Probe based confocal laser endomicroscopy (pCLE) is an advanced technique which provides imaging of gastrointestinal mucosa at subcellular resolution and, importantly, a valid tool for the evaluation of microvasculature during endoscopic examination. In order to assess intratumoral vascularization and the efficiency of blood flow in locally advanced gastric cancer, we examined 57 patients through pCLE imaging. The vascular alterations in gastric cancer were mainly characterized by leakage and by the presence of tortuous and large size vessels. Defects in blood flow were detected very rarely. No association between the angiogenic score and the gastric tumor site or histological type was observed. Interestingly, no correlation was also found with the tumor grading indicating that the vascular angiogenic anomalies in gastric cancer represent an early pathological event to be observed and detected. The majority of patients displayed unchanged vascular alterations following neoadjuvant chemotherapy and this positively correlated with stable or progressive disease, suggesting that an unaltered angiogenic score could per se be indicative of poor therapeutic efficacy. Different vascular parameters were evaluated by immunofluorescence using bioptic samples and the vessel density did not correlate with clinical staging, site or histologic type. Interestingly, only CD105, Multimerin-2 and GLUT1 were able to discriminate normal from tumoral gastric mucosa. Taken together, these findings indicate that functional and structural angiogenic parameters characteristic of tumor blood network were fully detectable by pCLE. Moreover, the evaluation of tumor vasculature by real-time assessment may provide useful information to achieve tailored therapeutic interventions for gastric cancer patients.

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Ydj1, a class B J-protein (Hsp40) in yeast, has two zinc finger domains in each monomer and belongs to an important co-chaperone family that plays crucial roles in cells, such as recognizing and binding partially folded proteins and assisting the Hsp70 chaperone family in protein folding. Yeast cells with ydj1 deletion were less efficient at coping with zinc stress than wild-type cells, and site-directed mutagenesis studies that impair or delete the zinc finger region have confirmed the importance of this region to the function of Ydj1; however, little is known about whether the presence of zinc is critical for the function of the protein. To gain insights into the effect of zinc on the structure and function of Ydj1 without having to modify its primary structure, a method was developed and optimized to quantify and remove the zinc from the protein. Recombinant Ydj1 was produced and purified, and its zinc content was determined by ICP-MS. The result showed that two zinc atoms were bound per monomer of protein, a good indicator that all sites were saturated. To optimize the removal of the bound zinc, variations on chelating agent (EDTA, EGTA, 1,10-phenanthroline), chelator concentration, reaction time, pH, and temperature were tested. These procedures had no effect on the overall secondary structure of the protein, since no significant changes in the circular dichroism spectrum were observed. The most significant removal (91 ± 2%, n = 3) of zinc was achieved using 1,10-phenanthroline (1 × 10-3 mol L-1) at 37°C with a pH 8.5 for 24 h. Zinc removal affected the stability of the protein, as observed by a thermal-induced unfolding assay showing that the temperature at the middle of the transition (Tm) decreased from 63 ± 1°C to 60 ± 1°C after Zn extraction. In addition, the effect on the ability of Ydj1 to protect a model protein (luciferase) against aggregation was completely abolished after the Zn removal procedure. The main conclusion is that zinc plays an important role in the stability and activity of Ydj1. Additionally, the results highlight the medical importance of chaperones, as altered zinc homeostasis is implicated in many diseases, such as neurodegenerative disorders.

Aims: Vein grafts are frequently used conduits for arterial reconstruction in patients with cardiovascular disease. Unfortunately, vein graft disease (VGD) causes diminished patency rates. Innate immune system components are known to contribute to VGD. However, the role of T cells has yet to be established. The purpose of this study was to investigate the role of T cells and T cell activation pathways via the T cell receptor (TCR), co-stimulation and bystander effect in VGD. Methods and results: Here, we show upon vein graft surgery in mice depleted of CD4+ T cells or CD8+ T cells, that CD8+ T cells are locally activated and have a major protective role for vein graft patency. In presence of CD8+ T cells vein grafts appear patent while CD8+ T cell depletion results in occluded vein grafts with increases apoptosis. Importantly, the protective effect of CD8+ T cells in VGD development was TCR and co-stimulation independent. This was demonstrated in vein grafts of OT-I mice, CD70-/-, CD80/86-/-, and CD70/80/86-/- mice compared to C57BL/6 mice. Interestingly, cytokines including IL-15, IL-18, IL-33, and TNF are up-regulated in vein grafts. These cytokines are co-operatively capable to activate CD8+ T cells in a bystander-mediated fashion, in contrast to CD4+ T cells. Conclusions: T cells are modulators of VGD with a specific protective role of CD8+ T cells, which are locally activated in vein grafts. CD8+ T cells may protect against occlusive lesions by providing survival signals, and concert their protection independent of TCR and co-stimulation signaling.

The aim of this study was to evaluate the outcomes of trabeculectomy with mitomycin C (MMC) in patients with Pseudoexfoliative Glaucoma (PXG) and compare the results with the outcomes of trabeculectomy without MMC in PXG and with MMC in Primary Open Angle Glaucoma (POAG). Ninety eyes (76 patients) submitted to trabeculectomy were included in a one-year retrospective study. Fifty-eight eyes with PXG were divided into group 1 (28 eyes) and group 2 (30 eyes), with and without MMC application respectively. Then, the group 1 results were compared with 32 eyes with POAG that performed trabeculectomy with MMC (group 3). Main outcome measures were intraocular pressure (IOP), number of IOP lowering medications, rate of bleb failure (encapsulation, flattening and/or vascularization) and the number of eyes submitted to surgical procedures after trabeculectomy (needling, 5-fluorouracil (5FU) or 2nd trabeculectomy). Results revealed that compared to trabeculectomy with MMC in POAG and trabeculectomy with MMC in PXG, trabeculectomy without MMC in PXG leads to higher IOP (preoperative mean ± standard deviation [SD] was 28.6 ± 5.4 mmHg in group 1, 32.2 ± 8.2 mmHg in group 2 and 26.1 ± 6.5 mmHg in group 3; and after one year was 13.9 ± 3.9 mmHg in group 1, 16.1 ± 5.9 mmHg in group 2 and 12.5 ± 4.0 mmHg in group 3); higher number of IOP lowering medications (preoperative mean ± SD was 3.1 ± 0.60 in group 1, 2.8 ± 0.81 in group 2 and 3.4 ± 0.76 in group 3; and after one year was 1.1 ± 1.1 in group 1, 1.1 ± 1.0 in group 2 and 0.33 ± 0.89 in group 3); higher prevalence of bleb failure (47% in group 1, 53% in group 2, and 18% in group 3); and increased participation in surgical procedures following trabeculectomy (47% in group 1, 57% in group 2, and 6% in group 3). We concluded that trabeculectomy without MMC in PXG had the worst surgical outcome. Thus, PXG appears to be a potential risk factor for filtration bleb failure. Therefore, it could be considered in surgical protocols of MMC application.

Hip arthroscopic surgery has become an increasingly common surgical technique to diagnose and treat various hip abnormalities. While increased efficacy has been reported, debate remains regarding appropriate surgical indications. Multiple factors including patient demographics, surgical procedure, and underlying disease have been associated with poor surgical outcomes. Preoperative diagnostic and treatment interventions including physical therapy and injections may affect surgical indications and outcomes.

There is a scarcity of data on pneumococcal serotypes carried by children in Ethiopia. We studied pneumococcal nasopharyngeal carriage rate, serotypes, and risk factors among children with community acquired pneumonia (CAP).

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.

Mutations of human BEST1, encoding a Ca2+-activated Cl- channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the "neck" and "aperture", respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.

The conservation and development of chicken have received considerable attention, but the admixture history of chicken breeds, especially Chinese indigenous breeds, has been poorly demonstrated. In this study, we aimed to evaluate the genetic diversity and population structure of eight chicken breeds (including conserved chicken breeds) from different geographic origin and to identify admixture within these breeds using a 600-K single-nucleotide polymorphism panel for genotyping. Using the genotype of 580,961 single-nucleotide polymorphism markers scored in 1,200 animals, we evaluated the genetic diversity (heterozygosity and proportion of polymorphic markers), linkage disequilibrium decay, population structure (principal component analysis and neighbor-joining tree), genetic differentiation (FST and genetic distance), and migration events (TreeMix and f-statistics) of the eight domesticated chicken breeds. The results of population analytical methods revealed patterns of hybridization that occurred after divergence in Tibetan chicken. We argue that chicken migration and admixture, followed by trade, have been important forces in shaping the genomic variation in modern Chinese chicken. Moreover, isolation by distance might play a critical role in shaping the genomic variation within Eurasia continent chicken breeds. Moreover, genetic information provided in this study is valuable resources for production applications (genomic prediction, and breeding strategy) and scientific research (genetic basis detection, studying evolution, or domestication).

Mass spectrometry imaging (MSI) is an analytical technique for the unlabeled and multiplex imaging of molecules in biological tissue sections. It therefore enables the spatial and molecular annotations of tissues complementary to histology. It has already been shown that MSI can guide subsequent material isolation technologies such as laser microdissection (LMD) to enable a more in-depth molecular characterization of MSI-highlighted tissue regions. However, with MSI now reaching spatial resolutions at the single-cell scale, there is a need for a precise co-registration between MSI and the LMD. As proof-of-principle, MSI of lipids was performed on a breast cancer tissue followed by a segmentation of the data to detect molecularly distinct segments within its tumor areas. After image processing of the segmentation results, the coordinates of the MSI-detected segments were passed to the LMD system by three co-registration steps. The errors of each co-registration step were quantified and the total error was found to be less than 13 μm. With this link established, MSI data can now accurately guide LMD to excise MSI-defined regions of interest for subsequent extract-based analyses. In our example, the excised tissue material was then subjected to ultrasensitive microproteomics in order to determine predominant molecular mechanisms in each of the MSI-highlighted intratumor segments. This work shows how the strengths of MSI, histology, and extract-based omics can be combined to enable a more comprehensive molecular characterization of in situ biological processes.

In pulmonary hypertension (PH), hypoxia represents both an outcome and a cause of exacerbation. We addressed the question whether hypoxia adaptation might affect the mechanisms underlying PH alleviation through phosphodiesterase-5 (PDE5) inhibition.

With reduced thymic activity, the population of naïve T cells in humans is maintained by homeostatic proliferation throughout adult life. In young adults, naïve CD4 T cells have enormous proliferative potential and plasticity to differentiate into different lineages. Here, we explored whether naïve CD4 T-cell aging is associated with a partial loss of this unbiased multipotency. We find that naïve CD4 T cells from older individuals have developed a propensity to develop into TH9 cells. Two major mechanisms contribute to this predisposition. First, responsiveness to transforming growth factor β (TGFβ) stimulation is enhanced with age due to an upregulation of the TGFβR3 receptor that results in increased expression of the transcription factor PU.1. Secondly, aged naïve CD4 T cells display altered transcription factor profiles in response to T-cell receptor stimulation, including enhanced expression of BATF and IRF4 and reduced expression of ID3 and BCL6. These transcription factors are involved in TH9 differentiation as well as IL9 transcription suggesting that the aging-associated changes in the transcription factor profile favor TH9 commitment.

Adipose tissue expansion involves angiogenesis to remodel its capillary network. The enzymemethionine aminopeptidase 2(MetAP2) promotes angiogenesis.MetAP2 inhibitors suppress angiogenesis and have potential anti-obesity effect. However, impairment in adipose tissue expansion is also linked with impaired glycemic control.This study investigated the effect of BL6, a MetAP2 inhibitor, on adipogenesis and glucose disposal.To test effect on angiogenesis, Human Umbilical Vein Endothelial Cells(HUVECs) were treated with BL6 for 24h to determine tube formation. Further, to test effect on adipogenesis and glucose disposal,3T3-L1 pre-adipocytes were treated with BL6(0 µM, 20µM, 50 µM or 100µM) during differentiation. Differentiated cells were stained with Oil Red O for determining lipid accumulation, and glucose uptake assay. Protein levels and RNA expression for key genes involved in the adipogenic cascade were determined.BL6 treatment of HUVECs dose dependently blocked angiogenesis. During differentiation of pre-adipocytes, 50μM and 100µM BL6 significantly reduced lipid accumulation. Treatment with 100µM BL6 significantly decreased expression of adipogenic genes. Interestingly, BL6 treatment dose dependently increased glucose uptake by 3T3-L1 cells.MetAP2 inhibitor blocks angiogenesis, attenuates adipogenesis, yet increases cellular glucose uptake. Collectively this proof of concept study supports a possible role for MetAP2 inhibitor BL6, as a putative anti-obesity therapeutic agent.

The aim of the present study was to assess different obesity indices, as well as their best cut-off point, to predict the occurrence of hypertension (HTN) in an Iranian population.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

Diabetes mellitus in Asia accounts for more than half of the global prevalence. There is a high prevalence of cardiovascular disease (CVD) in the region among people with type 2 diabetes mellitus (T2DM) and it is often associated with multiple risk factors including hypertension, renal disease and obesity. The early onset of T2DM and the eventual long disease duration portends an increasing proportion of the population to premature CVD. In addition to lowering blood glucose, sodium-glucose co-transporter-2 (SGLT-2) inhibitors exert favourable effects on multiple risk factors (including blood pressure, body weight and renal function) and provide an opportunity to reduce the risk of CVD in patients with T2DM. In this article, we consolidated the existing literature on SGLT-2 inhibitor use in Asian patients with T2DM and established contemporary guidance for clinicians. We extensively reviewed recommendations from international and regional guidelines, published data from clinical trials in the Asian population (dapagliflozin, canagliflozin, empagliflozin, ipragliflozin, luseogliflozin and tofogliflozin), CVD outcomes trials (EMPAREG-OUTCOME, CANVAS and DECLARE-TIMI 58) and real-world evidence studies (CVD-REAL, EASEL, CVD-REAL 2 and OBSERVE-4D). A series of clinical recommendations on the use of SGLT-2 inhibitors in Asian patients with T2DM was deliberated among experts with multiple rounds of review and voting. Based on the available evidence, we conclude that SGLT-2 inhibitors represent an evidence-based therapeutic option for the primary prevention of heart failure hospitalization and secondary prevention of CVD in patients with T2DM, and should be considered early on in the treatment algorithm for patients with multiple risk factors, or those with established CVD.

Mycobacteriophages are viruses that infect and kill mycobacteria. The peptidoglycan hydrolase, lysin A (LysA), coded by one of the most potent mycobacteriophages, D29, carries two catalytic domains at its N-terminus and a cell wall-binding domain at its C-terminus. Here, we have explored the importance of the centrally located lysozyme-like catalytic domain (LD) of LysA in phage physiology. We had previously identified an R198A substitution that causes inactivation of the LD when it is present alone on a polypeptide. Here, we show that upon incorporation of the same mutation (i.e. R350A) in full-length LysA, the protein demonstrates substantially reduced activity in vitro, even in the presence of the N-terminal catalytic domain, and has less efficient mycobacterial cell lysis ability when it is expressed in Mycobacterium smegmatis. These data suggest that an active LD is required for the full-length protein to function optimally. Moreover, a mutant D29 phage harbouring this substitution (D29R350A) in its LysA protein shows significantly delayed host M. smegmatis lysis. However, the mutant phage demonstrates an increase in burst size and plaque diameter. Taken together, our data show the importance of an intact LD region in D29 LysA PG hydrolase, and indicate an evolutionary advantage over other phages that lack such a domain in their endolysins.

Prolonged exposure to glucocorticoids (GCs) causes various metabolic derangements. These include obesity and insulin resistance, as inhibiting glucose utilization in adipose tissues is a major function of GCs. Although adipose tissue distribution and glucose homeostasis are sex-dependently regulated, it has not been evaluated whether GCs affect glucose metabolism and adipose tissue functions in a sex-dependent manner. In this study, high-dose corticosterone (rodent GC) treatment in C57BL/6J mice resulted in nonfasting hyperglycemia in male mice only, whereas both sexes displayed hyperinsulinemia with normal fasting glucose levels, indicative of insulin resistance. Metabolic testing using stable isotope-labeled glucose techniques revealed a sex-specific corticosterone-driven glucose intolerance. Corticosterone treatment increased adipose tissue mass in both sexes, which was reflected by elevated serum leptin levels. However, female mice showed more metabolically protective adaptations of adipose tissues than did male mice, demonstrated by higher serum total and high-molecular-weight adiponectin levels, more hyperplastic morphological changes, and a stronger increase in mRNA expression of adipogenic differentiation markers. Subsequently, in vitro studies in 3T3-L1 (white) and T37i (brown) adipocytes suggest that the increased leptin and adiponectin levels were mainly driven by the elevated insulin levels. In summary, this study demonstrates that GC-induced insulin resistance is more severe in male mice than in female mice, which can be partially explained by a sex-dependent adaptation of adipose tissues.