Cell Surface Characteristics Research Articles

Characterization of yeast cell surface displayed Lentinula edodes xylanase and its effects on the hydrolysis of wheat

The current study displayed a xylanase from Lentinula edodes on the surface of Pichia pastoris (sdLeXyn) and investigated its properties and effects on the wheat hydrolysis. Fluorescence microscope results showed that sdLeXyn was successfully anchored and displayed on the surface of P. pastoris X-33 cells. The highest activity of sdLeXyn was obtained at pH 3.0 and 50 °C. The sdLeXyn exhibited anti-high temperature property and showed broad temperature adaptability (>55% of the highest activity at 20–80 °C). The sdLeXyn was very stable at room temperature and could remain functionally stable at 50 °C for 3 h. The Km value was greater in sdLeXyn than that in free recombinant L. edodes xylanase. The sdLeXyn exhibited well resistance to Mn2+, Zn2+, Ca2+, Na+, Cu2+, Mg2+, K+, Ni2+ (1 mM and 5 mM) except Cu2+, which reduced the sdLeXyn activity by 54.5% at 5 mM dosage. The activity of sdLeXyn was increased by 42.6% by 5 mM Mn2+, 5 mM DTT, Trition X-100, and Tween 20 did not affect the activity of sdLeXyn, but SDS and EDTA slightly reduced it by 12.8% and 14.6%, respectively. The sdLeXyn could resist the degradation of pepsin, efficiently hydrolyzed wheat and reduced the viscosity of wheat hydrolysate. Current data indicate that the sdLeXyn has a potential as a feed additive to improve the utilization of wheat in poultry production.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for Twelve Antimicrobials (Biocides and Antibiotics) in Eight Strains of Listeria monocytogenes.

When selecting effective doses of antimicrobials, be they biocides or antibiotics, it is essential to know the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these substances. The present research determined the MICs and MBCs for three biocides, sodium hypochlorite (SH), benzalkonium chloride (BC), and peracetic acid (PAA), and nine antibiotics in eight strains of Listeria monocytogenes of varying serotypes. Marked intra-species differences were observed in the resistance of L. monocytogenes to the biocides and antibiotics. The MICs (ppm) for the biocides ranged between 1750 and 4500 for SH, 0.25 and 20.00 for BC, and 1050 and 1700 for PAA. Their MBCs (ppm) ranged from 2250 to 4500 for SH, 0.50 to 20.00 for BC, and 1150 to 1800 for PAA. The MICs (ppm) for antibiotics lay between 1 and 15 for ampicillin, 8 and 150 for cephalothin, 20 and 170 for cefoxitin, 0.05 and 0.20 for erythromycin, 4 and 50 for chloramphenicol, 3 and 100 for gentamicin, 2 and 15 for tetracycline, 2 and 80 for vancomycin, and 160 and 430 for fosfomycin. The corresponding MBCs (ppm) were from 5 to 20 for ampicillin, 9 to 160 for cephalothin, 70 to 200 for cefoxitin, 4 to 5 for erythromycin, 9 to 70 for chloramphenicol, 5 to 100 for gentamicin, 3 to 30 for tetracycline, 3 to 90 for vancomycin, and 160 to 450 for fosfomycin. Notably, erythromycin showed considerable efficacy, demonstrated by the low values for both MIC and MBC. Based on EUCAST and the CLSI criteria, all strains were susceptible to erythromycin. All strains were resistant to cephalothin, cefoxitin, gentamicin, and fosfomycin. Further values for resistance were 87.50% for ampicillin and vancomycin, 75.00% for tetracycline, and 62.50% for chloramphenicol. The high prevalence of antibiotic resistance is a matter for concern. A positive correlation was found between MIC and MBC values for most of the biocides and antibiotics. The higher the hydrophobicity of the cell surface, the higher the susceptibility to biocides, suggesting that surface characteristics of bacterial cells influence resistance to these compounds.

SCANNING ELECTRON MICROSCOPY OF INVASIVE DUCTAL BREAST CANCER

Aim. Establishing the surface characteristics of ductal breast cancer of different degree of differentiation and different histological structure of the invasive component in scanning electron microscopy.
 Material and Methods. The results of the study of invasive ductal breast cancer using light and scanning electron microscopy were analyzed. The degree of malignancy was determined according to a modified scheme of P. Scarff, H. Bloom and W. Richardson. Electron microscopic features of the cell surface of invasive ductal breast cancer during this study were compared in each case with its histological structure and degree of malignancy G.
 Results and Discussion. Histologically, tubular, trabecular, solid, alveolar structures and discrete (single) groups of tumor cells were diagnosed in the infiltrative component of invasive ductal breast carcinoma. In terms of the surface configuration of tumor cells, we have identified three cell variants. The first variant was characterized by the presence in most tumor cells numerous asymmetric microvilli with diffuse or focal distribution on the cell surface and was prevailed in the tubular structures of the infiltrative component of ductal breast carcinoma of G1 grade (p<0,05). The second variant of the surface configuration was characterized by a small number of asymmetric microvilli scattered throughout the cell surface and was diagnosed in a solid and trabecular invasive component of malignancies G2 and G3 grades. The third variant was characterized by the absence of microvilli on the surface in most tumor cells and was prevailed in the alveolar structures of the invasive component of G2 and G3 grades. It was found that the decrease in the grade of histological differentiation of invasive ductal breast cancer is associated with a decrease or absence of asymmetric microvilli on the cell surface. Tumors classified as G2 showed a wide range of surface configurations of cancer cells; in 75% [27,96-99,89] of cases G3 carcinoma was characterized by the absence of microvilli on the surface in most tumor cells, i.e., there was a III variant surface configuration of cells.
 Conclusions. Scanning electron microscopy makes it possible to provide information in a nanometer scale about the surface features of tumor cells, and the identification of phenotype features between tumor cells can be prognostically important in the course of breast cancer.

PD-L1 Expression on Circulating Tumour-Derived Microvesicles as a Complementary Tool for Stratification of High-Grade Serous Ovarian Cancer Patients.

Simple SummaryPD-1/PD-L1 axis blockade immunotherapy leverages on assessment of tumour PD-L1 status by immunohistochemistry for identifying cancer patients’ who would benefit most from this therapy. Because some PD-L1 negative cancer patients also benefit from the treatment, it has been postulated that spatial heterogeneity of PD-L1 expression in tumour tissue may reduce the predictive value of the test. Here we demonstrate for the first time the presence of PD-L1+ circulating tumor–derived microvesicles in the blood stream of high grade serous ovarian cancer patients and propose the assessment of tumor-derived circulating PD-L1+ microvesicles to be inserted in predictive algorithms for complementing tumour PD-L1 status.Background: Ovarian cancer (OC) has recently attracted attention for the use of PD-1/PD-L1 axis blocking agents, with durable activity reported only in a subset of patients. The most used biomarker for sensitivity to the PD-1/PD-L1 axis blockade is tumour PD-L1 status by immunohistochemistry. However, patient stratification using this method suffers from intrinsic heterogeneity of OC, likely contributing to the unsatisfactory results obtained so far. Cells communicate with each other by releasing microvesicles (MVs) that carry parental cell surface features. Thus, we hypothesised that PD-L1+ tumour cells (TC) and infiltrating PD-L1+ leukocytes should shed MVs carrying surface PD-L1 that may serve as a proxy for the whole tumour PD-L1 status. Results: We showed for the first time the presence of measurable amounts of TC- and leukocyte-derived PD-L1+ MVs (range: 1.4–178.8 MVs/μL and 6.2–504.8 MVs/μL, respectively) in the plasma of high-grade serous OC (HGSOC) patients (n = 63), using a sensitive flow cytometry platform. The concentration of PD-L1+ MVs of either origin did not associate with the PD-L1 status of TCs and leukocytes in the tumour biopsies, suggesting that the circulating PD-L1+ MVs also included ones from locations not selected for immunohistochemistry analysis and represented the PD-L1 status of the whole tumour mass. In this study, we also describe the serendipitous discovery of circulating PD-L1+ MVs of platelet origin (10.3–2409.6 MVs/μL). Conclusions: The enumeration of circulating PD-L1+ MVs in HGSOC patients may provide a novel direction for assessing the tumour PD-L1 status and contribute to HGSOC patient stratification for immunotherapy interventions. The presence of circulating PD-L1+ MVs of platelet origin, a finding not yet reported in HGSOC patients, warrants further studies.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Selective Adsorption of Amino Acids in Crystals of Monohydrocalcite Induced by the Facultative Anaerobic Enterobacter ludwigii SYB1

The morphology, crystal structure, and elemental composition of biominerals are commonly different from chemically synthesized minerals, but the reasons for these are not fully understood. A facultative anaerobic bacterium, Enterobacter ludwigii SYB1, is used in experiments to document the hydrochemistry, mineral crystallization, and cell surface characteristics of biomineralization. It was found that carbonate anhydrase and ammonia production were major factors influencing the alkalinity and saturation of the closed biosystem. X-ray diffraction (XRD) spectra showed that calcite, monohydrocalcite (MHC), and dypingite formed in samples with bacterial cells. It was also found that the (222) plane of MHC was the preferred orientation compared to standard data. Scanning transmission electron microscopy (STEM) analysis of cell slices provides direct evidence of concentrated calcium and magnesium ions on the surface of extracellular polymeric substances (EPS). In addition, high-resolution transmission electron microscopy (HRTEM) showed that crystallized nanoparticles were formed within the EPS. Thus, the mechanism of the biomineralization induced by E. ludwigii SYB1 can be divided into three stages: (i) the production of carbonate anhydrase and ammonia increases the alkalinity and saturation state of the milieu, (ii) free calcium and magnesium ions are adsorbed and chelated onto EPS, and (iii) nanominerals crystallize and grow within the EPS. Seventeen kinds of amino acids were identified within both biotic MHC and the EPS of SYB1, while the percentages of glutamic and aspartic acid in MHC increased significantly (p < 0.05). Furthermore, the adsorption energy was calculated for various amino acids on seven diffracted crystal faces, with preferential adsorption demonstrated on (111) and (222) faces. At the same time, the lowest adsorption energy was always that of glutamic and aspartic acid for the same crystal plane. These results suggest that aspartic and glutamic acid always mix preferentially in the crystal lattice of MHC and that differential adsorption of amino acids on crystal planes can lead to their preferred orientation. Moreover, the mixing of amino acids in the mineral structure may also have a certain influence on the mineral lattice dislocations, thus enhancing the thermodynamic characteristics.

Selected Kefir Water from Malaysia Attenuates Hydrogen Peroxide-Induced Oxidative Stress by Upregulating Endogenous Antioxidant Levels in SH-SY5Y Neuroblastoma Cells.

Kefir, a fermented probiotic drink was tested for its potential anti-oxidative, anti-apoptotic, and neuroprotective effects to attenuate cellular oxidative stress on human SH-SY5Y neuroblastoma cells. Here, the antioxidant potentials of the six different kefir water samples were analysed by total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) assays, whereas the anti-apoptotic activity on hydrogen peroxide (H2O2) induced SH-SY5Y cells was examined using MTT, AO/PI double staining, and PI/Annexin V-FITC assays. The surface and internal morphological features of SH-SY5Y cells were studied using scanning and transmission electron microscopy. The results indicate that Kefir B showed the higher TPC (1.96 ± 0.54 µg GAE/µL), TFC (1.09 ± 0.02 µg CAT eq/µL), FRAP (19.68 ± 0.11 mM FRAP eq/50 µL), and DPPH (0.45 ± 0.06 mg/mL) activities compared to the other kefir samples. The MTT and PI/Annexin V-FITC assays showed that Kefir B pre-treatment at 10 mg/mL for 48 h resulted in greater cytoprotection (97.04%), and a significantly lower percentage of necrotic cells (7.79%), respectively. The Kefir B pre-treatment also resulted in greater protection to cytoplasmic and cytoskeleton inclusion, along with the conservation of the surface morphological features and the overall integrity of SH-SY5Y cells. Our findings indicate that the anti-oxidative, anti-apoptosis, and neuroprotective effects of kefir were mediated via the upregulation of SOD and catalase, as well as the modulation of apoptotic genes (Tp73, Bax, and Bcl-2).

X-Ray Photoelectron Spectroscopy on Microbial Cell Surfaces: A Forgotten Method for the Characterization of Microorganisms Encapsulated With Surface-Engineered Shells.

Encapsulation of single microbial cells by surface-engineered shells has great potential for the protection of yeasts and bacteria against harsh environmental conditions, such as elevated temperatures, UV light, extreme pH values, and antimicrobials. Encapsulation with functionalized shells can also alter the surface characteristics of cells in a way that can make them more suitable to perform their function in complex environments, including bio-reactors, bio-fuel production, biosensors, and the human body. Surface-engineered shells bear as an advantage above genetically-engineered microorganisms that the protection and functionalization added are temporary and disappear upon microbial growth, ultimately breaking a shell. Therewith, the danger of creating a “super-bug,” resistant to all known antimicrobial measures does not exist for surface-engineered shells. Encapsulating shells around single microorganisms are predominantly characterized by electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, particulate micro-electrophoresis, nitrogen adsorption-desorption isotherms, and X-ray diffraction. It is amazing that X-ray Photoelectron Spectroscopy (XPS) is forgotten as a method to characterize encapsulated yeasts and bacteria. XPS was introduced several decades ago to characterize the elemental composition of microbial cell surfaces. Microbial sample preparation requires freeze-drying which leaves microorganisms intact. Freeze-dried microorganisms form a powder that can be easily pressed in small cups, suitable for insertion in the high vacuum of an XPS machine and obtaining high resolution spectra. Typically, XPS measures carbon, nitrogen, oxygen and phosphorus as the most common elements in microbial cell surfaces. Models exist to transform these compositions into well-known, biochemical cell surface components, including proteins, polysaccharides, chitin, glucan, teichoic acid, peptidoglycan, and hydrocarbon like components. Moreover, elemental surface compositions of many different microbial strains and species in freeze-dried conditions, related with zeta potentials of microbial cells, measured in a hydrated state. Relationships between elemental surface compositions measured using XPS in vacuum with characteristics measured in a hydrated state have been taken as a validation of microbial cell surface XPS. Despite the merits of microbial cell surface XPS, XPS has seldom been applied to characterize the many different types of surface-engineered shells around yeasts and bacteria currently described in the literature. In this review, we aim to advocate the use of XPS as a forgotten method for microbial cell surface characterization, for use on surface-engineered shells encapsulating microorganisms.

Imaging Mass Cytometry to Assess the Pathobiology of Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Long-term survival after lung transplantation remains limited, mostly because of Chronic Lung Allograft Dysfunction (CLAD), which has two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our tools for assessing lung allograft cells are limited by lung tissue autofluorescence. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a mass spectroscopic technology that allows high-resolution, multi-dimensional characterization of lung cell surface and intracellular proteins at a single cell level, using heavy metal-tagged antibodies. <h3>Methods</h3> Explanted lung tissue was obtained at the time of retransplantation from one BOS and one RAS patient. One donor lung sample was used as control (figure 1A-C). Paraffin-embedded lung tissue was sectioned and stained with 24 heavy-metal-tagged antibodies selected to identify structural and immune proteins of interest. Three 1.5mm² regions of interest in each sample were ablated and data was analyzed using MCD viewer and HistoCAT software. <h3>Results</h3> Visualization of individual antibody binding patterns showed differences between samples, such as greater E-cadherin staining (epithelial cells) in airways of normal lung compared to those of CLAD (figures 1D-F). Spatial distribution of B and T cells within a lymphoid aggregate in the RAS sample is shown (figure 1G-J). We also used an exploratory tSNE strategy to create a 2D representation of cells clustered according to the similarity of their antibody-binding in 24-dimensional space (figure 1K). Precise localization of specific cell populations such as B cell and memory T cell subsets can then be achieved by first identifying them on the tSNE plot and then highlighting them on the IMC image (figures 1L-N). <h3>Conclusion</h3> Our results indicate that IMC is a powerful tool for precise localization of cells within lung tissue samples, allowing both hypothesis-driven comparisons as well as exploratory analyses.

Dimethyl phthalate damages Staphylococcus aureus by changing the cell structure, inducing oxidative stress and inhibiting energy metabolism

Dimethyl phthalate (DMP), used as a plasticizer in industrial products, exists widely in air, water and soil. Staphylococcus aureus is a typical model organism representing Gram-positive bacteria. The molecular mechanisms of DMP toxicology in S. aureus were researched by proteomic and transcriptomic analyses. The results showed that the cell wall, membrane and cell surface characteristics were damaged and the growth was inhibited in S. aureus by DMP. Oxidative stress was induced by DMP in S. aureus. The activities of succinic dehydrogenase (SDH) and ATPase were changed by DMP, which could impact energy metabolism. Based on proteomic and transcriptomic analyses, the oxidative phosphorylation pathway was enhanced and the glycolysis/gluconeogenesis and pentose phosphate pathways were inhibited in S. aureus exposed to DMP. The results of real-time reverse transcription quantitative PCR (RT-qPCR) further confirmed the results of the proteomic and transcriptomic analyses. Lactic acid, pyruvic acid and glucose were reduced by DMP in S. aureus, which suggested that DMP could inhibit energy metabolism. The results indicated that DMP damaged the cell wall and membrane, induced oxidative stress, and inhibited energy metabolism and activation in S. aureus.

Metataxonomic analysis of microbiota from Pakistani dromedary camelids milk and characterization of a newly isolated Lactobacillus fermentum strain with probiotic and bio-yogurt starter traits.

This study was undertaken to investigate the starter and probiotic potential of lactic acid bacteria isolated from dromedarian camel's milk using both culture-dependent and -independent approaches and metataxonomic analysis. Strains of lactic acid bacteria recovered were examined in vitro for tolerance to gastric acidity, bile, and lysozyme. Bile salt hydrolysis, serum cholesterol-lowering, oxalate degradation, proteolytic activity, exopolysaccharide production, and cell surface characteristics necessary for colonizing intestinal mucosa were also evaluated. A single strain of the species, Lactobacillus fermentum named NPL280, was selected through multivariate analysis as it harbored potential probiotic advantages and fulfilled safety criteria. The strain assimilated cholesterol, degraded oxalate, produced exopolysaccharides, and proved to be a proficient alternate yogurt starter with good viability in stored bio-yogurt. A sensorial analysis of the prepared bio-yogurt was also found to be exemplary. We conclude that the indigenous L. fermentum strain NPL280 has the desired traits of a starter and adjunct probiotic culture for dairy products.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Characterization of Cell Surface Properties of 14 Vaginal &amp;lt;i&amp;gt;Lactobacillus&amp;lt;/i&amp;gt; Strains as Potential Probiotics

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Human-origin Lactobacillus is a preferable source of probiotic bacteria. This study screened 14 vaginal Lactobacillus strains as probiotic candidates by investigating probiotic-related cell surface characteristics including cell surface hydrophobicity (CSH), Lewis acidity/basicity, autoaggregation, and biofilm formation. Moderate to high CSH and autoaggregation, high basicity and low acidity were prevalent in the 14 tested strains. Biofilm formation varied in a large range among the 14 tested strains. CSH showed a high correlation with Lewis acidity and autoaggregation, while Lewis acidity was highly correlated with autoaggregation and biofilm formation. Four strains were selected as promising probiotic strains. This study was the first one to compare antibiotic sensitivity between biofilm-forming cells and planktonic cells of Lactobacillus species, and found that biofilm-forming cells of a L. fermentum strain had a significantly higher survival rate than planktonic cells in cefotaxime, cefmetazole and tetracycline, but were as sensitive to oxacillin and ampicillin as planktonic cells were.

Patient-derived and artificial ascites have minor effects on MeT-5A mesothelial cells and do not facilitate ovarian cancer cell adhesion.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

Homologous Recombination in Clostridioides difficile Mediates Diversification of Cell Surface Features and Transport Systems.

Illness caused by the pathogen Clostridioides difficile is widespread and can range in severity from mild diarrhea to sepsis and death. Strains of C. difficile isolated from human infections exhibit great genetic diversity, leading to the hypothesis that the genetic background of the infecting strain at least partially determines a patient's clinical course. However, although certain strains of C. difficile have been suggested to be associated with increased severity, strain typing alone has proved insufficient to explain infection severity. The limited explanatory power of strain typing has been hypothesized to be due to genetic variation within strain types, as well as genetic elements shared between strain types. Homologous recombination is an evolutionary mechanism that can result in large genetic differences between two otherwise clonal isolates, and also lead to convergent genotypes in distantly related strains. More than 400 C. difficile genomes were analyzed here to assess the effect of homologous recombination within and between C. difficile clades. Almost three-quarters of single nucleotide variants in the C. difficile phylogeny are predicted to be due to homologous recombination events. Furthermore, recombination events were enriched in genes previously reported to be important to virulence and host-pathogen interactions, such as flagella, cell wall proteins, and sugar transport and metabolism. Thus, by exploring the landscape of homologous recombination in C. difficile, we identified genetic loci whose elevated rates of recombination mediated diversification, making them strong candidates for being mediators of host-pathogen interaction in diverse strains of C. difficileIMPORTANCE Infections with C. difficile result in up to half a million illnesses and tens of thousands of deaths annually in the United States. The severity of C. difficile illness is dependent on both host and bacterial factors. Studying the evolutionary history of C. difficile pathogens is important for understanding the variation in pathogenicity of these bacteria. This study examines the extent and targets of homologous recombination, a mechanism by which distant strains of bacteria can share genetic material, in hundreds of C. difficile strains and identifies hot spots of realized recombination events. The results of this analysis reveal the importance of homologous recombination in the diversification of genetic loci in C. difficile that are significant in its pathogenicity and host interactions, such as flagellar construction, cell wall proteins, and sugar transport and metabolism.

Dispirotripiperazine-core compounds, their biological activity with a focus on broad antiviral property, and perspectives in drug design (mini-review).

Viruses are obligate intracellular parasites and have evolved to enter the host cell. To gain access they come into contact with the host cell through an initial adhesion, and some viruses from different genus may use heparan sulfate proteoglycans for it. The successful inhibition of this early event of the infection by synthetic molecules has always been an attractive target for medicinal chemists. Numerous reports have yielded insights into the function of compounds based on the dispirotripiperazine scaffold. Analysis suggests that this is a structural requirement for inhibiting the interactions between viruses and cell-surface heparan sulfate proteoglycans, thus preventing virus entry and replication. This review summarizes our current knowledge about the early history of development, synthesis, structure-activity relationships and antiviral evaluation of dispirotripiperazine-based compounds and where they are going in the future.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.71 Role of caveolae in T-tubule biogenesis

Caveolae, 60-70 nm flask-shaped membrane invaginations of the plasma membrane, are abundant features of muscle cell surface. Caveolin 3, the muscle-specific isoform has been proposed to participate in the process leading to the development of T-tubules, specialised membrane invaginations involved in excitation-contraction coupling. It has been suggested that caveolae could fuse together and provide the substratum for membrane bending and curvature sensing proteins to tubulate lipid membranes. The importance of caveolae and caveolins in skeletal muscle is underscored by the fact that defects in caveolae function and organisation have been reported in several myopathies and that mutations in CAV3 cause a family of autosomal-dominant neuromuscular diseases termed caveolinopathies although to date the pathophysiological mechanisms remain poorly understood.

Peptide Probes of Colistin Resistance Discovered via Chemically Enhanced Phage Display.

Colistin is an antibiotic of last resort used to treat infections caused by multidrug-resistant Gram-negative bacterial pathogens. The recent surge in reported cases of colistin-resistant infections urgently calls for fast and reliable diagnostic methods, which can be used for the facile detection and proper treatment of these challenging infections. A major mechanism of colistin resistance involves phosphoethanolamine (PE) modification of lipopolysaccharide (LPS), the molecular target of colistin. This LPS modification mechanism has been recently reported to be transferrable via a plasmid-carried mcr-1 gene, which is particularly concerning as it may readily confer colistin resistance to a wide array of bacterial pathogens. To develop molecular tools to allow facile detection of colistin resistance, we have herein enlisted a novel phage library that incorporates dynamic covalent warheads to recognize PE modifications on bacterial cells. Screening of this chemically modified phage library against colistin-resistant pathogens revealed a number of peptide probes that readily differentiate colistin-resistant bacterial strains from their colistin-susceptible counterparts. With a fluorophore label, these peptide probes selectively stain colistin-resistant bacteria at sub-to-low micromolar concentrations. The bacterial staining is minimally inhibited by the presence of serum proteins or even blood serum. Mechanistic studies indicate that our peptide probes bind colistin-resistant bacteria primarily by targeting PE-modified lipids. However, some species-specific features of the cell surface can also contribute to the peptides' association to bacterial cells. Further elucidation of such cell surface features may give molecular probes with improved species and strain specificity, which will enable bacterial infection diagnosis with high precision.

Functional probiotics of lactic acid bacteria from Hu sheep milk

BackgroundProbiotics are being considered as valuable microorganisms related to human health. Hu sheep is referred as one of the important sheep breeds in China. Goat milk produced by Hu sheep is characterized with high nutritional value and hypoallergenic in nature. Particularly, this milk contains plenty of milk prebiotic and probiotic bacteria. This study was aimed to scrutinize more bacterial strains from Hu sheep milk with potential probiotic activity.ResultsBased on 16S rRNA sequence analysis, pool of forty bacterial strains were identified and evaluated their antimicrobial activities against Staphylococcus aureus, enterohemorrhagic Escherichia coli (EHEC), Salmonella typhimurium, Listeria monocytogenes enterotoxigenic E. coli (ETEC) and Aeromonas caviae. Four out of these isolated strains demonstrated their efficient bacteriostatic ability and potential healthy properties. We also examined the safety aspects of these bacterial candidates including three Lactococcus lactis strains (named as HSM-1, HSM-10, and HSM-18) and one Leuconostoc lactis strain (HSM-14), and were further evaluated via in vitro tests, including antimicrobial activity, cell surface characteristics (hydrophobicity, co-aggregation, and self-aggregation), heat treatment, antibiotic susceptibility, simulated transport tolerance in the gastrointestinal tract, and acid/bile tolerance. The obtained results revealed that HSM-1, HSM-10, HSM-14, and HSM-18 showed high survival rate at different conditions for example low pH, presence of bovine bile and demonstrated high hydrophobicity. Moreover, HSM-14 had an advantage over other strains in terms of gastrointestinal tract tolerance, antimicrobial activities against pathogens, and these results were significantly better than other bacterial candidates.ConclusionHu sheep milk as a source of exploration of potential lactic acid bacteria (LAB) probiotics open the new horizon of probiotics usage from unconventional milk sources. The selected LAB strains are excellent probiotic candidates which can be used for animal husbandry in the future. Rationale of the study was to utilize Hu sheep milk as a source of potential probiotic LABs. The study has contributed to the establishment of a complete bacterial resource pool by exploring the Hu sheep milk microflora.

Bioaccumulation of Ni(II) on growing cells of Bacillus sp.: Response surface modeling and mechanistic insight

Abstract The growing cells of Bacillus cereus M 16 1 , depending on the growth phase of culture was found to bioaccumulate Ni(II) from its aqueous solution to the extent of 80% wherein surface binding ( ∼ 60%) dominated over intracellular accumulation ( ∼ 20%). No growth was observed in the metal ion concentration beyond 50 mg L − 1 . The hydroxyl, amine, carboxylate and phosphate groups of cell surface were the most important moieties in chemical interactions with Ni(II) ions as measured by FTIR. The surface characteristics of cells were investigated meticulously by FE-SEM equipped with EDXA indicating conspicuous changes due to metal accumulation. The potential effects of the process variables [initial pH (A), temperature (B), inoculum volume (C) and medium volume (D)] were optimized using Response Surface Methodology (RSM) followed by Box–Behnken design to study the bioaccumulation phenomenon. The maximum Ni(II) accumulation was noted at 6.5, 32.5 °C, 2.5% and 50 mL corresponding to pH, temperature, inoculum volume and medium volume, respectively. The most significant factors including one independent variable (D) and two quadratic effects (B2 and D2) were identified by ANOVA. The experimental response under these optimum conditions was found to be 78.76% which closely matched the yield (78.52%) predicted by the statistical model with R 2 = 0 . 8790 .