Phytochemical profile,antimicrobial, and anti-quorum sensing properties of fruit stalks of Prunus avium L.

The aim of this study is to investigate the phytochemical contents and antibacterial properties of 2-year Prunus avium L. standard cultivars [Cristalina (Cr), 0900 Ziraat (Zr)] and to elucidate the mechanism of action of the extracts on the quorum sensing (QS) system by using homology modelling and molecular docking. Phenolic contents of methanol extract of Cr and Zr stalks were detected by HPLC. As a result, catechin hydrate (6364·67–8127·93 µg g⁻¹) and chlorogenic acid (998·81–1273·4 µg g⁻¹) were found to be the highest in stalk extracts in the two varieties in 2017. All extracts had inhibitory effect on Gram-positive bacteria. Stalk extract of Zr showed higher inhibition rate (86%) on swarming motility. Stalk samples of Zr collected in 2017 and 2018 also reduced biofilm formation by 75 and 73%, respectively. The computational analysis revealed that one of the major component of the extracts, chlorogenic acid, was able to bind to the QS system receptors, LasR, RhlR, and PqsR. Therefore, the mechanism of decreasing the production of virulence factors by the extracts might be through inhibiting these receptors and thus interfering with the QS system.     

The effect of bovine serum albumin and fetal calf serum on sperm quality,DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 10⁶ sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5 mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P < 0.01). The supplementation of BSA and FCS had a protective effect on motility (P < 0.05), plasma membrane integrity (P < 0.01) and acrosomal integrity (P < 0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P < 0.001). Although supplementation of BSA and FCS caused significant (P < 0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P < 0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.     

Identification of Molecular Markers Associated with Alteration of Receptor-Binding Specificity in a Novel Genotype of Highly Pathogenic Avian Influenza A(H5N1) Viruses Detected in Cambodia in 2013

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.     

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

Overexpression of SIRT1, a NAD⁺-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.     

Identification of Cellular Targets in Human Intrahepatic Cholangiocarcinoma Using Laser Microdissection and Accurate Mass and Time Tag Proteomics

Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.Cholangiocarcinoma (CCA),¹ which arises from the hepatic bile ducts, is the primary cancer accounting for ∼10–15% of all hepatobiliary malignancies. CCA is categorized by the International Classification of Diseases for Oncology into intrahepatic and extrahepatic forms, the latter including perihilar, hilar, and distal bile duct CCA. The global incidence rate of intrahepatic cholangiocarcinoma (ICC) has increased by 2–6% during recent decades, whereas the annual incidence rate of the more common, extrahepatic form has remained relatively stable (1, 2). The diagnosis of ICC remains particularly challenging because the disease can mimic metastasis to the liver or hepatocellular carcinoma (HCC). The only potentially curative treatment options available at present are surgical. Unfortunately, the majority of patients are diagnosed at an advanced, unresectable stage because of the initially silent clinical characteristics of this malignancy. The prognosis of ICC is therefore devastating with survival of less than 24 months following diagnosis. Although several risk factors have been reported as contributing to the development of the disease, most cases of ICC occur in the absence of known etiological factors (3–5). As a consequence, considerable efforts have been made to identify reliable markers to enable the early detection of biliary cancers and provide new insights into the pathogenesis of this deadly disease (6). Recent studies have focused on the cytokines and growth factors (7, 8) produced by CCA cells as well as on the proteomics analysis of serum and bile (9, 10). Follow-up studies are ongoing to determine the sensitivity and specificity of the markers that have emerged from these investigations.Tumor tissue is undeniably the most appropriate resource to investigate tumor-specific signals. Indeed, the determination of alterations to the protein profiles of ICC may offer opportunities to identify tumor-specific markers. To date, proteomics studies performed using tumor tissues have mainly tried to identify proteins with a differential expression between HCC and ICC to prevent the misclassification of these pathologies (11, 12). Furthermore, these studies, performed on total liver homogenates, were not appropriate to detect proteins with altered expression in tumorous cholangiocytes. Indeed, ICC tumor cells are essentially embedded in an abundant stroma containing inflammatory cells and fibroblasts, which may impair the detection of proteins displaying tumor-specific expression patterns (13). In summary, because no proteomics studies have so far been performed on isolated cholangiocytes, any proteome alterations in the setting of ICC remain a matter of speculation.Laser microdissection (LM) has emerged as a suitable tool to selectively extract cells of interest from their natural environment. This technology has been used extensively to profile global gene expression in purified tumor cells (14–16). It has also been used successfully in experiments designed to discover protein biomarkers through mass spectrometry-based proteomics studies (17–19). However, working with such small samples challenges conventional proteomics techniques in terms of sensitivity and the precision of quantitation.One particularly efficient method for proteome analysis is the so-called accurate mass and time (AMT) tag approach where high performance liquid chromatography (HPLC) and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FTMS) work in synergy to achieve a broad proteome coverage even for small sample amounts (20, 21). A recent report demonstrated the significant advantage of the AMT tag method over more conventional proteomics technologies in enabling significantly broader proteome coverage using the minute protein quantities available from tissue microdissection (22).In the present study, we used an AMT tag strategy to determine the differential protein profile between tumorous and non-tumorous microdissected cholangiocytes to define specific proteome alterations of human ICC. Among proteins with altered expression in ICC, we detected a panel of proteins conforming to potential tumorigenic pathways that could be candidates as therapeutic targets and tumor markers for this lethal disease.     

Glycine betaine: A widely reported osmolyte induces differential and selective conformational stability and enhances aggregation in some proteins in the presence of surfactants

In this study, we extensively report the effect of glycine betaine during the refolding of partially folded bovine α‐lactalbumin (α‐LA) in presence of hexadecyl trimethyl ammonium bromide (HTAB), and Ribonuclease A (RNAse A) in presence of sodium dodecyl sulfate (SDS) by different complementary biophysical, light scattering, and microscopic techniques. Though a substantial refolding/compaction was observed in both the studied proteins, the fluorescence studies contradicted the finding obtained from circular dichroism spectroscopy (CD) in case of α‐LA. CD stopped flow showed extensive presence of intermediates during the refolding of proteins which could potentially lead to aggregation. The aggregates as observed in dynamic light scattering (DLS), in α‐LA were massive as compared to RNAse A and was directly proportional to betaine concentration. The zeta potential confirmed that the aggregates are a direct manifestation of strong aggregating and/or immense preferential excluding tendency of GB and not because of charge neutralization; however a possible role of conformational change as observed in FTIR spectroscopy cannot be completely ruled out. In contrary though RNAse A showed a substantial refolding, the final state of the folded protein was significantly different from the native state. These findings for α‐LA and RNAse A were further supported by electron microscopic and thermodynamic studies. We thus propose that betaine has a strong macromolecular excluding tendency, primarily directed to shield the hydrophobic exposure either by refolding or aggregation, and depending on the hydrophobicity of the proteins, the functional restoration of the protein is manifested. © 2012 Wiley Periodicals, Inc. Biopolymers 97:933–949, 2012.     

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.