simultaneously, with the help of chaperones, it also regulates folding attempts for misfolded proteins. The broad range of CHIP substrates and their associations with multiple pathologies make it a key molecule to work upon and focus for future therapeutic interventions. E3 ubiquitin ligase CHIP interacts and degrades many protein inclusions formed in neurodegenerative diseases. The presence of CHIP at various nodes of cellular protein-protein interaction network presents this molecule as a potential candidate for further research. In this review, we have explored a wide range of functionality of CHIP inside cells by a detailed presentation of its co-chaperone, E3 and E4 enzyme like functions, with central focus on its protein quality control roles in neurodegenerative diseases. We have also raised many unexplored but expected fundamental questions regarding CHIP functions, which generate hopes for its future applications in research, as well as drug discovery.Cells regularly synthesize new proteins to replace old and abnormal proteins for normal cellular functions. Two significant protein quality control pathways inside the cellular  The guidance of chaperones assists immature polypeptides to acquire a functional macromolecular structure and move towards appropriate cellular localization to perform pre-defined functions  and autophagy. These two systems either work independently or in collaboration with each other  and other proteins to tag misfolded or improperly folded proteins for their removal from the cells Edkins, . UPS invAutophagy is another widely explored pathway that helps in clearance of the cytoplasmic bulk of misfolded proteins, by using lysosomal proteolysis  is profoundly involved in most of the above-described quality control pathways , with the cytogenetic band at location p13.3 on chromosome number 16. The pre-mRNA consists of seven exons and six introns within 2760 base pairs of the CHIP/STUB1 gene. As represented in Figure Interaction of CHIP with heat shock proteins occurs because of its TPR domain; this interaction of CHIP with C-terminus of Hsc70 or Hsp70 does not have any interference in its binding with misfolded proteins. Apart from TPR domain, its adjacent positively charged region is also significantly responsible for this interaction , giving an idea of its modulation, under stress conditions  are only modified functionally, instead of being degraded by any of these degradation pathways , estrogen alpha receptor (ERα) and androgen receptor (AR), using UPS  and neuronal enzymes like neuronal nitric oxide synthase , includes removal of mutant MKKS protein from the cell by CHIP  via the mechanism of autophagy. The autophagic flux of many substrates in the cell is controlled by CHIP, as observed in CHIP knockdown cells, where it regulates autophagic marker proteins such as p62 and microtubule-associated protein 1A/1B-light chain 3 , includes the roles of E3 ubiquitin ligase CHIP. It mediates polyubiquitination of Hsp70/Hsc70, which preferentially promotes degradation of SOD1 via 26S proteasome, as shown in Figure Parkinson’s disease also involves the formation of various proteotoxic inclusions inside the neurons, which may later result in dementia or in extreme cases, the death of patients  protein levels in the cells. Ubiquitination of SirT6 at K170 position by CHIP E3 ubiquitin ligase stabilizes the protein, which is rather degraded by some unknown E3 ubiquitin ligase. Such kind of positive regulation of SirT6 by CHIP provides a novel example where these two arms of DNA and protein quality maintenance systems cooperatively regulate homeostasis and aging processes  are special class of proteases which can release ubiquitin from substrate proteins by breaking the peptide bonds in between substrate and ubiquitin molecules. Regulating DUBs activities is assumed to have some possible therapeutic benefits. Ataxin-3, one of the most studied DUB, found to have a direct interaction with CHIP along with an E2 enzyme Ube2W in degradation of its substrates. An important mechanism of regulation of DUB ataxin-3 by its ubiquitination is also present inside the cells. CHIP has shown to have the capacity to ubiquitinate ataxin-3 and in turn may have a regulatory effect on this neuroprotective DUB enzyme (Scaglione et al., Many years of research has established CHIP, as a unique molecule, which has a large number of interacting partners. An interaction network of such a wide variety of substrates enables this E3 ligase enzyme to take part, directly or indirectly, in an array of cellular functions including metabolism, signaling, proliferation and apoptosis. The structure of CHIP, consisting of both TPR and U-box domain, is also a crucial factor in deciding its functional capabilities. Performing the roles of co-chaperone and E3 ligase simultaneously needs enormous molecular capabilities, and its advantageous structure provides it an extra edge in performing these dual functions. It is still a matter of investigation how exactly these decisions are being made by CHIP, inside the cells. A major question yet to be answered is, how CHIP, in combination with other chaperones recognizes the abnormal and to be degraded substrates inside the cytoplasm. Further work in this direction will indeed aid in understanding the possible ways of identifying cellular proteins on the basis of their structural and physiological features. Several studies have been done to explore the methods of discriminating the abnormal proteins from the healthy ones, whether they are to be modified, modulated or degraded. But, a clear answer is still far from our current understanding. Major focus of research over CHIP has been given to its roles in signaling, apoptosis and cell proliferation. But, a line of evidence clearly indicates that this molecule has equally important contribution in maintaining the cellular homeostasis and providing protection from various stresses, which are major factors causing protein aggregation and related neurodegenerative diseases. Few possible developmental and aging-related roles of CHIP has also been observed; still a lot of research is needed to be done to understand the physiological importance of CHIP in neurodevelopmental and aging processes.Maintaining a healthy proteome is a prerequisite of a living cell. To achieve this, cells are evolved with chaperone machinery and quality control pathways that maintain proteins into proper conformation and protect cells from toxicity generated in various pathological conditions (Yerbury et al., Although, enough reports have been made to establish protective roles of E3 ubiquitin ligases in various neurodegenerative diseases, still no notable success has been registered so far to treat these life-threatening neuronal disorders. In one of our previous review articles, we have selected few such E3 ligases which have shown cellular protection against these pathological conditions (Upadhyay et al., VJ, AU, AA and RM, executed complete drawing of figures, AK provided critical inputs. AM formulated the entire concept of manuscript and designed the initial draft of figures. All authors reviewed the manuscript.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.


Virulence, Ikram et al. have compiled an extensive proteomic bioinformatics study of characteristic mutations in the hepatitis E virus (HEV) genome. This study showed genotype-specific prevalence of respective mutations and demonstrated the selective pressure imposed upon the virus by both host immune defense and antivirals.In the recent issue of omics-analyses  and bioinformatics became crucial when it became apparent that genetic variants of viruses were associated to pathogenesis and disease progression. The first article in terms of virus diversity and course of disease was published by Pierre Lépine in 1938 who reported on the evolution of different rabies virus strains and their association to different characteristics of infectivity and virulence.The evolutionary significance of virus infections has been a subject of discussion for decades;Long thought to be a healthcare burden exclusive to developing countries with sporadic cases in industrialized countries confined to travel-borne infections, HEV is now being increasingly recognized as an emerging disease globally.Viral adaptation as a response to host innate and adaptive immune response and selective pressure by antiviral treatment is critical to ensure continued viral fitness and replication competency. Studies on chronic infections with hepatitis B virus (HBV) and C virus (HCV) are prime examples of how viral evolution can overcome host immune defenses and antiviral therapy. For example numerous immune-driven adaptations within nonstructural regions of HCV have been described, including genotype-specific HLA-associated escape mutations.in vitro and in situ.Unfortunately, we are far from having reached this goal in the control of HEV. As a single-strand positive-sense RNA virus, HEV genome replication is extremely error-prone since HEV RNA-dependent RNA polymerase lacks proofreading activity and thus generates mutations rapidly during transcription, undoubtedly contributing to viral evolution. A plethora of mutations within the three HEV open reading frames (ORFs) have been described In light of the accumulating descriptions of patient-specific HEV proteome alterations, there is an urgent need to analyze their global prevalence and assess their association to clinical outcomes. Undoubtedly, the optimal antiviral treatment ensuring sustained virological response is a personalized treatment strategy that targets individual host and viral factors. Individualized therapy could be optimized if treatment-naive viral quasispecies and functional mutations have been identified and assessed before treatment starts. This in turn requires a comprehensive analysis of the true prevalence and significance of relevant mutations described so far.et al. at the onset of their study was to identify which effects specific HEV variants can have on susceptibility, pathogenesis of infection and treatment outcome.in vitro data for mutations in the HEV ORF1 and ORF2 regions the investigators identified 20 characteristic amino acid variants (17 in ORF1 and 3 in ORF2). In a next step they assessed the prevalence of the 20 mutations within 406 full-length ORF1 and ORF2 sequences of different HEV genotypes, while taking into account their association with hepatic failure, treatment failure, chronicity and altered immunoreactivity. The authors made the astounding realization that specific mutations  were conserved and by contrast the remaining analyzed variations showed a high degree of variability. The latter includes two notable mutations T735I and G1634R/K that were frequent among the major human pathogenic HEV genotypes 1, 3, and 4 and correlated with liver failure and ribavirin therapy failure, respectively. Furthermore, two amino acid residues Leu477 and Leu613 within the capsid protein (ORF2) known to be involved in formation of neutralization-sensitive epitopes were mutated with considerably high frequency in HEV genotype 1 and 3 implicating that immune evasion is a common strategy in HEV infection.In their article under discussion, Ikram and colleagues employed elegant large-scale proteomic bioinformatics to shed new light on previously described HEV mutations associated with ribavirin treatment failure, chronic infection, hepatic failure and altered immunoreactivity. They carefully assessed the global prevalence of characteristic mutations among different HEV genotypes , their impact on B an T cell epitope reactivity and their genotype-specific evolution.in silico SNP algorithms to predict whether the investigated mutations were deleterious and caused protein destabilization, respectively. Their finding indicates that most ORF1 mutations and 1 of 3 ORF2 mutations are indeed deleterious and that notably two ORF2 substitutions  can destabilize the capsid protein structure.Next, the investigators utilized The authors went on to investigate whether the described mutations resulted in altered B and T cell epitope antigenicity based on the hypothesis that acquisition of escape mutations may hide the virus from host defenses. Through a combination of epitope prediction programs and online databases B and T cell epitopes (class MHCI and MHCII) of HEV genotype 1, 3 and 4 consensus sequences were explored. Intriguingly, overlap of epitopes and mutation sites were found for many of the studied substitutions. Subsequent evaluation revealed reduced antigenicity of mutated epitopes as compared to wild-type with only few having retained or increased antigenicity. These findings strongly imply that, similarly to HBV and HCV, HEV may profit from altered epitopes via decreased B and T cell recognition.Finally, Ikram and colleagues mapped the evolution of characteristic mutations for each HEV genotype. Notably, the investigators found that especially the mutations T735I, the ribavirin failure-associated G1643R/K and leucine substitutions L477T and L613T in ORF2 developed earlier in HEV genotype 1 than 3 and thus evolution of HEV-1 may have occurred earlier than HEV-3 and -4.Taken together, Ikram and colleagues have addressed an important issue of virus infection, namely virus evolution and the relevance of characteristic mutations in the viral genome. The in-depth approach using large-scale proteomic bioinformatics to characterize the intra-genotypic prevalence and evolution of HEV variants and their implications for clinical outcomes and host immune evasion is a prime example of how bioinformatics can be a valuable tool to better understand the functions of viral adaptation. As shown also for other viruses , the course of antiviral treatment can be adjusted and optimized if host and viral factors are assessed for each individual patient. The high mutation rate of HEV, the frequent emergence of ribavirin failure-associated quasispecies and the ability of HEV to hide from host immune defense are factors that underline the necessity to profile and study characteristic viral adaptations. There is therefore need to invest in modern techniques since the powerful next generation sequencing approaches  and digital epidemiology including intensified molecular surveillance and proteomic bioinformatics will provide us with the necessary tools to identify and discriminate characteristic and relevant variations from polymorphisms in the viral genome. More detailed knowledge about virus evolution and diversity will help us not only to understand the pathophysiology of virus infection and virus-host interactions but also to better manage infected patients. Accordingly, the findings from Ikram and colleagues add new insights to our knowledge of the complex processes of viral evolution, viral diversity, and selection of HEV quasispecies. We are still at the early stages of characterizing viral diversity and its association with pathogenesis, immune defense, and therapy efficacy. The paper under discussion is an excellent example of how to explore virus diversity and its impact on the course of infection using sophisticated methods. Only then can the burden of serious hepatitis E be lifted.


To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health. The impact of the mammalian gut microbiome on health and disease, especially its response to diet, has gained increasing attention in recent years. Previous studies have demonstrated that the community membership and diversity of bacterial communities shift rapidly with diet-specific responses in mice  and the availability of metabolic functions  . Unlike To better understand the dynamics of gut viruses alongside bacterial communities, we characterized the concurrent responses of bacterial and viral intestinal communities in mice that underwent a dietary perturbation. Mice were fed a purified low-fat (LF) or high-milkfat (MF) diet, switched to the alternate diet, and returned to the original diet for a washout period to study the resistance of community responses. The selection of the high-refined sugar, MF diet was based on its approximation of Western consumption . It is an=3 per group) were fed purified high fiber, LF  or purified high-refined sugar, MF diet  (ad libitum after weaning (~3-4 weeks of age). At 8–9 weeks of age  the diets of the mice were disturbed with a switched diet for 3 weeks, followed by a return to their original diets for three weeks. Stool samples were collected weekly. All animal protocols and experiments were approved by the Institutional Animal Care and Use Committee at the University of Chicago.C57BL/6 mice were bred and maintained under standard 12:12 h light/dark conditions at the University of Chicago. Co-housed female mice from mixed litters (atories)  ad libitm-filtered supernatants of stool slurries followed by purification on an iodixanol  density gradient. DNA representing IND was obtained from VLPs collected from stool pellet slurries  incubated anaerobically with mitomycin C for 18 h at 37 °C. Owing to low sample yield, it was necessary to amplify DNA from viral fractions (VLP and IND) with whole-genome amplification . DNA isolation, extraction, and quantification methods are described in more detail in Stool was collected weekly from mice, from which DNA was directly extracted for 16S ribosomal RNA (rRNA) gene amplification and for bacterial (BAC) metagenomic libraries. Viruses in the stool were targeted in two separate fractions: virus-like particles (VLPs) and inducible prophages (IND) present in gut bacteria. DNA originating from virus-like particles (VLP) was isolated from 0.22 μAmplified products of the V4–V5 region of the 16S rRNA gene originating from the BAC fraction were sequenced with Illumina MiSeq  using barcoded primers 515F/806R (The two groups of mice were fed LF or MF diet for 4–5 weeks prior to the start of the experiment (Day 0), followed by 3 weeks of the other diet , and 3 weeks of their initial baseline diet , all associated metagenomes were cumulatively assembled, providing deeper sequencing depth for assembly. The resulting assembled contigs from each fraction were combined, and contigs sharing >99% sequence similarity were clustered, with the longest contig selected as representative. The sequencing efforts for this study purposefully targeted deep sequencing of DNA from viral fractions , and we E-value⩽1e–5), viruses contained with NCBI RefSeq (Release 66)  and MetaVir2 . The majority of contigs, though abundant in our samples, could not be annotated against available databases. Among the total 117 460 contigs, 23 811 (21%) could be associated with functional gene annotations and 21 119 (19%) could be associated with taxonomic classifications in MG-RAST , percent identity minimum ⩾60%, minimum Metagenomes from samples originating from BAC fractions were dominated by bacteria-associated annotations, with the most abundant phyla being Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, though hits to the bacteriophage taxon Caudovirales were also present . CommuniP=0.009) and a significant increase in Clostridiales  in 16S rRNA gene amplicons 3 weeks after the switch to the MF diet (Day 22) (P=0.0029) in 16S rRNA gene amplicons  were obtained. For both baseline diets, fecal bacterial communities were predominantly composed of members of the phyla Firmicutes ) and Bacteroidetes , (Day 22) . In contmplicons  was obsemplicons , we did mplicons .P=0.0058, P=0.0085, Alpha diversity of the fecal bacterial communities fluctuated throughout the experiment with distinct responses for each diet treatment. Bacterial communities from baseline LF mice showed a maximum increase in alpha diversity by Day 22, the end of the switch to MF diet, and were significantly more diverse than the baseline MF group on the same day , 22 and 43 to Day 0 communities . With only one exception , bacterial communities on days 1, 22 and 43 were not significantly different relative to Day 0, as measured by clustering of communities by either 16S rRNA amplicons or BAC metagenomes , Table 4P=0.009) (P<0.05) and returned to initial levels by the end of the washout period . VLP counts of baseline MF stool showed no clear pattern in response to diet.Direct counts of VLPs  isolated from stool confirmed the distinct responses of viruses under baseline MF and LF conditions. VLP counts differed significantly among treatment groups at the start of the experiment  , with baTo identify functions associated with the response of baseline MF viral communities, contigs whose abundances changed significantly over the course of the experiment were identified . The mosn=7) were associated with Firmicutes, mainly with the taxa Bacillus and Clostridium, and contained genes associated with phage integration and excision. In Group II, Caudovirales-associated contigs were identified, mainly those similar to Siphoviridae and Myoviridae and with annotations to phage with hosts Lactobacillus, Streptococcus, Staphylococcus, Clostridium and Listeria.The predicted taxonomic origin associated with these contigs varied and included both bacterial hosts and bacteriophages . The majThe relative abundances of Group II contigs are observed to share consistent trends in the baseline MF IND samples, increasing in abundance on Day 1 relative to baseline Day 0 , decreasing in abundances after the diet disturbance  and increasing when returned to the baseline diet  . These tClostridia and Bacillus) and Bacteroidetes as well as Caudovirales. The abundances of these contigs in both viral fractions were correlated to 16S rRNA genes- or BAC-associated sequences that shared similarity to sequences associated with Bacillales, Lactobacillales and Clostridiales. Module II contained contigs from Group I and II and was similar between VLP and IND fractions . Module II contigs were similar to phages previously known to be associated with Firmicutes (Bacilli) as well as Caudiovirales (Siphoviridae). In both fractions, these contigs were significantly correlated to 16S rRNA amplicons or BAC contigs that were similar to sequences associated with Erysipelotrichales and Clostridiales.To explore potential interactions with bacterial hosts, we tested co-occurrence relationships between these Group I and II contigs and bacterial taxa using BAC-associated contig abundances and 16S rRNA amplicons see . Co-occuRecent insights into the microbial processes and pathways that contribute to gut microbiome stability and resilience have focused on bacterial responses to diet in the gut system  and a smaller proportion of reads from the bacterial fraction (7–9%). To complement the relatively shallow sequencing depth of metagenomes from the bacterial fraction , 16S rRNA amplicon sequencing was performed to provide additional information about the structure of the bacterial gut community throughout the experiment. Notably, in our assembled reference, the large majority of sequences could not be classified against known sequences. Many of these unknown sequences originated from viral fractions, highlighting the need to develop improved references for characterizing these communities. These sequences reveal a significant community in these microbiomes that are uncharacterized and that are overlooked by relying on previously characterized reference genomes. Overall, among viral contigs, we observed that viruses were largely temperate, evidenced in the overlapping presence of similar contigs in both IND and VLP fractions  and consThe combination of the three fractions served as an effective strategy for assessing changes in community diversity at coarse time resolution . Together with weekly 16S rRNA bacterial community data, we could explore responses of the bacterial and viral community structures, as well as functional responses, to diet changes, specifically, their resistance to diet disturbances. As the low viral biomass available in mouse stool required the use of whole-genome amplification, we expect biases in quantification of sequence abundance. Nevertheless, our biological replicates exhibited similar responses, suggesting that any amplification biases are minimal or are similar across samples. Despite these challenges, the resulting sequencing catalog generated from this effort is an important reference for future studies.P =0.0039). In baseline LF mice, the mean relative abundance of this taxon decreased, though not significantly, when on the MF diet . These diet-specific changes associated with Turicibacterales have also been reported by others, including reduced relative abundances of Turicibacter in mice fed a high-fat diet . These dietary differences may have contributed to the contrasting changes in bacterial diversity depending on how certain bacterial members responded to these specific dietary components. In BAC metagenomes, we observed significantly different bacterial communities but not different functional profiles on Days 0, 22 and 43 of the experiment. The resistance of these gut functions to diet changes likely contributes to overall stability, where various gut bacteria likely share redundant functions.We observed significantly different bacterial communities in response to diet shifts, and this result is consistent with previous reports that have indicated diet-specific changes in gut bacterial communities  and washout communities (Day 43). In contrast to bacterial communities that were similar before and after the diet shift, virus communities were significantly different, most profoundly observed in the baseline MF IND and VLP metagenomes. This result, supported by direct counts of VLPs, contrasts with previous reports of similar bacterial and viral trends in response to a diet shift (To identify potential drivers in the baseline MF virus fractions that were associated with the observed unique responses, we identified contigs that were observed to change significantly in relative abundance over the experiment in VLP and IND fractions. The most significant functions were associated with known phage-associated sequences, and these contigs were grouped into two representative clusters based on their abundances in baseline MF viral fractions. The majority of these contigs were similar to sequences originating from phages associated with Firmicutes, both Clostridia and Bacilli. Firmicutes have previously been identified as a dominant division responding to diet in the gut microbiome (To our knowledge, this is the first demonstration that viral communities can respond to specific diet perturbations independently and that bacteria and viruses in the same mice may have unique resistance to disturbances. The contrasting responses of the baseline MF viral communities relative to their counterpart LF viral communities suggest that historical contingency—not just the current diet treatment of the host—may have a key role in community development; for example, some factor during the early life of the baseline MF mice may have constrained their microbiomes' responses to the LF diet and to the reversion to MF. Importantly, our experimental design cannot rule out the environmental differences resulting from housing the baseline LF and MF mice in different cages, often a problem due to the logistics of housing in mammalian studies (


Adiponectin and its signaling through the receptors AdipoR1 and AdipoR2 rank highly in the ongoing search for the holy grail that might improve insulin sensitivity, modulating inflammatory responses, and regulating energy metabolism in patients with obesity and/or Type 2 Diabetes (T2D). Adiponectin is secreted by adipose tissue and binds to its ubiquitously expressed receptors AdipoR1 and AdiopoR2. Over-expression of adiponectin promotes insulin action and improves insulin resistance as well as suppressing inflammatory responses. Over 15 years ago, Philipp Scherer and colleagues demonstrated that intraperitoneal injection of full-length adiponectin into mice reduced plasma glucose levels and suppressed gluconeogenesis, revealing the liver as a major target tissue of adiponectin Molecular Metabolism, Holland and colleagues studied transgenic mouse models that over-express AdipoR1 and AdipoR2 in adipose tissue and liver to assess the impact of AdipoR signaling on glucose and lipid metabolism In this issue of In their previous work, Holland and colleagues demonstrated that ceramide metabolism predominantly influenced the insulin-sensitizing effects of adiponectin on its primary target tissue, the liver In an attempt to decipher the molecular mechanisms that would link increased adiponectin-receptor-signaling with improved glucose homeostasis, the key fuel sensing kinase AMP-activated protein kinase (AMPK) appeared to be a logical target. Indeed, it was shown more than a decade ago that binding of adiponectin to its receptors AdipoR1 and AdipoR2 results in the activation of AMPK, which stimulates glucose utilization, suppressing hepatic gluconeogenesis and de novo lipogenesis, and promotes fatty acid oxidation Holland and colleagues have previously shown, however, that the activation of ceramidase downstream of adiponectin binding to its receptors is independent of the activation of AMPK Why is hydrolysis of ceramides beneficial when regulating insulin sensitivity? Ceramides are an important key factor in understanding regulation of weight gain and glucose intolerance as they increase during obesity and promote insulin resistance. Ceramides are synthesized by six ceramide synthase enzymes (CerS). Overexpression of CerS6, for example, correlates with insulin resistance, and CerS6-deficient (CerS6D/D) mice exhibit reduced C16:0 ceramides and are protected from high-fat-diet-induced obesity and glucose intolerance However, a key question remains: Is the activation of AMPK upon adiponectin signaling dependent on S1P being produced or will AMPK also be activated independently of ceramidase activation? If both act independently, and both events result in insulin-sensitizing, anti-inflammatory and anti-apoptotic actions, what is the reason for this parallel signaling? Holland and colleagues present further in this issue that in WT mice, injection of AdipoRon, an adiponectin receptor agonist, resulted in a severe increase in ceramidase activity from liver lysates, indicating that adiponectin agonism may be a beneficial therapeutic for the treatment of hyperglycemia and function via a ceramide-lowering mechanism.Can the positive effects of adiponectin binding to its receptors be used in treating obese and insulin resistant patients as well as patients with obesity induced non-alcoholic fatty liver disease (NAFLD) or the more severe non-alcoholic steatohepatitis (NASH)? One challenge that remains is that the Kd for small molecules like AdipoRon binding to AdipoR1 and AdipoR2 is much higher than that of the full-length domain of adiponectin While the search for new drugs to treat T2D and fatty liver disease continues, one intervention that also improves insulin resistance and decreases fatty liver is physical exercise. It has been shown that AMPK is markedly up-regulated in skeletal muscle upon exercise


Macrophages are traditionally viewed as a key component of the immunity defense system. Recent studies have identified resident macrophages in multiple organs including the heart, in which the cells perform their crucial role on tissue repair after myocardial infarction (MI). The cardiac-specific macrophages interdigitate with cardiomyocytes particularly at the atrioventricular node region. The integrative communication between macrophage and cardiomyocytes can modulate the contractile function of the heart. Coordinated control of intracellular calcium signaling and intercellular electrical conduction via the syncytium network underlie the synchronized beating of the heart. In this review article, we introduce the concept the syncytium calcium signaling in the cardiomyocytes can modulate gene expression in the resident macrophages and their integration with the cardiomyocytes. The cardiac macrophages originate from bone marrow stem cells, migrate to local via vessel, and settle down as a naturalization process in heart. As the macrophages perform on regulating electrical conduction, and accomplish post MI non-scared completed regeneration or partial regeneration with fibrotic scar at different stage of postnatal development, we understand that multiple functions of cardiac macrophage should carry on with diverse linages. The naturalization process in heart of macrophages to the cardiomyocytes serves important roles to control of electrical signaling and calcium-dependent contractile function of the heart. As a major component in the first line of immunity defense, macrophages are distributed in almost every tissues, including cardiac macrophages in the heart, cerebellum microglia cells in the brain, hepatic Kupffer cells in the liver, alveolar macrophages in the lungs, and Langerhans cells in the epidermis. Although macrophage functions in the immune system have been investigated extensively –3, the tThe cardiac macrophages developed from bone marrow stem cells (plus spleen stem cells as well in mouse), migrated through cardiac vessel from circulated blood, and settled down with polarization in heart could comprehend as a naturalization process in heart. The macrophages play crucial role on regulating electrical conduction by associating with AV node , and theA recent study by Hulsmans et al. showed that resident macrophages were enriched in human and mouse atrioventricular (AV) node and can regulate the electrophysiological activity of cardiomyocytes through the gap-junction protein, connexin 43 (Cx43), at the “linking” portion of the conducting cardiomyocyte and the macrophage , 11. ThiUsing GFP labelled cardiac macrophages, cardiomyocytes located in the lower nodal or AV bundle were frequently interspersed with macrophages that have an elongated, spindle-shaped appearance , 13. TheIn heart tissue, Cx43 is located on the intercalated discs responsible for electrical conduction through neighboring cardiomyocytes, and is essential for the synchronized contraction of the heart. The Cx43-mediated cell–cell linkage provides the connection between resident macrophages and cardiomyocytes  and formCx3cr1 Cx43/−− mice developed severe 1st degree and 2nd degree AV block. With CSF1 deletion in the macrophages, the CSF1op mice could develop non-matured macrophages with a lack of normal function and exhibited 1st degree and 2nd degree AV block. Deletion of macrophage Cd11b [Cd11bDTR mice produced 1st degree, 2nd degree, and even life threatening 3rd degree AV block [Although more detailed information on the mechanisms of how resident macrophages facilitate the conduction system in the heart remained to be explored, the physiological function of the macrophages on maintaining AV conduction were supported by several animal model studies where AV blocks were introduced through loss-of-function investigations. With deletion of Cx43 in the macrophages, the ge Cd11b , 14 in tAV block . These tThe recent study by Hulsmans et al.  revealedRationally, cell surface antigens on specified macrophages would be critical for the cell migration, polarization summarized as naturalization in heart although the related molecular mechanisms are still under investigation. These cardiac macrophage antigens plus intracellular markers of the cell are utilized biomarkers for us to discover function of macrophages in cardiac tissues. The cell surface antigens distribute on monocyte-macrophage include CCR2/CD192, CD64/FcγR1, CX3CR1 and Mac3 –9. Some In adult mammals, cardiac macrophages origin from their bone marrow as well as spleen in mouse. While how macrophages differentiating from monocyte during embryonic development is still a mystery (discussed below), many information are discovered from myocardial infarction mouse model. For tracking the macrophage differentiation and settlement in heart after MI, many gating strategies employed with combined different antigens. The combination Ly6ChighCCR2highCX3CR1lowCD62 L+ used to examines classical monocytes , and MHCSynchronized contractile function of the heart is essential to life. Exactly how resident macrophages in the heart evolved as a fail-safe way to guarantee robust cardiac output under physiological and pathological conditions remains an important area of research.Calcium (Ca) ions are important second messengers modulating many cellular functions. In the heart, entry of extracellular Ca via Ca channels located on the plasma membrane triggers opening of the ryanodine receptor (RyR) located in the sarcoplasmic reticulum (SR) through Ca-induced Ca release (CICR) –27. The Synchronized elevation of intracellular Ca triggers contraction of the actin-myosin apparatus by diastolic depolarization, and the crosstalk of electrical conduction between neighboring cardiomyocytes via the interconnection of their intercalated discs through the connexin complex. Longitudinal flow of Ca signaling via the syncytium network characterizes the heart as an efficient circulation pump.Although we normally consider that macrophages function as cells in the front line of the immune system, these macrophages also play critical roles in many other aspects, including cardiac electrical activity, wound repair, embryonic development, and many more –3. All tRecent research demonstrated that Ca may contribute to modulation of gene expression in the macrophage. Using monocyte-derived macrophages (MDMs) from patients with chronic obstructive pulmonary disease (COPD), Provost et al. showed that extracellular Ca could enhance phagocytosis and cytokine secretion associated with IL-8, TNF-α, and macrophage inflammatory protein (MIP) subunits MIP-1a and MIP-1b . AdditioExtracellular Ca influxes through plasma membrane Ca channels take the responsibility for the cytoplasmic phagosomal oxidative reaction and inflammatory cytokine reaction , 40, 42.Although the Ca-dependent manner of macrophage function was discovered in monocyte-derived macrophages or macrophage-like U937 and MH-S cells, it is possible that the resident macrophages would behavior according to Ca levels in the micro-environment of their niche in the heart tissue.Macrophages are developed and differentiated from the mononuclear phagocyte system (MPS) , 50. WhiThe characteristics of macrophages with deletion of CSF-1 in the mouse model pinpoint many critical functions of macrophages in somatic differentiation and the development of the pancreas and nervous system in mammal , 54. Gen−) anions [The mononuclear phagocyte lineage differentiating progress is under the control of macrophage CSF, however, no research has reported the direct involvement of Ca signaling with CSF. An earlier data revealed that the concentration of cytosolic Ca pre-incubated with granulocyte–macrophage CSFs can effectively activate an oxidative burst of granulocytes measured with the production of intracellular superoxide (O2) anions . Release) anions . These d) anions . It is pIn the heart, CICR and syncytium cell–cell communication underlie the synchronized contractions of the cardiomyocytes to drive blood circulation throughout the entire body Fig. a, b. EleThe concept of resident macrophages facilitating electrical conduction in the heart raises many interesting subjects that should be explored further about the role of macrophages in other cardiac functions such as how pre-mononuclear phagocytes differentiate along with conducting cardiomyocytes, what principle role they play during co-developmental architecture, how these resident macrophages function in adult heart, what maintains their role in continuous contracting tissue as a non-contractile cells, and whether anchoring proteins and extracellular matrix proteins are required to direct and connect resident macrophage to conducting cardiomyocyte.It should not be a coincidence that there is both a Ca-dependency of macrophages and CICR dependency of cardiomyocytes for contraction. The intracellular Ca in both cells should provide coordination for their integration, and the extracellular Ca should provide a micro-environment for homeostasis. The syncytium Ca signaling would allow for a more efficient macrophage niche within the cardiomyocytes and consequently for the synchronized contraction of the heart.


The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of β-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/β-catenin–TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming. Stem cells have, by definition, the ability to self-renew, i.e., give rise to at least one identical daughter cell, as well as to differentiate into the myriad of specialized cell types from early embryonic stages of development until adult age. These hallmark characteristics of stem cells have sparked a widespread interest in their biology, which fueled formidable research efforts worldwide.Stem cells can be classified according to their origin. Their provenance also specifies their biological competence. As such, stem cells can be derived from embryonic or adult tissues and are thus termed embryonic stem cells (ESCs) or somatic stem cells (SSCs), respectively. With regard to biological competence, ESCs can differentiate and contribute to any of the three germ layers  and are therefore pluripotent , whereasSelf-renewing mouse pluripotent ESCs (mESCs) were first derived from the inner cell mass (ICM) of pre-implantation blastocysts in the early 1980s . The estThe ability to proliferate without limit and to differentiate towards any cell type of the body render ESCs as a limitless supply of specific cell types for basic research and transplantation therapies for many conditions, ranging from heart disease to neurodegenerative disorders. Furthermore, ESCs harbor an important potential not only for basic and translational research, but also for the improvement of pre-clinical safety and efficacy testing of novel pharmaceutical drugs.Despite the vast and wondrous application potential of human ESCs (hESCs), their use remains very controversial due to their embryonic provenance. Moreover, histocompatibility issues are a major hindrance to the application of hESCs in regenerative medicine and cell transplantation therapies .In 2006, a ground-breaking study by Shinya Yamanaka demonstrated that somatic cells can be reprogrammed to a pluripotent “ESC-like” state by overexpressing the pluripotency-inducing transcription factors OCT4, SOX2, c-MYC and KLF4. As a result, induced pluripotent stem cells (iPSCs) were born . One greAltogether, the potential and impact of ESCs and iPSCs in basic and translational/clinical research are considerable. The possibilities for their application have been increasing steadily, indicating their full potential is yet to be known. Nevertheless, our understanding of the key pathways that rule self-renewal and differentiation of pluripotent stem cells remains far from complete. The signaling pathways and transcription factors that control early developmental stages are central in the regulation of the pluripotent state of ESCs and iPSCs. Therefore, it is not surprising that key features of the pluripotent state are governed by pathways such as leukemia inhibitory factor (LIF), sonic hedgehog, fibroblast growth factor (FGF), Nodal/ bone morphogenetic protein (BMP) and Wnt . Indeed,In this review, we will focus on the biological functions of the canonical Wnt pathway during early mammalian development, as well as its indispensable function on the regulation of ESC self-renewal, cell cycle and somatic cell reprogramming.Wnt signaling is an evolutionarily-conserved pathway, which regulates multiple cellular processes including proliferation, cell polarity, stem cell self-renewal, differentiation and organogenesis during both embryogenesis and adult tissue homeostasis . Its derDrosophila melanogaster, and it was named Wingless (Wg) due to its role as an essential morphogen for wing development ..99].As such, the short G1 phase has been thought to serve as a pluripotency protective mechanism, which narrows the time window during which ES cells can be coerced to differentiate. Additionally, a significant lengthening of the cell cycle accompanies differentiation. Specifically, the G1 phase becomes enlarged as the once pluripotent cells come under regulation of the p53 and Rb-dependent cell cycle checkpoints. Recently, it has been demonstrated that stem cells show a certain level of specificity as to which differentiation cues are permitted in the early or late G1 phase. Specifically, human ESCs seem to only commit to neuroectoderm differentiation while in the late G1 phase, whereas commitment towards the endoderm cell fate is permitted during the early G1 phase . This dic-Myc in mouse embryonic stem cells maintained in 2i conditions has been shown to induce a phenotype resembling the naturally-occurring embryonic diapause, which can occur during development of several invertebrate and mammalian species, including mice, as a strategy to improve reproductive fitness. Importantly, c-MYC depletion-induced arrest can be rescued without loss of pluripotency by re-expression of exogenous c-MYC or by withdrawal of pharmacological inhibition of the transcription factor [Several recent studies have shown that in certain conditions, a lengthening of the cell cycle does not necessarily correlate with a loss of potency. Deletion of n factor .Interestingly, a very recent study from Huurne et al. may have induced a tectonic shift in our understanding of the regulation of the pluripotent cell cycle by challenging the notion that mESC lack typical G1 regulation, making use of bromodeoxyuridine and propidium iodide stainings and flow cytometric analysis to determine cell cycle distribution of mESC maintained in 2i and serum conditions. Their flow cytometry results point out important differences in the cell cycle profile of both culture conditions: in serum mESCs, the number of cells found in S phase is considerably higher than that of 2i mESCs, while the number of G1 phase cells is increased in 2i mESCs. Additionally, making use of the FUCCI reporter, Huurne et al. determined the G1 phase of 2i cells to be “markedly longer” than serum mESCs, along with a shorter S and G2 phase .A short G1 phase may not be an inherent feature of stem cell biology, but rather a characteristic that arises from culture conditions and their similarity with different stages of embryonic development. Huurne further contributed to understanding the mechanisms of differential regulation of the cell cycle in 2i and serum conditions by demonstrating that ERK pathway activity induces a decrease in hypo-phosphorylated RB and loss of the G1 restriction point culminating in a condensation of the G1 phase. Ink4/Arf tumor suppressor locus to drive expression of p16Ink4a and p19Arf has been described in the same study. Moreover, Wnt activity in mESCs was shown to result in increased nuclear p53 and depressed c-MYC expression [The Wnt signaling pathway is known to support maintenance of pluripotency and to be importantly involved in controlling somatic cell reprogramming ,103. It pression . These rpression .Much is yet to be known about the pluripotent cell cycle and its functions. It is, however, clear that it is a highly dynamic cellular function, which, like an octopus, reaches far and wide to exert some level of control over each and every aspect of the pluripotent cell’s life. Wnt signaling seems to also have a dynamic role, and the outcomes of its activation may depend not only on the strength of its signal , but also on the developmental/pluripotency state of the cell  . RegardlThe technology to induce murine and human somatic cells to revert to a pluripotent state (iPSCs) by way of transduction of several transcription factors  has created the possibility of generating individualized pluripotent cells for patient-specific therapy or drug screening ,105. RepThe current reprogramming methods remain very inefficient and yield a high number of cells that fail to undergo complete reprogramming ,109. ThuGrowth factors and signaling pathways are the main external players in maintaining self-renewal and potency of pluripotent cells in vitro . As descPORCN gene and therefore defective in Wnt secretion, fail to reprogram to an induced pluripotent state. Defects in reprogramming of FDH fibroblasts can be rescued by ectopic activation of Wnt signaling or by overexpression of PORCN [Nevertheless, several recent studies have focused their attention on the central importance of the Wnt/β-catenin pathway as both an essential element to achieve reprogramming and on how its modulation affects the efficiency of the reprogramming process itself. Indeed, canonical Wnt signaling has been shown to be an important player in both fusion-mediated and factor-induced reprogramming in mouse and human somatic cells ,110 Fig. The useof PORCN . In order to investigate which specific Wnt ligand may be directly implicated during somatic reprogramming, Kimura et al. performed a population expression profile of cells enriched for undergoing reprogramming . Their rDkk1 promoter and reduces its expression, which generates an increase in the levels of β-catenin and activation of its target genes. The crosstalk between NANOG and the Wnt pathway is essential for reprogramming since inhibition of the Wnt pathway in NANOG-overexpressing cells impairs the generation of reprogrammed cells [Wnt pathway activation is one of the main factors implicated in maintaining mouse and human naive ESC pluripotency and therefore in the expression of the transcriptional pluripotent network. However, it has been shown that a reverse crosstalk from the pluripotency network towards the Wnt pathway exists, as well. NANOG, one of the main components of the ESC transcriptional core  and whosed cells .In addition to Wnt proteins, timing and levels of Wnt activation during mouse somatic cell reprogramming influence the appropriate induction of the pluripotent state. Two independent reports have shown that, in order to achieve reprogramming, Wnt signaling must be repressed during the earliest stages of the process, while its activation is a requirement during the late steps of reprogramming ,112. IntThe central effect of β-catenin stabilization on rescuing the repressive influence of TCF3 on the self-renewal capacity of mouse ESC has stimulated the interest in studying the role of TCF/LEF factors in somatic reprogramming. Consequently, it has been demonstrated that in pluripotent stem cells, TCF1 and LEF1 act mainly as Wnt transcriptional activators, while TCF3 and TCF4 act as transcriptional repressors . In accoc-Myc, is one of the four Yamanaka factors capable of inducing reprogramming [c-Myc expression [c-Myc along reprogramming events.However, the mechanism or the identity of the downstream reprogrammer(s) that is (are) activated by β-catenin/TCF signaling is still not entirely known. Increased levels of β-catenin by addition of CHIR99021 during induction of pluripotent stem cells or by overexpression of β-catenin in cell-fusion-induced reprogramming reduce cell proliferation, ruling out a pro-proliferative function of the Wnt pathway during somatic cell reprogramming ,119. Actgramming . Howeverpression ,110,119,Nanog, Esrrb, Dppa3 and Zpf42, among others [TCF3 has been shown to repress the expression of several pluripotency-related genes. The absence of TCF3 opens the chromatin by increasing the levels of acetylated H3 and Histone 3 Lysine 4 trimethylation in both pluripotent cells and somatic cells undergoing reprogramming ,122. Furg others ,81,112. In conclusion, several reports have already shown that even though Wnt/β-catenin pathway activation itself does not induce reprogramming to pluripotency; its endogenous autocrine–paracrine activity in somatic cells is necessary and enough to sustain cell fusion-dependent or direct reprogramming. External abolishment of somatic Wnt activity abrogates cellular reprogramming, and vice versa, activation of Wnt pathway increases reprogramming efficiency.Axin2 can be detected in the morula or pre-implantation blastocyst, transcriptional activity of the Wnt pathway seems to be dispensable for development until the blastocyst stage. This is demonstrated by the absence of developmental defects in β-catenin knockout (KO) pre-implantation blastocysts. To solve this paradox, further investigations should ensue, making use of embryonic stem cells.The canonical Wnt pathway plays major and critical roles during embryonic development of vertebrate organisms. An intricate control over its activity is required for the correct unfolding of the events that encompass the very beginning of life. One such role is governing the correct specification of the different cellular lineages, which will give rise to the different tissues and organs of the adult organism. Specifically, its activation drives differentiation and commitment to the meso-endodermal lineage during the post-implantation blastocyst stage. However, whether the Wnt signaling pathway also regulates the specification of extraembryonic tissues remains to be further studied. The importance of the Wnt pathway during the earliest stages of embryonic development remains unclear. Although the presence of β-catenin in the nucleus and expression of the Wnt target gene Wnt signaling is essential for the maintenance of the homogeneous pluripotent naive state in ESCs. However, β-catenin KO ESCs maintain naive pluripotency in vitro as long as they are cultured in the presence of LIF. However, when deprived of this factor, β-catenin null cells rapidly exit naive pluripotency. On the other hand, inducing Wnt signaling in the absence of LIF supports the maintenance of the naive pluripotent state. As such, the Wnt and LIF pathways seem apparently redundant in the maintenance of naive pluripotency, and further studies should be performed in order to understand whether other pathways could be involved in the maintenance of in vitro naive pluripotency and early embryonic development in β-catenin KO mouse models.Due to their ability to replicate indefinitely and their capacity to differentiate towards all embryonic lineages, iPSCs and ESCs hold a valuable potential for application in regenerative medicine. However, among other reasons, their use in regenerative therapies is not possible at the moment due to unamenable concerns regarding the tumor initiating capacity of pluripotent stem cells. The Wnt pathway has been recently shown to regulate both pluripotency and proliferation in mESCs. These cellular functions are seemingly regulated by different TCF/LEF Wnt-dependent transcription factors that govern discrete gene programs. This non-redundant nature of TCF/LEF transcription factors could potentially be exploited in order to achieve the modulation of particular beneficial Wnt-dependent cellular outcomes without concomitant modulation of undesired functions, i.e. maintaining iPSC/ESC self-renewal without oncogenic potential. As such, further investigation into the roles of Wnt signaling in the pluripotent cell cycle needs to be performed in the hopes of obtaining safer pluripotent stem cells, finally unlocking the grail of regenerative medicine.


The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types  Adenoviruses (Ad) are the most commonly used vectors in human clinical trials and approximately 75% of these trials are directed to cancer treatment . Replicacis for viral DNA replication. A cis-acting packaging signal, required for encapsidation of the genome, is located near the left ITR. The Ad genome can be roughly divided into two sets of genes: the early region genes  that are expressed before DNA replication and the late region genes (L1 to L5) that are expressed to high levels after DNA replication. First generation Ad (FGAd) vectors typically have foreign DNA inserted in place of early region 1 (E1). E1-deleted vectors are replication deficient and are propagated in E1 complementing cells such as 293 [in vivo because of acute and chronic toxicity secondary to low levels of viral gene expression from the vector backbone [Ad have a non-enveloped icosahedral capsid containing a linear double-stranded DNA genome of ~30–40 kb. Up to now, at least 55 different human Ad (species A–G) have been reported and they are generally associated in immunocompetent patients with subclinical or self-limiting diseases of the airways, eyes, and gastrointestinal tract. Ad vectors most commonly used for clinical trials and experimental gene therapy applications, including helper-dependent adenoviral (HDAd) vectors, are derived from serotypes 2 (Ad2) and 5 (Ad5) of subgroup C . Moreover, the liver is a very attractive target for gene therapy because it is the affected organ in several genetic and acquired diseases and it can be used as a factory organ for systemic delivery through the circulation of vector-encoded therapeutic proteins. Inherited liver diseases are logical disease targets but several studies have also uncovered the opportunity to treat non-Mendelian diseases by liver-directed gene therapy. Expressing specific genes into hepatocytes can induce immune tolerance towards antigens that may be exploited for treatment of the deleterious consequences of immune response  or autoimmune disorders ,19,20. FSeveral examples of liver-directed gene therapy using HDAd in monogenic disease animal models have clearly shown long term transgene expression and phenotypic correction in the absence of chronic toxicity, thus supporting the potential of HDAd for clinical applications ,23,24,25The activation of the acute inflammatory response by systemic Ad injection is multifactorial and is observed in both rodents and nonhuman primates given comparable (on a per kg basis) systemic high doses of Ad vectors. However, mice are much more tolerant than nonhuman primates to high vector doses ,35,40. Dv integrins) [in vitro infection, it does not apply to in vivo infection, at least in the liver. Ad5-mediated hepatocyte transduction occurs independently of viral association with CAR and integrins [in vivo: mutations in the hexon protein or FX ablation by warfarin treatment [In recent years, new and important knowledge has been gained on Ad-host interactions and their role in activation of the innate immunity. According to the early model of the 1990s, Ad5 infection is dependent upon receptors for attachment  and entry (αtegrins) ,42,43. Wntegrins ,45. Follntegrins ,47,48,49ntegrins   ,76,77,78The observation that administration of polyinosine, as well as other polyanionic ligands, into mice prior to Ad intravenous injection drastically reduces Ad accumulation in Kupffer cells and increases hepatocyte gene transfer ,80, has The presence of endothelium fenestrations in the liver permits transduction of hepatocytes by Ad vectors whereas other tissues that lack such fenestrations are poorly transduced ,85. Neveex vivo [Systemic administration of high doses of Ad vectors likely results in widespread transduction of a large number of various extrahepatic cell types  which are also important barriers to efficient hepatocyte transduction. Over 90% of Ad vectors bind to human erythrocytes ex vivo  through ex vivo , in nonhex vivo ,91, and ex vivo .Given the potential of HDAd vectors for gene therapy, several groups have investigated various strategies to overcome the obstacle of acute toxicity. Because the severity of the acute response is dose-dependent and appears to correlate with extrahepatic systemic vector dissemination, one of these strategies aimed at targeting the vector to the liver thereby allowing the use of lower vector doses. This has been accomplished using physical approaches aiming at preferential hepatocyte transduction. One such strategy was to deliver the HDAd into surgically isolated livers to limit systemic exposure and resulted in greater hepatocyte transduction compared to intravenous injection and no chronic toxicity in nonhuman primates . A folloTo overcome the limitations due to the invasiveness of the surgical delivery method, a minimally invasive, percutaneous balloon occlusion catheter-based method was then developed to achieve preferential hepatocyte transduction. In this method, a sausage-shaped balloon is inflated in the inferior vena cava to occlude the hepatic venous outflow and the HDAd is injected directly into the liver via the hepatic artery. This vector delivery method resulted in high levels of transgene expression compared to peripheral intravenous injection in nonhuman primates using clinically relevant low doses ,29. FurtIn another approach, effective dose reduction was achieved by direct injections of HDAd into the liver parenchyma that resulted in improved efficacy and reduced toxicity. Direct vector injections into liver parenchyma is a relatively simple and flexible technique that is similar to the procedure accomplished routinely for liver biopsies and is performed in humans for treatment of hepatic metastases . HoweverKupffer cell depletion by pre-treatment with gadolinium chloride, dichloromethylene biphosponate, or clodronate lyposomes prior to vector injection result in enhanced hepatocyte transduction ,100,101,Genetic or chemical modifications or a combination of these changes to the viral capsid is another strategy that has been investigated to evade Kupffer cell uptake. Given that Ad-Kupffer cell interaction is mediated by the hypervariable loops of the virus hexon protein, Ad vectors have been genetically modified to disrupt such interactions. A chimeric vector in which the hypervariable region of Ad5 is replaced with that of Ad6 showed reduced Kupffer cell uptake, higher levels of liver transduction, and reduced hepatotoxicity compared to Ad5 vector . The hypIn addition, “masking” the viral capsid has also been reported to attenuate the severity of the innate inflammatory response. Systemic administration of non-specific PEGylated Ad into mice resulted in 50%–70% reduction of serum IL-6 compared to unPEGylated vector without compromising hepatic transduction ,110,111.11 vp/kg of a HDAd expressing factor VIII (FVIII) [There was a single case of intravascular administration of an HDAd vector into a human patient; however, this study has not been published in a peer-reviewed format and much of the details are not available. From what is known, a hemophilia A patient received by intravenous injection 4.3 × 10 (FVIII) . This su (FVIII) . ex vivo clinical trial to treat anemia in patients with chronic kidney failure [ex vivo with an HDAd expressing erythropoietin (EPO), and implanted under local anesthesia in the subcutaneous tissue in an autologous manner. To achieve the pre-determined blood levels of EPO, a precise number of HDAd transduced cells was implanted. There were no adverse events in this trial and hemoglobin levels were sustained for up to one year after a single treatment with the HDAd transduced cells [HDAd has recently been used in an  failure . In thised cells . This trHDAd are attractive vectors for gene therapy particularly given their ability to drive long-term transgene expression with no chronic toxicity and with low risk of insertional carcinogenesis. To date, in small and large models, including nonhuman primates, HDAd transduced hepatocytes  are not eliminated by the immune system and result in multi-year transgene expression. Nevertheless, whether this holds true for humans is not known at the present time, particularly in consideration of the outcomes of the AAV clinical trials for hemophilia B that resulted in a CTL immune response against AAV-transduced hepatocytes ,116.The dose-dependent activation of the innate inflammatory response by viral capsids remains an important concern for those applications requiring systemic intravenous injection of high vector dose to achieve clinically relevant benefits. A significant amount of data on vector biodistribution, toxicity, and efficacy has been generated through studies performed in small animal models and clearly these studies have led to increased knowledge in the field. However, the translational relevance of several of these findings requires further validation in larger animal models that more closely predict human outcomes. The multitude of host factors interacting with Ad particles has highlighted a series of unforeseen and intriguing mechanisms that illustrate the complexity of Ad vector-host interactions. Importantly, these studies have identified potential molecules that could be targeted to avoid vector scavenging and degradation by macrophages with the goal of achieving efficient gene transfer to hepatocytes or tumors. Pharmacological approaches directed towards these targets in combination with advancements in vector targeting or de-targeting, or with physical methods, such as balloon catheter-assisted delivery, would result in more efficient and safer gene delivery with potential for clinical applications.


Efforts have been made by the transplant community to expand the deceased donor pool in paediatric liver transplantation (LT). The growing experience on donation after circulatory death (DCD) for adult LT has encouraged its use also in children, albeit in selective cases, opening new perspectives for paediatric patients. Even though there has recently been a slight increase in the number of DCD livers transplanted in children, with satisfactory graft and patient outcomes, the use of DCD grafts in paediatric recipients is still controversial due to morbid outcomes associated with DCD grafts. In this context, recent advances in the optimization of donor support by extracorporeal membrane oxygenation and in the graft preservation by liver machine perfusion could find application in order to expand the donor pool in paediatric LT. In the present study we review the current literature on DCD liver grafts transplanted in children and on the use of extracorporeal donor support and liver perfusion machines in paediatrics, with the aim of defining the current status and future perspectives of paediatric LT.  Organ shortage is the main challenge in liver transplantation (LT) and the discrepancy between the number of waiting list patients and potential donor offers is greater in the paediatric field due to the scarcity of size-matched donors/grafts. The paediatric waiting list mortality rate nowadays is about 10%; however mortality appears to be highest in children younger than 6 years . FurtherIn the last few decades, there has been a great interest in donation after circulatory death (DCD), the use of which has rapidly increased . DCD donAlthough the use of DCD livers in adult recipients is increasing, paediatric experience is limited. The use of DCD grafts in paediatric candidates for LT remains controversial and mainly single-center experiences have been reported. Nevertheless, paediatric DCD donors could constitute an important portion of the paediatric donor pool, since withdrawal of life-sustaining therapy accounts for 30–65% of deaths of patients in paediatric intensive therapy units (ITU) . MoreoveRecent advances in the optimization of donor stability with extracorporeal membrane oxygenation (ECMO) and the improvement of organ preservation with perfusion machine might also start a new era in graft availability for children, as these technological advances may help to minimize the morbid complications associated with conventional DCD grafts as well as to expand the safe utilization of extended-criteria DBD. Here we have reviewed the current literature on DCD liver grafts transplanted in children and the use of extracorporeal donor support and liver perfusion machine in paediatric donors. Our aim was to define the current status and future perspectives of paediatric LT. paediatric transplantation, liver transplantation in children, donation after cardiac death, donation after circulatory death, non–heart-beating donors, liver splitting, partial liver graft, reduced liver graft, extracorporeal donor support, extracorporeal membrane oxygenation, organ preservation, normothermic regional perfusion, normothermic machine perfusion, hypothermic machine perfusion, and sub-normothermic machine perfusion. All papers published in English between January 1995 and October 2017 and focusing on the following topics were retrieved: (1) donation after circulatory death used in paediatric LT (recipient age defined <18 years); (2) extracorporeal donor support used for paediatric LT; (3) liver perfusion machine used in paediatric LT; (4) splitting liver during perfusion machine. Studies of nonliver organs were excluded, as were studies where ECMO was used as a support for recipients awaiting LT or after transplantation. The selected papers included randomized controlled trials, conference consensus reports, meta-analyses, review articles, and pilot studies and case reports. Given the heterogeneity of the published papers and data, the present paper is in the form of a conventional review where important aspects of the aforementioned studies are highlighted.Two authors of this manuscript  independently searched the PUBMED/MEDLINE database with the following key words:We identified 48 relevant records on the use of DCD grafts in children, 41 of which were excluded as ineligible for the following reasons: 19 studies included non-LT from DCD donor to paediatric recipients, 18 reports focused on the ethical aspects and the implementation policy of paediatric DCD donation, and 4 studies included LT from paediatric DCD donor to adult recipients; the remaining 7 records, focusing on DCD grafts used in paediatric LT recipients, were included in the review –24.Regarding the use of ECMO in paediatric LT, 19 records were identified, 17 of which were excluded because ECMO was used as bridge therapy while awaiting LT or as a support for complications after LT . Therefore, only 2 records on the use of ECMO in liver donors for paediatric recipients were eligible , 26.Out of 10 papers selected on the use of liver machine perfusion in human LT, none included paediatric recipients; therefore all of them were excluded. Finally, since split liver represents the most common type of graft used in paediatric LT, we considered 2 reports on the split liver procedure during machine perfusion, even if the grafts were not transplanted, in order to open new prospective on the machine perfusion applicability in children , 28.Donation after circulatory death (DCD) involves donors with permanent absence of respiration and circulation, which have been defined by the Maastricht classification of uncontrolled DCD donors  and controlled DCD donors  . Due to Despite the growing experience with DCD grafts in adult LT in recent years, the limitation of the use of DCD livers in paediatric LT recipients might be related to the inferior graft outcomes and higher complication rates reported in adults as well as the scarcity of DCD donors with adequate size-match. In the United Network of Organ Sharing (UNOS) database, the use of paediatric DCD donors represented 0.4% of the whole paediatric donor pool in 1995–2005, this figure increasing to 8–10% in 2006–2016 , 1817, 1p = 0.3). The authors concluded that paediatric recipients of DCD organs appeared to have results similar to those of patients transplanted with DBD organs, but the small size of the population was insufficient to draw solid conclusions.In 2006, a UNOS report on DCD paediatric transplantation described the largest experience till then of DCD liver grafts in children . Out of Later, the Birmingham group described in different series 7 children transplanted with DCD liver grafts from paediatric and young adult donors –22. In 2 in vivo into the biliary tree during organ procurement and tissue plasminogen activator was infused into the donor hepatic artery at the back table. These data suggest that the good outcomes were also possibly related to the large experience of the center in adult DCD transplantation, leading to technical refinements of DCD organ retrieval and optimization of logistic planning to minimize the cold ischemia time.In 2010, Hong et al. from the University of California Los Angeles (UCLA) compared the long-term outcome of 7 children transplanted with DCD paediatric livers to that of 21 children transplanted with DBD paediatric grafts . At 10-yFinally, a recent report from van Rijn et al. described the long-term outcome of 20 paediatric DCD liver grafts, 3 of which (15%) were transplanted in children . In thisAs recently Schlegel et al.  reportedIn summary, 50 DCD liver grafts transplanted in paediatric recipients have been reported so far. The DCD donors were all classified as Maastricht category III with a median donor age of 10 years (0.3 months–64 years) and a median donor WIT of 15 10–25) minutes. Notably, the donor WIT was defined in 5 reports –23, 25 a minutes.n = 1; 2%), ischemic cholangiopathy , anastomotic biliary stricture , hepatic artery thrombosis , and portal vein thrombosis . Despite the small numbers, altogether these findings suggest that children transplanted with DCD livers present satisfactory graft and patient outcomes at medium-to-long-term follow-up, with different features when compared to adult DCD LT. In particular, the incidence of ischemic cholangiopathy seems to be significantly lower in children than in adult DCD recipients, which may reflect the superselective nature of the grafts [Except for one (2%) child who died, all recipients were reported to be alive with a median follow-up of 4.7 years (1.7 months–10 years). The retransplantation rate was 6%. A total of 8 (16%) recipients presented postoperative complications including PNF  is used to provide cardiac and pulmonary support when heart perfusion and lung oxygenation are inadequate to sustain life .The use of ECMO in organ donation has been implemented in the last decades worldwide, and it is currently used in hemodynamically unstable DBD donors or in DCD donors to improve organ preservation for transplantation and to expand the donor pool , 38. AftIn paediatrics, the use of grafts from donors supported by ECMO prior to organ procurement has been reported primarily in kidney transplantation , 47, whiRecently, Assalino et al. reported two cases of in situ liver splitting in DBD young donors  in whom refractory cardiac arrest and hemodynamic instability were supported by venoarterial ECMO; after the in situ liver splitting, two children were transplanted with a left lateral segment graft and two adults with an extended-right lobe graft with uneventful postoperative course . AlthougIn recent years, there has been increasing interest in the possible use of liver machine perfusion to minimize the effects of organ preservation and to resuscitate suboptimal donor grafts. Recent trials have shown that normothermic or hypothermic machine perfusion of liver grafts is associated with short-term superiority over cold storage in terms of early biochemical liver function (lower peak serum aspartate aminotransferase), decrease of early graft dysfunction, and reduced rate of discarded organ , 48, 49.Recently, normothermic machine perfusion (NMP) showed a useful role in reducing ischemia/reperfusion liver damage and in “resuscitating” organs from extended-criteria donors such as DCD . NMP in The application of extracorporeal liver perfusion in children has not yet been explored probably because paediatric LT recipients are usually transplanted with “optimal grafts” deriving from (a) split livers, which are normally obtained from “ideal donors”  or (b) whole grafts harvested from young size-matched donors. Therefore, the use of machine perfusion to improve the “graft quality” might not be considered necessary in the paediatric transplantation setting.So far, the largest application of NMP is in DCD grafts, but no cases of DCD livers perfused on NMP have been reported in children. Thus, what is not documented in the literature is the potential paediatric donor offers as DCD grafts, even from very young and small donors, whose use could be increased and safer preservation of these organs could be optimized by machine perfusion.Recently, there have been some reports on the technical feasibility of splitting during liver machine perfusion , 28. BroLater, four pig livers were split during NMP in order to verify the effectiveness and stability of NMP system . During These two reports proved the technical feasibility of splitting during liver machine perfusion and could lead to new perspectives in the application of NMP in paediatric LT. Though not proven by clinical outcomes, these options open up new possibilities for paediatric patients where even marginal liver grafts that are well preserved could be used for small children. How far could we go? Potentially, splitting the liver during NMP could combine the advantages of in situ and ex situ splitting techniques by reducing prolonged CIT and minimizing the ischemia-reperfusion injury to the partial grafts, influencing split liver outcomes . FurtherGreat efforts have been made by the transplant community to expand the donor pool for paediatric LT. The recent advances in transplantation of extended-donor criteria grafts in adult recipients have led to the selective use of these grafts also in children, opening new perspectives for paediatric LT. In the last few years, an increasing number of DCD grafts have been transplanted in children, with satisfactory graft and patient outcomes at medium and long terms and a low incidence of morbidity. A careful donor selection and short donor WIT and CIT are all mandatory for good outcomes of DCD grafts in paediatric candidates, and any additional donor risk factor should be avoided. The use of DCD liver grafts in children could be increased by the donor support with ECMO to optimize the graft quality.In paediatric LT like for adults, graft preservation on machine perfusion could permit assessing the viability of the organ and reducing the ischemia-reperfusion injury. Moreover, the technical feasibility of splitting liver during NMP could lead to new applications: splitting livers during machine perfusion could increase the number of partial grafts available for paediatric recipients and change the logistics of splitting. The use of new technologies for donor optimization and graft preservation could find selected applications in paediatric LT, but much work still needs to be done.


Thus,it is imperative to recognize early risk factors for the development ofpostoperative pulmonary complications (PPC) and to focus on the adoption of measuresto prevent them from occurring.Among these measures, recent evidence points to some generally defined strategies,such as intraoperative protective mechanical ventilation, that may help minimize theoccurrence of PPC. Other methods include the rational use of the fraction ofinspired oxygen (FiO2), tidal volume (Vt) and positive end-expiratorypressure (PEEP).Postoperative pulmonary complications are an important cause of hospital morbidityand mortality and are known to be associated with longer hospital stays and higherlong-term mortality rates.is believed to be the best tool for the preoperative identification of patients atrisk of developing PPCs,4 g PPCs,4 .2 may lead to directpulmonary toxicity and the development of interstitial fibrosis, reabsorptionatelectasis and tracheobronchitis. In addition, hyperoxia is associated with increased productionof reactive oxygen species, which cause damage to cellular structures in animalmodels. In a randomizedclinical trial in patients undergoing abdominal surgery, the use of highFiO2 (80%) in the perioperative period was not associated with anincrease in the rates of pulmonary complications and hospital mortality compared toa low FiO2 group (30%), although mortality at 30 days was statisticallyhigher in the subgroup of patients who underwent colorectal surgery using a highFiO2 strategy.In humans, the indiscriminate use of high FiO2 >150mmHg), there were higher mortality rates in the intensive care unit, includingcases of circulatory shock, hepatic dysfunction and bacteremia, compared to a grouptreated with the conservative strategy (PaO2 70 - 100mmHg).Recently, data from a randomized clinical trial that assessed the role of hyperoxiain the outcomes of critically ill patients brought further controversy to thedeleterious effects of hyperoxia, although the study was terminated early due torecruitment difficulties. In a group of critically ill patients treated with ahyperoxic strategy  level above 92% isrecommended in non-obese surgical patients with healthy lungs undergoing openabdominal surgery.Thus, the lowest possible FiO although such relationships were not effectively demonstratedin a clinical trial using computed tomography of the chest. However, as was already robustlydemonstrated in critically ill patients, the use of low Vt values is associated with a reduction inlung injuries induced by mechanical ventilation and has been consistently describedas more appropriate for pulmonary protection during the intraoperativeperiod. This rationaleis based on three large randomized clinical trials that demonstrated thatintraoperative ventilation with a Vt of 6 - 8mL/kg PBW prevents the development ofPPC in patients undergoing elective surgery.-14Historically, high Vt values (up to 15mL/kg predicted body weight - PBW) were usedduring the anesthetic act in order to increase the end-expiratory lung volume and toreduce the incidence of atelectasis, Althoughthere are no randomized clinical trials evaluating this strategy in the context ofintraoperative surgical patients, a recent meta-analysis of individual data hasshown that intraoperative ventilation in patients undergoing elective surgeries withhigh distending pressure values, as well as changes in PEEP values that promote anincrease in distending pressure, is associated with the development ofPPCs.In addition, there is currently an association between higher distending pressurevalues (defined by the difference between the plateau pressure and the PEEP), whichcorrespond to the Vt values corrected for complacency of the respiratory system, andworse clinical outcomes in patients with acute respiratory distresssyndrome.-14 Further evidence is still needed to recommendintraoperative ventilation based on distending pressure. However, it is worth notingthat the potential deleterious effect of high distending pressures in this scenarioshould be avoided, suggesting that the plateau and PEEP pressures should beroutinely monitored during the intraoperative period.Thus, it is believed that patients with healthy lungs subjected to intraoperativeventilation during open abdominal surgery benefit from a Vt of 6 to 8mL/kgPBW. On the other hand, the strategy ofintraoperative permissive atelectasis, in which PEEP levels are kept low withoutalveolar recruitment maneuvers, aims to minimize stress on the pulmonaryepithelium.The use of PEEP during intraoperative mechanical ventilation is based on the idea ofmaintaining open alveoli during the respiratory cycle and on the opening ofatelectatic areas due to mechanical ventilation and the anesthetic act. However, a recently published randomized controlledtrial compared mechanical ventilation with a Vt of 8mL/kg PBW and a low PEEPstrategy (≤ 2cmH2O) without alveolar recruitment maneuvers to ahigh PEEP strategy (PEEP 12cmH2O) with alveolar recruitment maneuvers innon-obese patients undergoing elective open abdominal surgery. There were no notabledifferences in PPCs between the two groups. However, the high PEEP group had higherrates of intraoperative arterial hypotension and a greater need for vasoactive drugscompared to the low PEEP group.Currently, there is evidence that the use of PEEP can reduce atelectasis, improvecompliance without increasing dead space, and maintain the end expiratory volume inobese and non-obese patients under general anesthesia.2O without the use of alveolar recruitment maneuvers. In cases ofhypoxemia with no response to increased FiO2 and PEEP, alveolarrecruitment maneuvers based on the gradual increase in the Vt may beused.Thus, it is believed that patients with healthy lungs undergoing mechanicalventilation during open abdominal surgery benefit from PEEP values of up to2cmH2 values, appears to be the best strategy available forminimizing complications and improving clinical outcomes (The adoption of protective intraoperative ventilatory strategies is critical to thereduction of postoperative pulmonary complications. Currently, based on the bestscientific evidence available, the use of low Vt values, which is associated withlow PEEP and FiOoutcomes .


Dear Editor,In vitro studies have suggested that the envelope glycoprotein  of EBOV mediating virus entry may also directly contribute to the viral pathogenesis, as cell surface expression of GP1,2 can induce adherent cell rounding and detachment and sterically mask some cell surface molecules, such as β1-integrin  are required  is one of the most virulent pathogens to humans. Recently, the largest-ever outbreak of Ebola virus disease (EVD) in West Africa, 2013–2016, resulted in the unprecedented damage to human health and social economy. However, there is currently no licensed vaccine or antiviral available against EVD. Clinically, EBOV infection can result in exaggerated inflammatory responses and multiorgan damage, although the pathogenesis (including the inflammatory pathogenesis) and the viral virulence factor(s) involved in the excessive inflammation induction and viral pathogenicity are largely unclear. in vitro and in vivo, we designed an experiment of adenovirus-mediated gene delivery to cultured cells and mice  to utilize the N-terminal signal peptide (SP) and C-terminal TMD of TVA as previously described  were respectively injected into the adductor magnus muscles of BALB/c mice with local hair depilated. Intramuscular route was chosen for real-time in situ observation; additionally, the infection through broken skins and muscles is likely one of the main EBOV transmission routes  or 72 hpi (n = 6), the injected muscles and controls were dissected for further analyses. Firstly, EGFP signals could be detected in the muscles inoculated with viruses under an animal imaging system  and tissue damage, inflammation is also a significant pathologic effect mediated by Emuc, reminiscent of the excessive inflammation during EBOV infection in clinic  to activate inflammatory reaction of immune cells in vitro .in vivo gene transfer by adenoviral vectors may represent a safe and convenient experimental model which is especially valuable in the physiological function elucidation of the individual virulent proteins encoded by highly dangerous pathogens including EBOV and the development of antivirals specifically targeting the certain virulent proteins such as Emuc identified in this study. The full-length GP of EBOV has been generally used as the immunogen in vaccine researches Supplementary material 2 (PDF 293 kb)Below is the link to the electronic supplementary material.