Month: July 2020

The intestinal microflora plays an important not only in establishing immune tolerance but also in the development of inflammatory bowel disease and obesity. While studies of the microbiome have mostly focused on commensal bacteria, several species of fungi are also major constituents of the mammalian gastrointestinal system, with highest fungal concentrations in the colon. It is estimated that fungi are detectable in all gastrointestinal segments of about 70% of healthy adults, with Candida spp. being predominant. Candida albicans is a commensal fungus of the human gastro-intestinal tract, capable of causing life-threatening opportunistic fungal infections. C. albicans is considered opportunistic pathogen or a “pathobiont”, a resident microbe with pathogenic potential yet harmless under normal conditions. Gut-colonizing Candida can cause candidaemia, but mucosal damage and neutropenia are required for Candida dissemination from the colon. An increase in fungal load and Candida species was observed in patients suffering from Crohn’s disease. The C-type lectin Dectin-1, which recognizes cell wall b1,3- glucan, is the major receptor involved in antifungal immune responses. While essential during antifungal immune responses during systemic C. albicans infections in mice, lack of Dectin-1 does not affect gastrointestinal colonization by C. albicans. Dectin-1 knockout mice are more sensitive to chemically-induced colitis, presumably due to diminished immune surveillance of endogenous fungal species. The contribution of fungi to the development of IBD is further illustrated by human polymorphisms in Dectin-1 associated with ulcerative colitis and its signaling molecule CARD9. The latter has been associated with both UC and Crohn’s disease. Serum antibodies against yeast cell wall components are predictive for Crohn’s disease and ulcerative colitis. Influenza A virus is an enveloped virus containing eight negative-sense RNA gene segments. It is the causative agent of seasonal epidemics of respiratory illness as well as occasional pandemics, the most recent of which occurred in 2009. IAV is pleomorphic, producing virions of spherical and filamentous morphology. Strains that produce predominantly spherical or ovoid virions have typically been passaged many times within laboratory substrates, while filament-producing strains occur in primary or low passage isolates. Filaments are of variable length and can be up to 30 mm long. Herein, we NVP-BKM120 define filaments as any virion 300 nm in length or longer. Studies performed using reverse genetics systems have identified the M1 matrix protein as the major genetic determinant of virion morphology, however portions of the viral nucleoprotein as well as the cytoplasmic tails of the M2 ion channel, hemagglutinin and neuraminidase proteins have been shown to affect virion morphology as well. Early observations showed that the filamentous morphology is gradually lost upon continued passage in embryonated chicken eggs in favor of a more spherical morphology.

The newly identified GLI1/DNMTs axis set a bridge between Hh signaling pathway and epigenetics, which would help to elucidate the underling molecular mechanism in the development of PC, and may provide new therapeutic targets or biomarkers for earlier diagnosis. PE, together with PPE, are peculiar mycobacterial proteins over-represented in pathogenic mycobacterial species. Despite lacking typical secretion signals, both PE and PPE are secreted or located in the mycobacterial envelope. PE proteins can be divided into three distinct subfamilies, of which the most abundant is represented by PE_PGRS. All PE proteins are characterized by an N-terminal highly conserved domain of about 110 amino acids, named PE after the conserved signature motif Pro-Glu present near the N-terminus. In PE_PGRS proteins, the PE domain is followed by a C-terminal domain with a highly variable Gly-Ala rich sequence, which has been suggested to be involved in antigenic variation. In the other PE proteins the PE domain can be followed by an unrelated Cterminal domain, or the PE domain represents the entire protein. In the latter case the PE-encoding gene is usually in tandem with a PPE-encoding gene, and at least in one case the PE and PPE domains encoded by the coupled genes have been shown to interact. Hardly any of the about 100 PE proteins encoded by the Mycobacterium tuberculosis genome have been associated with a physiological function, with the exceptions of LipY, whose C-terminal domain shows lipase activity, PE_PGRS11, which was recently shown to encode a functional phosphoglycerate mutase and PE_PGRS33, which might be involved in induction of macrophage necrosis and apoptosis through interaction with Toll-like receptor 2. We recently showed that PE_PGRS33 is surface exposed when expressed in Mycobacterium smegmatis and that its PE domain contains the information necessary for the surface exposure. Chimeric proteins based on this PE domain were expressed on the M. smegmatis and Mycobacterium bovis BCG cell surface, and this feature was used to develop a surface delivery system to express heterologous antigen on M. bovis BCG envelope and increase its immunogenic potential. As already mentioned, although most PE and PPE proteins lack classical secretion signals, many are exported to the mycobacterial surface, suggesting the involvement of a novel secretory pathway. PLX-4720 Interestingly, in Mycobacterium marinum their secretion has recently been linked to ESX-5, a member of a novel family of secretion systems typical of mycobacteria. PE are exported mycobacterial proteins characterized by a well conserved N-terminal domain of about 100 amino acids that we recently showed to be required for their export. The lack in the PE domains of clear secretion signals led to the hypothesis that these proteins might be secreted through a new type of mycobacterial-specific secretion system. Indeed, it was recently showed that in M. marinum PE protein secretion is abrogated in mutants lacking the type VII secretion system ESX-5.

Different PE_PGRS mutant proteins were expressed in different model organisms to study their localization: i) M. smegmatis, whose chromosome encodes neither ESX-5 nor PE_PGRS proteins, but was previously shown to be able to export PE_PGRS33 and chimeric proteins based on its PE domain ; ii) M. marinum, whose chromosome encodes ESX-5 and many PE_PGRS proteins, but not an apparent PE_PGRS33 orthologue, and iii) M. bovis BCG and M. tuberculosis, whose chromosomes encodes both ESX-5 and PE_PGRS33. In different genetic backgrounds to investigate the conservation of the PE-exporting pathway. As expected, the protein lacking the entire PE domain was not able to translocate and was exclusively found in the bacterial cytoplasm. The same expression profile was found in the mutant protein missing the first 30 amino acids of the PE domain, suggesting that this portion of the protein is essential for the translocation. Surprisingly, mutation of the SF or of the PE (+)-JQ1 abmole bioscience conserved residues did not result in any clear phenotype with the exception of a strong instability of the resulting proteins in M. marinum. It is worth noting that PE_PGRS33 was found in Genapol extracts of M. marinum, but not in those of the other tested mycobacterial species suggesting a weaker association of this protein to the cell wall in this species. Of course it is still possible that using different experimental procedures PE_PGRS33 and/or some of the mutant protein might be extracted with this detergent even in M. smegmatis, M. tuberculosis or M. bovis BCG. These results confirm previous proteomics data on Genapol extracts from M. marinum as compared to M. tuberculosis or M. smegmatis. Moreover, the size of PE_PGRS33 in M. marinum Genapol extracts showed a molecular weight lower than that predicted for the entire protein, suggesting a maturation process, as we recently reported for LipY, another PE protein not belonging to the PE_PGRS family. In that case the protein was also processed in M. tuberculosis, but only when bacteria were grown inside macrophages, suggesting that still unknown factors only expressed during infection are required for full export and maturation of PE proteins in M. tuberculosis. For this reason, PE_PGRS33 might also be expected to be processed in M. tuberculosis during growth inside macrophages. In M. smegmatis some of the recombinant proteins showed multiple bands. However, since bands of the same size were present in both cytoplasmic and cell wall fractions, they were likely not due to processing during translocation but rather the result of degradation during cell lysis. The lack of processing observed in M. smegmatis might be due to the absence of ESX-5 in this species. Finally, the release of PE_PGRS33 was totally abrogated in the M. marinum ESX-5 mutant, confirming that PE protein translocation in M. marinum is dependent on this secretion system.

Therefore, it is desirable to develop natural drugs that have cholesterol-lowering effect comparable to statins, but could be tolerated well by the patients. Astragalus polysaccharides, an extract of Radix Astragali, is one of the main efficacious principles. APS-I and APSII are well known to be the major structural components of APS. APS-I consists of arabinose and glucose in a molar ratio of 1:3.45, while APS-II consists of rhamnose, arabinose and glucose in a molar ratio of 1:6.25:17.86. In China, APS has been extensively used to treat viral infection, acute myocarditis, glomerulonephritis, diabetes, tumor, and many other illnesses, with no toxic record in clinic. Previous reports indicated that some dietary soluble polysaccharides lower plasma cholesterol via reduction of intestinal cholesterol absorption or interference with the enterohepatic circulation of bile acids. However, the cholesterol-lowering effect of APS has not been well studied and underlying mechanisms behind are still elusive. Hence, we are here to determine how APS regulates plasma cholesterol and the cholesterol metabolic pathways in hyperlipidemia hamsters. Inhibiting HMG-CoA reductase activity and increasing hepatic LDLR expression are primary mechanisms of statins therapy for hyperlipidemia. Here, we identify a new cholesterol-lowering drug, APS, that effectively reduces plasma cholesterol to levels comparable to that of statins, but may work through different mechanisms. In our study, a 45.8% reduction of TC, 30% reduction of TG, and 47.4% reduction of LDL-C were achieved in hyperlipidemic hamsters after 3-month treatment of APS, similar to statins that lowered LDL-C by 56.5%. Previous study by Wu CZ et al. showed Ogi-Keishi-Gomotsu-To-Ka-Kojin, a Chinese medicine composite that contains a small amount of APS may also exert a hypolipidemic effect in hamsters. Yet they did not make clear which ingredient really works, as Panax ginseng, a crude drug of OKGK was also reported to show antihypercholesterolemic action in human, chicken and rat. The small intestines are implicated in regulating cholesterol homeostasis through affecting cholesterol absorption. An inhibition of intestinal absorption BI-D1870 S6 Kinase? inhibitor results in lower levels of circulating cholesterol. As ezetimibe and sitosterol, which act directly at the level of intestine, lower plasma cholesterol by inhibiting intestinal fractional cholesterol absorption. Our results revealed that APS led to a profound reduction in intestinal cholesterol absorption and a markedly accelerated rate of fecal neutral sterol excretion. Correlation between cholesterol absorption rates and plasma TC or LDL-C concentrations were also observed, suggesting a hitherto unknown mechanism that APS reduces plasma cholesterol by interfering with the intestinal cholesterol absorption.

In addition, it is advisable to test protein orthologs of LY2157299 different origin, including distantly related or unrelated species. At this point, analysis of the primary and secondary structure of both the encoding mRNA and the translated polypeptide may anticipate downstream problems. There is a plethora of freely available software and databases for identifying protein families and sequence conservation patterns, putative signal peptides, lipoboxes, glycosylation, phosphorylation and other posttranslational modifications, transmembrane domains, and unfolded/disordered regions. Protein location within the cell, i.e. cytoplasmic, periplasmic, or extracellular, provides an indication of the requirements of the protein for proper folding, including disulfide bond formation and the need for special chaperons in each cellular compartment. Further prediction of the secondary structure content can give clues about possible protein domains and motifs, a characterisation which may prove useful for chopping full-length multi-domain proteins into globular moieties. In general, successful recombinant protein expression depends on the removal of wild-type SP, lipoboxes, posttranslational signals, low-complexity regions, hydrophobic residues at the protein termini and membrane spanning regions, while conserving the boundaries of globular domains. In parallel, cDNA characterisation is important in designing the cloning strategy and identifying potential problems at the transcriptional and translational levels. Although these processes are affected by a number of exo- and endo-nucleases, the stability of the resulting mRNA is critical in protein expression experiments. mRNA can be protected by introducing sequences at the 59 untranslated regions and stem loop structures at the 39 UTRs. The GC base content may affect levels of expression and can be easily determined by sequence analysis software. Rare codons, especially consecutive ones, are frequently found in heterologous genes and may lead to translational errors due to ribosomal stalling. Such codon bias can be remedied by replacing selected codons or, if necessary, by overall gene optimisation using appropriate software. Once the above requirements are fulfilled, the gene can be inserted into the vector by directional cloning using restriction enzymes that do not cut within the gene sequence. Efficiency of translation termination can be increased by introducing strong stop codons at the end of the translated gene. No expression system is generic for all target proteins, so both bacterial and eukaryotic systems need to be explored. Escherichia coli provides the cheapest expression host, and it is the most widely used but its machinery is not as sophisticated as that of eukaryotic hosts, and it cannot always express well folded proteins of variable origin. Other alternatives often need to be tested, including bacterial systems such as Bacillus subtilis and more advanced eukaryotic systems such as the yeasts Pichia pastoris and Saccharomyces cerevisiae, the baculovirus expression system in insect cells, mammalian cells, or cell-free systems using prokaryotic extracts, which have highly variable costefficiency ratios.