Month: November 2020

Implantation of blastocysts is a well-organized process regulated by multiple growth factors and cytokines. We demonstrated the facilitatory effects of key growth factors to promote blastocyst outgrowth. The trophectoderm cells of blastocysts differentiate during embryonic development to form the invasive trophoblasts that mediate implantation of embryos into the uterine wall. The outgrowth of trophoblast cells from cultured blastocysts is believed to reflect the proper differentiation of the embryo, important for trophoblast invasion of the endometrial stroma during implantation in utero. Although blastocyst transfer is effective to select the best quality embryos with high implantation potential, overall implantation rate is,30%, suggesting human embryo transfer might be improved. Due to the low amount of liquid in the uterine cavity, factors included in the transfer media could be retained in high concentrations. Indeed, embryo transfer in medium containing hyaluronan is effective in improving implantation rates in patients with recurrent implantation failure.Hyaluronan is the major glycosaminoglycan present in LDK378 follicular, oviductal and uterine fluids and presumably promotes embryo–endometrial interactions during the initial phases of implantation. Because key growth factors promoted blastocyst outgrowth in vitro, future supplementation of embryo transfer media with key growth factors could also promote implantation during embryo transfer. Generating an autologous patient-specific embryonic stem cell line from SCNT embryos holds great promise for the treatment of degenerative human diseases. Successful derivation of embryonic stem cell lines following SCNT has been reported in mouse, rabbit, and non-human primates. However, the efficiency for the production of embryonic stem cell lines following SCNT is still low, particularly when adult somatic cells were used as the donor karyoplasts. Although many embryonic stem cell lines have been derived from surplus human blastocysts, no human cell-lines have been generated following SCNT. Among the many compounding factors, suboptimal in vitro culture condition contributes to the poor embryonic development of reconstructed embryos following SCNT. The present study represents an attempt to optimize the culture conditions for the development of human SCNT embryos. Although no blastocyst was obtained following fibroblast nuclear transfer, there was a trend to an augmented development of reconstructed embryos cultured with media containing autocrine/paracrine growth factors. Results from the present study provide the basis for future use of autocrine/paracrine factors to facilitate the derivation of patient-specific embryonic stem cells. In conclusion, the present study demonstrated the utility of growth factor supplementation for optimal human early embryo development and blastocyst outgrowth. The findings may allow the design of better conditions for individual human embryo cultures, for estimating their developmental potentials using secretory products, and for the inclusion of growth factors in embryo transfer media to promote implantation. Although the present experimental design is based on the supplementation of endogenous growth factors diluted during assisted reproductive procedures, future studies on the potential side effects of these paracrine/autocrine factors on chromosomal numbers, genomic integrity and epigenetic modifications are essential before clinical use. The gut microbiota performs necessary metabolic functions such as production of short chain fatty acids and synthesis of vitamins. It also influences the maturation of the immune system after birth, which is clearly illustrated in studies of germ-free animals. GF mice have fewer intestinal dendritic cells and mice with a restricted microbiota have less plasmacytoid DCs.

Another possibility was that a delay on the improvement of MELD-Na scores occurred after the LPS level decreased by the buffering of LPS-binding substance produced in the remission phase. As suggested by previous studies, we presume that higher LPS BMS-354825 inquirer levels were due to the production of LPS surpassed the phagocytic ability of kupffer cells rather than the decrease binding capacity of LPS-binding substances in acute phase. As disease progressed, the buffering system of LPS binding substances was activated and reached the peak level. Thus, it is possible that the liver injury induced by LPS was establish in the progression and peak phase with fluctuating lower levels of LPS-binding substances. Although our data was generated prospectively with control subjects, several limitations in this study are worth noting: 1) Patients included in the analysis were those who achieved spontaneous remission within 12 weeks on supportive care. The study result may not be applied to patients with prolonged peak phases and worsening disease activity or received additional intervention on top of supportive care; 2) Data analysis excluded patients expired during the study. Thus, the scale or levels of LPS in patients with severe liver necrosis remains uncertain. 3) For the feasibility of the study, we use healthy individuals as controls, which were less desirable than using CHB patients without ACHBLF. 4) A number of patients were not analyzed due to the intervention during the study period as required by the standard of care, such as antibiotic treatments for sepsis and antiviral use if patient consented to it. These patients were excluded because antibiotic may affect the gut flora and antiviral may influence the LPS level, which was reported by Koh et al in patients with CHB or hepatitis C during antiviral treatment. Due to the limited numbers of patients that were analyzed in this study, a future trial with the larger sample size is warranted to confirm our findings. In conclusion, the peak levels of LPS occurred during the severe necrosis phase in ACHBLF patients. The abnormal distributions of LPS levels among different phases were statistically significant in ACHBLF when compared to the controls. The highest MELD-Na mean scores in the ACHBLF group were observed in the peak phase and in parallel with the peak level of LPS. MELD-Na scores were correlated with LPS on progression phase and peak phase. Our data demonstrated the dynamic changes of LPS in ACHBLF as well as the relationship between LPS levels and the disease severity indicated by MELD-Na scores. These findings are important and may serve as the concept for the future development of therapeutic agents with the capacity to reduce LPS production or improve LPS clearance, which may have a significant impact on the clinical outcome of patients with ACHBLF. Early embryonic development from fertilization to implantation takes place in the oviduct and uterus without direct cell-to-cell contact with reproductive tract tissues until the final stage. During transit through oviduct and uterus, cells in preimplantation embryos undergo division, differentiation, and apoptosis. Early studies using animal models demonstrated enhanced embryonic development and survival when the volume of culture media was reduced or when early embryos were cultured in groups to increase concentrations of locally secreted factors. In addition, promotion of blastocyst formation and inhibition of apoptosis were found when culture media for animal embryos were supplemented with individual growth factors, including insulin-like growth factor-I, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, brain-derived growth factors, artemin, colony stimulating factor 1, glial cell-line derived neurotrophic factor.

The first prospective randomized control trial for antiviral use in ACHBLF patients was published by Grag et al in 2011, which demonstrated that tenofovir could safely reduce short term mortality in these patients. Our study was concluded prior to the first aforementioned randomized control trial. Although antiviral treatment may provide some short term survival benefits, many patients died despite the significant reduction of HBV DNA. Thus, our data remained relevant for the understanding of disease mechanism and the future development of novel intervention. Previous studies have demonstrated that endotoxinemia and delayed clearance of LPS in the circulation resulted in the development of ACLF in alcoholic liver disease. Although Han et al proposed that LPS played an important role in ACHBLF as a secondary liver injury on top of the CHB infection in animal models. The changes of LPS levels and their roles on disease severity in patients with ACHBLF were not fully explored. Our study showed that baseline LPS levels in ACHBLF patients did not differ from those in the healthy controls. However, significant elevation in LPS levels was observed in the peak phase of ACHBLF when compared to those in the progression or remission phase. The abnormal distributions of LPS levels among different phases were statistically significant in ACHBLF. In addition, the changes in LPS levels were correlated with MELD-Na scores in the progression and the peak phase. To our knowledge, this is by far the first study in which detailed the dynamic changes of LPS levels in different phases of ACHBLF. Since MELD-Na scores were correlated with LPS levels in the progression and the peak phase, our data pointed to the direction of the secondary injury from LPS in chronic liver disease leading to liver failure, which was proposed by Han et al. in the study from animal model. Further studies with histology correlation to LPS are needed to confirm if the severity of liver injury actually is directly correlated with LPS levels in ACHBLF patients. The findings in this study also implied a possible therapeutic intervention for ACHBLF by removing LPS from the serum. Several studies done by Adachi et al observed that there was a positive correlation between the occurrence of bacterial translocation from the gut to portal system and liver dysfunction in alcoholic hepatitis. Li et al demonstrated that elevation of endotoxin levels in the circulation from translocation of gut flora occurred during acute flares in patients with chronic hepatitis. It is possible that the elevation of LPS level in CHB patients was due to bacterial translocations from the gut to portal circulation GSK1120212 resulting in endotoxemia in the early phase of ACHBLF. On the other hand, the liver dysfunction in the early stage of ACHBLF probably further induced bacterial translocation from the gut leading to higher level of endotoxemia. In addition, in patients with liver dysfunction, the uptake of endotoxin by hepatic and Kupffer cells were compromised as compared to normal physical conditions, resulting in higher circulating levels of LPS. High levels of LPS then induced the aggravations of liver injury through the LPS-MD-2/TLR4/NF-kb signal pathway and further negatively impacted on KC and hepatic clearance of endotoxin. Thus, it is expected that the peak level of LPS was observed during the peak phase of ACHBLF. In our study, the dynamic changes of LPS were paralleled with the changes of TBil and MELD-Na in different phases of ACHBLF. The changes in LPS levels were correlated with MELD-Na scores in the progression and the peak phase, further indicated that the worsen disease severity was the result of LPS induced liver injury. The MELD-Na scores were not correlated with LPS levels in the remission phase.

This has likely occurred for CA3 in the present study. Obviously, the kidney has a major influence on urine content and approximately 70% of the proteins in urine originate from this organ. Since most proteins identified in this study are not liver-specific, we investigated whether potential kidney injury by APAP could have been a confounding factor. No signs of kidney injury were observed after APAP treatment as determined by histology and the absence of kidney injury markers. We, therefore, assume that the proteins found in urine after APAP-induced liver injury were not the result of kidney injury, but were released from liver into blood and subsequently excreted by the kidney. Most of the proteins identified in this study were only found in mice with high plasma ALT values and do not seem to be suitable as biomarker. Urinary CA3 and SOD1 showed a good correlation with plasma ALT and probably are also leakage markers of injured hepatocytes. The advantage over plasma ALT is that these markers can be measured in patients non-invasively. CaM proved to be the most promising biomarker, because the protein was found in urine of mice treated with a high dose of APAP that did not show elevated plasma ALT levels. This was also observed in urine samples of human APAP intoxicants. Although plasma ALT levels were not increased in these patients, plasma APAP concentrations were high enough that liver injury was a concern as indicated by the Rumack-Matthew normogram. These data indicate that CaM has potential as predictive biomarker for acute DILI and that a mechanism of hepatocyte release other than leakage may be involved. Most of the proteins that we detected in urine are involved in intracellular processes related to APAP-induced liver injury. These process are not specific to APAP and, accordingly, the biomarkers identified in this study are most likely not specific to APAP, but rather to acute hepatocellular injury. In line with this, urinary CaM concentration was also increased in human cases of DILI not caused by APAP. Since oxidative stress, mitochondrial damage and disrupted calcium homeostasis play an important role in APAP-mediated hepatotoxicity, it is not exceptional that we identified SOD1 and CaM as proteins with biomarker potential. The involvement of superoxide dismutases in APAP-induced liver injury has previously been demonstrated by the increased toxicity of APAP in mice with reduced activity of SOD2. The exact role of SOD1 in APAP-mediated hepatotoxicity remains controversial as both protective and damaging effects have been reported, but SOD1 nitration and reduction in SOD1 activity appear to be involved. A role for CaM in APAP-induced liver injury has not been clearly described; however, CaM does play a key role in maintaining intracellular calcium balance. Binding of NAPQI to mitochondrial proteins can cause mitochondrial permeability transition, after which mitochondrial Ca2+ is released into the cytosol. The cytosolic Ca2+ concentration is tightly regulated and any excess Ca2+ will be effluxed via the plasma membrane Ca2+ LEE011 1211441-98-3 ATPase transporter, using CaM as ultimate cofactor. However, the peroxynitrite formed during APAPinduced oxidative stress can oxidize specific methionine positions of CaM, after which CaM is no longer able to activate PMCA, which results in reduced excretion of cytosolic Ca2+. Previous studies showed decreased activity of PMCA during APAP-induced liver injury. With sustained high cytosolic Ca2+ concentrations, Ca2+ will be translocated to the nucleus by CaM, where it will cause DNA fragmentation and ultimately lead to cell death. CaM is thus involved in the initiating events of APAPinduced liver injury and may, therefore, be a potential early biomarker.

Under physiological conditions and they are capable of absorbing water, which is essential for optimal wound healing. The previously reported strong photobactericidal effect of O2-producing nanofiber materials led us to test a similar approach for the photoinactivation of viruses. We used polyomaviruses as models for non-enveloped viruses and baculoviruses as models for enveloped viruses. The capsid proteins of non-enveloped viruses and the envelope glycoproteins encoded by enveloped viruses enable the viruses to cross plasma membranes into cells and deliver their genetic material to the cell nucleus, resulting in viral gene expression. These proteins are responsible for cell surface receptor recognition and for subsequent interactions with cellular structures, leading to the disassembly of virus particles and the release of genetic information. Therefore, oxidative damage to virion surface proteins via photooxidation of readily oxidizable amino acids by O2 may be an effective way to prevent infection. Polyomaviruses, small tumorogenic non-enveloped DNA viruses, have a wide range of hosts, including humans. Two human polyomaviruses, JCV and BKV, which were discovered in 1971, cause progressive multifocal leukoencephalopathy and nephropathy, respectively, in immunosuppressed patients. Since 2007, six new human polyomaviruses have been identified. Merkel cell polyomavirus, which was described in 2008, is suspected to cause the majority of the cases of Merkel cell carcinoma, a rare but aggressive form of human skin cancer. Baculoviruses, which are large enveloped DNA viruses, are insect pathogens that have been widely used to produce recombinant proteins in cultured insect cells. Baculovirus envelope proteins are also able to mediate entry into human and other mammalian cells and, thus, facilitate the expression of recombinant genes under the transcriptional control of a mammalian promoter. The Autographa californica multiple nuclear polyhedrosis virus, which was used in our experiments, enters cells via a low pH-dependent CPI-613 endocytic pathway. During endocytosis, the major envelope glycoprotein GP64 mediates low pH-triggered membrane fusion. Singlet oxygen generated in close proximity to living eukaryotic or bacterial cells has been shown to have strong cytotoxic effects. It is well established that the main targets of O2 are cytoplasmic membrane proteins. Integrated proteins that cross the lipid bilayer and peripheral proteins associated with the cell surface have important, often indispensable physiological functions, and damaging these proteins quickly leads to cell death. Exposure of proteins to O2 can result in oxidation of side-chains, formation of crosslinked/aggregated species, protein unfolding or conformational changes. Aromatic amino acids and sulphur-containing amino acids are direct targets of O2. Other O2 targets include unsaturated lipids in the cytoplasmic membrane, which can be oxidized to form lipid hydroperoxides. Oxidation of cholesterol by O2 results in the formation of a number of readily distinguishable oxidation products, especially hydroperoxides. Enveloped viruses possess a lipid bilayer envelope derived from cellular membranes and embedded with viral proteins. These viral surface proteins are often glycosylated and play a crucial role in cell receptor recognition and viral entry into host cells. Therefore, enveloped viruses might be affected by O2 in a manner similar to bacterial and animal cells. Indeed, we showed that the baculoviruses, as representative enveloped viruses, were efficiently inactivated when applied to the surface of the nanofiber textiles doped with 1% TPP and exposed to visible light for 30 minutes. Similar effects may also be expected for other enveloped viruses.