Fractalkine/CX3CR1 axis is critical for neuroprotection induced by hypoxic postconditioning against cerebral ischemic injury.

Microglial activation-mediated neuroinflammation is a major contributor to neuronal damage after cerebral ischemia. The Fractalkine (FKN)/CX3C chemokine receptor 1 (CX3CR1) axis plays a critical role in regulating microglial activation and neuroinflammation. The aim of this study is to ascertain the role and mechanism of FKN/CX3CR1 axis in hypoxic postconditioning (HPC)-induced anti-inflammatory and neuroprotective effects on transient global cerebral ischemia (tGCI). We found that HPC suppressed microglial activation and alleviated neuroinflammation in hippocampal CA1 after tGCI. Meanwhile, HPC upregulated the expression of FKN and CX3CR1 in neurons, but it downregulated the expression of CX3CR1 in glial cells after tGCI. In addition, the overexpression of FKN induced by the administration of FKN-carried lentivirus reduced microglial activation and inhibited neuroinflammation in CA1 after tGCI. Furthermore, silencing CX3CR1 with CX3CRi-carried lentivirus in CA1 after tGCI suppressed microglial activation and neuroinflammation and exerted neuroprotective effects. Finally, the overexpression of FKN caused a marked increase of neuronal CX3CR1 receptors, upregulated the phosphorylation of Akt, and reduced neuronal loss of rats in CA1 after tGCI. These findings demonstrated that HPC protected against neuronal damage in CA1 of tGCI rats through inhibiting microglial activation and activating Akt signaling pathway via FKN/CX3CR1 axis.

Constructing hollow core-shell Z-scheme heterojunction CdS@CoTiO3 nanorods for enhancing the photocatalytic degradation of 2,4-DCP and TC.

Constructing Z-scheme heterojunctions incorporating an exquisite hollow structure is an effective performance regulation strategy for the realization of high quantum efficiency and a strong redox ability over photocatalysts. Herein, we report the delicate design and preparation of a core-shell hollow CdS@CoTiO3 Z-scheme heterojunction with a CdS nanoparticle (NP)-constructed outer shell supported on a CoTiO3 nanorod (NR) inner shell. The in situ growth synthetic method led to a tightly connected interface for the heterojunction between CdS and CoTiO3, which shortened the transport distance of photoinduced charges from the interface to the surface. The promoted charge carrier separation efficiency and the retained strong redox capacity caused by the Z-scheme photoinduced charge-transfer mechanism were mainly responsible for the boosted photocatalytic performance. Additionally, the well-designed core-shell structure afforded a larger interfacial area by the multiple direction contact between CdS and CoTiO3, ensuring sufficient channels for efficient charge transfer, and thus further boosting the photocatalytic activity. As an efficient photocatalyst, the optimized CdS@CoTiO3 nanohybrids displayed excellent 2,4-dichlorophenol (2,4-DCP) and tetracycline (TC) degradation efficiencies of 91.3% and 91.8%, respectively. This study presents a Z-scheme heterojunction based on ecofriendly CoTiO3, which could be valuable for the development of metal perovskite photocatalysts for application in environmental remediation, and also demonstrated the tremendous potential of integrating a Z-scheme heterojunction with the morphology design of photocatalyts.

Characterization of O-mannosylated proteins profiling in bacillus Calmette-Guérin via gel-based and gel-free approaches.

Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs observed in bacteria, which is crucial for functional regulations of many prokaryotic and eukaryotic organisms. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been recognized as an indispensable tool in the global fight against tuberculosis (TB) worldwide over several decades. Nevertheless, analysis of glycoprotein profiles of BCG has not been clearly investigated. In this study, we performed O-mannosylated protein analysis in BCG bacteria using gel-based and gel-free approaches. In total, 1,670 hexosylated peptides derived from 754 mannosylated proteins were identified. Furthermore, 20 novel protein products supported by 78 unique peptides not annotated in the BCG database were detected. Additionally, the translational start sites of 384 proteins were confirmed, and 78 proteins were validated through the extension of translational start sites based on N-terminus-derived peptides. The bioinformatic analysis of the O-mannosylated proteins was performed and the expression profiles of four randomly selected proteins were validated through Western blotting. A number of proteins involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis, oxidative phosphorylation, and two-component system, are discussed. Taken together, these results offer the first O-mannosylated protein analysis of a member of mycobacteria reported to date by using complementary gel-based and gel-free approaches. Some of the proteins identified in this study have important roles involved in metabolic pathways, which could provide insight into the immune molecular mechanisms of this recognized vaccine strain.

Evaluation of novel obesity- and lipid-related indices as predictors of abnormal glucose tolerance in Chinese women with polycystic ovary syndrome.

PURPOSE: We aimed to evaluate the performance of Chinese visceral adiposity index (CVAI), visceral adiposity index (VAI), lipid accumulation product (LAP), triglyceride glucose (TyG) as indices in screening abnormal glucose tolerance (AGT) in Chinese women with polycystic ovary syndrome (PCOS), using the oral glucose tolerance test (OGTT) as a reference test. In addition, we essentially compared the abilities of these indices with body mass index (BMI), waist circumference (WC), fasting plasma glucose (FPG). MATERIALS AND METHODS: All 1113 PCOS patients evaluated in this study underwent OGTTs. The 2-h post-oral glucose load (2 h-PG) level was used to categorize subjects into two groups: those having AGT or normal glucose tolerance (NGT) levels. RESULTS: A statistically significant positive correlation between levels of 2 h-PG and FPG, BMI, WC, LAP, VAI, CVAI, TyG, (P < 0.05), was observed. The strongest correlation was found between the levels of 2 h-PG and CVAI (r = 0.47). The CVAI provided the highest area under the receiver-operating characteristic curve (AUC) for AGT, followed by LAP, BMI, TyG, VAI, WC, and FPG. The CVAI of 32.61 (with AUC: 0.76, sensitivity: 73%, specificity: 70%, positive preductive value (PPV): 0.41, negative predictive value (NPV): 0.90) was found to be the cut-off point for AGT in Chinese women with PCOS. CONCLUSIONS: CVAI may not reliably detect AGT in Chinese women with PCOS. However, it is suitable as a first screening indicator to guide physicians to ordering OGTT.

Urinary proteomic analysis to identify a potential protein biomarker panel for the diagnosis of tuberculosis.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the primary causes of death worldwide. Rapid and accurate diagnosis of TB is one of the most direct means to reduce the incidence of TB. In this study, urinary proteomic profiling of TB patients and non-TB individual controls (HCs) was performed, and differentially expressed urinary proteins between TB and HCs were compared and exclusively expressed proteins in TB patients were selected to establish a clinically useful disease marker panel. In total, these top 11 targeted proteins with 265 peptides were scheduled for multiple reaction monitoring validation analysis by using urine samples from 52 TB patients and 52 HCs. The result demonstrated that a three-protein combination out of the five-protein panel (namely P22352, Q9P121, P15151, Q13291, and Q8NDA2) exhibited sensitivity rate of 82.7% in the diagnosis of TB. Furthermore, the three-protein combination could differentiate TB from the latent tuberculosis (LTB) effectively, which exhibited specificity rate of 92.3% for the diagnosis of TB from the LTB category. Although more numbers of clinical samples are required for further verification, the results provided preliminary evidence that this "three-protein combination" out of the five-protein panel could probably be a novel TB diagnostic biomarker in clinical application.

Urinary metabolomic analysis to identify potential markers for the diagnosis of tuberculosis and latent tuberculosis.

Tuberculosis (TB) is a serious infectious disease with high infection and mortality rates. 5%-10% of the latent tuberculosis infections (LTBI) are likely to develop into active TB, and there are currently no clinical biomarkers that can distinguish between LTBI, active TB and other non-tuberculosis populations. Therefore, it is necessary to develop rapid diagnostic methods for active TB and LTBI. In this study, urinary metabolome of 30 active TB samples and the same number of LTBI and non-TB control samples were identified and analyzed by UPLC-Q Exactive MS. In total, 3744 metabolite components were obtained in ESI- mode and 4086 in ESI + mode. Orthogonal partial least square discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA) showed that there were significant differences among LTBI, active TB and non-TB. Six differential metabolites were screened in positive and negative mode, 3-hexenoic acid, glutathione (GSH), glycochenodeoxycholate-3-sulfate, N-[4'-hydroxy-(E)-cinnamoyl]-l-aspartic acid, deoxyribose 5-phosphate and histamine. The overlapping pathways differential metabolites involved were mainly related to immune regulation and urea cycle. The results showed that the urine metabolism of TB patients was disordered and many metabolic pathways changed. Multivariate statistical analysis revealed that GSH and histamine were selected as potential molecular markers, with area under curve of receiver operating characteristic curve over 0.75. Among the multiple differential metabolites, GSH and histamine changed to varying degrees in active TB, LTBI and the non-TB control group. The levels of GSH and histamine in 48 urinary samples were measured by ELISA in validation phase, and the result in our study provided the potential for non-invasive biomarkers of TB.

Polypeptide LTX-315 reverses the cisplatin chemoresistance of ovarian cancer cells via regulating Beclin-1/PI3K/mTOR signaling pathway.

OBJECTIVE: Polypeptide LTX-315 induces immunogenic cell death, thus having the potential to improve the effect of anticancer treatment. However, the function of LTX-315 in reversing chemoresistance in ovarian cancer (OC) still remains elusive. Our study aims to decipher the effect of LTX-315 on reversing the chemoresistance of OC cells and explore its mechanism. METHODS: SKOV3, A2780, SKOV3/DDP, and A2780/DDP cells (cisplatin [DDP]-resistant cells] were treated with different concentrations of LTX-315 (10 and 20 µmol/L), respectively. Cell counting kit-8 assay, Transwell assay, and flow cytometry were used to assess cell viability, migration, invasion, apoptosis rate, and cell cycle of the cells. Western blot was performed to examine the expression of cleaved caspase 3, caspase 3, cleaved Poly (ADP-ribose) polymerase (PARP), PARP, Bax, Bcl-2, Beclin-1, p-Akt, Akt, p-mammalian target of rapamycin (mTOR), and mTOR. Furthermore, OC cells were treated with autophagy inhibitor 3-methyladenine (3-MA), and "rescue experiments" were performed. RESULTS: DDP-resistant OC cell models were established, and LTX-315 treatment resulted in lower IC50 of DDP. In OC cells treated with LTX-315, the viability, migration, invasion and the expression of Bcl-2 of were repressed, but the apoptotic rate and the expression of cleaved caspase 3, cleaved PARP and Bax were increased, and the cell cycle was arrested. Moreover, LTX-315 promoted Beclin-1 expression level and inhibited p-Akt and p-mTOR expression levels, whereas 3-MA could partially reverse the biological effects of LTX-315 on OC cells. CONCLUSION: LTX-315 can inhibit the resistance of OC cells to DDP in vitro and plays a role by regulating Beclin-1/phosphatidylinositol-3-kinase/mTOR signaling pathway.

Frequency-Domain Fusing Convolutional Neural Network: A Unified Architecture Improving Effect of Domain Adaptation for Fault Diagnosis.

In recent years, transfer learning has been widely applied in fault diagnosis for solving the problem of inconsistent distribution of the original training dataset and the online-collecting testing dataset. In particular, the domain adaptation method can solve the problem of the unlabeled testing dataset in transfer learning. Moreover, Convolutional Neural Network (CNN) is the most widely used network among existing domain adaptation approaches due to its powerful feature extraction capability. However, network designing is too empirical, and there is no network designing principle from the frequency domain. In this paper, we propose a unified convolutional neural network architecture from a frequency domain perspective for a domain adaptation named Frequency-domain Fusing Convolutional Neural Network (FFCNN). The method of FFCNN contains two parts, frequency-domain fusing layer and feature extractor. The frequency-domain fusing layer uses convolution operations to filter signals at different frequency bands and combines them into new input signals. These signals are input to the feature extractor to extract features and make domain adaptation. We apply FFCNN for three domain adaptation methods, and the diagnosis accuracy is improved compared to the typical CNN.

Effect of Deubiquitinase Ovarian Tumor Domain-Containing Protein 5 (OTUD5) on Radiosensitivity of Cervical Cancer by Regulating the Ubiquitination of Akt and its Mechanism.

BACKGROUND The aim of this study was to investigate the role of deubiquitinase [ovarian tumor domain-containing protein 5 (OTUD5)] in regulating Akt ubiquitination and its effect on the radiosensitivity of cervical cancer. MATERIAL AND METHODS Cervical cancer C33A cells were cultured, and then 2 groups of cells (overexpressed cells and silenced cells) were established by overexpressing and silencing OTUD5 gene. Next, quantitative polymerase chain reaction (qPCR) was employed to detect the expression level of OTUD5 in cells in each group. Co-immunoprecipitation and Western blot (WB) analysis were applied to measure the expression level of phosphorylated protein kinase B (Akt) and the level of ubiquitination. The sensitivity of cells to radiotherapy in each group was detected via clone-forming efficiency assay. After that, Statistical Product and Service Solutions (SPSS) 17.0 software was employed for analyses. The t test, one-way analysis of variance (ANOVA), and p test were used. P<0.05 suggested that a difference was statistically significant. RESULTS The levels of phosphorylated Akt and ubiquitination in OTUD5-overexpressed C33A cells were lower than those in the OTUD5-silenced group and control group. The sensitivity of OTUD5-overexpressed C33A cells to radiotherapy was higher than that of the OTUD5-silenced group and control group. CONCLUSIONS OTUD5 affects the radiosensitivity of cervical cancer through the regulation of Akt deubiquitination.

Proteogenomic Analysis and Discovery of Immune Antigens in Mycobacterium vaccae.

Tuberculosis (TB) is one of the leading causes of death worldwide, especially in developing countries. Neonatal BCG vaccination occurs in various regions, but the level of protection varies in different populations. Recently, Mycobacterium vaccae is found to be an immunomodulating therapeutic agent that could confer a significant level of protection against TB. It is the only vaccine in a phase III trial from WHO to assess its efficacy and safety in preventing TB disease in people with latent TB infection. However, the mechanism of immunotherapy of M. vaccae remains poorly understood. In this study, the full genome of M. vaccae was obtained by next-generation sequencing technology, and a proteogenomic approach was successfully applied to further perform genome annotation using high resolution and high accuracy MS data. A total of 3,387 proteins (22,508 unique peptides) were identified, and 581 proteins annotated as hypothetical proteins in the genome database were confirmed. Furthermore, 38 novel protein products not annotated at the genome level were detected and validated. Additionally, the translational start sites of 445 proteins were confirmed, and 98 proteins were validated through extension of their translational start sites based on N terminus-derived peptides. The physicochemical characteristics of the identified proteins were determined. Thirty-five immunogenic proteins of M. vaccae were identified by immunoproteomic analysis, and 20 of them were selected to be expressed and validated by Western blot for immunoreactivity to serum from patients infected with M. tuberculosis The results revealed that eight of them showed strong specific reactive signals on the immunoblots. Furthermore, cellular immune response was further examined and one protein displayed a higher cellular immune level in pulmonary TB patients. Twelve identified immunogenic proteins have orthologous in H37Rv and BCG. This is the first study to obtain the full genome and annotation of M. vaccae using a proteogenomic approach, and some immunogenic proteins that were validated by immunoproteomic analysis could contribute to the understanding of the mechanism of M. vaccae immunotherapy.

Lowly-expressed lncRNA GAS5 facilitates progression of ovarian cancer through targeting miR-196-5p and thereby regulating HOXA5.

PURPOSE: This investigation was aimed at extrapolating whether and how lncRNA GAS5, miR-196a-5p and HOXA5 altered progression of ovarian cancer (OA). METHOD: Totally 195 pairs of OA tissues and adjacent normal tissues were collected. Also si-GAS5, pcDNA-GAS5, miR-196a-5p mimic, miR-196a-5p inhibitor and negative control (NC) were, respectively, transfected into OA cells. Besides, dual-luciferase reporter gene assay was performed to validate the targeted relationships between GAS5 and miR-196a-5p, as well as between miR-196a-5p and HOXA5. The impacts of GAS5, miR-196a-5p and HOXA5 on viability, proliferation and apoptosis of OA cells were appraised via conduction of colony formation assay, MTT assay and flow cytometry assay. RESULT: Lower GAS5 expression and higher miR-196a-5p expression were associated with larger tumor size (≥5 cm) and more advanced FIGO stage (III-IV) of OA patients (P < 0.05). Transfection of si-GAS5, miR-196a-5p mimic or si-HOXA5 conferred OA cells with stronger viability, faster proliferation and smaller percentage of apoptosis (P < 0.05). After injecting mice models with si-GAS5, miR-196a-5p mimic or si-HOXA5, a larger tumor size was also observed within the rats (P < 0.05). GAS5 was indicated to directly target miR-196a-5p and modify its expression, and the targeted relationship also seemed to exist between miR-196a-5p and HOXA5 (P < 0.05). The HOXA5 was found to reverse the effects imposed by miR-196a-5p on viability, proliferation and apoptosis of OA cells (P < 0.05). CONCLUSION: LncRNA GAS5 depressed OA development by targeting miR-196a-5p and thereby down-regulating HOXA5 expression, providing substance for developing lncRNA-based strategies to treat OA.

Fucoxanthin and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Synergistically Promotes Apoptosis of Human Cervical Cancer Cells by Targeting PI3K/Akt/NF-κB Signaling Pathway.

BACKGROUND Fucoxanthin is a carotenoid present in the chloroplasts of brown seaweeds. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that selectively induces apoptosis in many tumor cells and is an attractive candidate for antitumor therapies. MATERIAL AND METHODS After human cervical cancer cell lines HeLa, SiHa, and CaSki were treated with fucoxanthin or TRAIL. Cell viability was determined by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) method. Apoptosis was measured by flow cytometry (FCM). Protein expression of phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt), phosphated Akt (p-Akt), NF-κB nuclear factor-k-gene binding (NF-κB). Phosphated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (p-IκBα), was measured by Western blot analysis. mRNA expression of Bax and Bcl2 was measured by RNA preparation and quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS In the present study, the effectiveness in terms of apoptosis was as follows: TRAIL plus fucoxanthin>fucoxanthin>TRAIL, indicating the combination of fucoxanthin and TRAIL, produced a strong synergistic effect on apoptosis in human cervical cancer cells. Additionally, we found that upstream signaling PI3K/Akt and NF-κB pathways-mediated cell apoptosis was activated by TRAIL and suppressed by fucoxanthin. By using PI3K and NF-κB inhibitors LY49002 and PDTC, we found that fucoxanthin- or TRAIL-induced apoptosis of human cervical cancer cells was obviously down-regulated. CONCLUSIONS Taken together, these findings suggest that fucoxanthin and TRAIL increased the apoptosis in human cervical cancer cells by targeting the PI3K/Akt/NF-κB signaling pathway.

Proteomic and transcriptomic studies of HBV-associated liver fibrosis of an AAV-HBV-infected mouse model.

BACKGROUND: Human hepatitis B virus (HBV) infection is an important public health issue in the Asia-Pacific region and is associated with chronic hepatitis, liver fibrosis, cirrhosis and even liver cancer. However, the underlying mechanisms of HBV-associated liver fibrosis remain incompletely understood. RESULTS: In the present study, proteomic and transcriptomic approaches as well as biological network analyses were performed to investigate the differentially expressed molecular signature and key regulatory networks that were associated with HBV-mediated liver fibrosis. RNA sequencing and 2DE-MALDI-TOF/TOF were performed on liver tissue samples obtained from HBV-infected C57BL/6 mouse generated via AAV8-HBV virus. The results showed that 322 genes and 173 proteins were differentially expressed, and 28 HBV-specific proteins were identified by comprehensive proteomic and transcriptomic analysis. GO analysis indicated that the differentially expressed proteins were predominantly involved in oxidative stress, which plays a key role in HBV-related liver fibrosis. Importantly, CAT, PRDX1, GSTP1, NXN and BLVRB were shown to be associated with oxidative stress among the differentially expressed proteins. The most striking results were validated by Western blot and RT-qPCR. The RIG-I like receptor signaling pathway was found to be the major signal pathway that changed during HBV-related fibrosis. CONCLUSIONS: This study provides novel insights into HBV-associated liver fibrosis and reveals the significant role of oxidative stress in liver fibrosis. Furthermore, CAT, BLVRB, NXN, PRDX1, and IDH1 may be candidates for detection of liver fibrosis or therapeutic targets for the treatment of liver fibrosis.

Association of toll-like receptor gene polymorphisms and its interaction with HPV infection in determining the susceptibility of cervical cancer in Chinese Han population.

The aim of the study is to investigate the association of several single-nucleotide polymorphisms (SNPs) within Toll-like receptors (TLRs) gene and additional gene-gene and gene-human papillomavirus (HPV) infection interaction with cervical cancer risk. A total of 1262 participants are selected, including 420 cervical cancer patients and 842 control participants. Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among five SNPs within TLR gene and HPV infection. Logistic regression was performed to calculate the ORs (95 %CI) for association of five SNPs within TLR gene and additional gene-HPV infection interaction with cervical cancer risk. Cervical cancer risk was significantly higher in carriers of the T allele of rs3775290 within TLR2 gene, the G allele of rs7873784 within TLR4 gene, and the A allele of rs352140 within TLR9 gene than those with wild genotype; adjusted ORs (95 %CI) were 1.78 (1.20-2.24), 1.65 (1.23-2.12), and 1.70 (1.16-2.31). However, we did not find any significant association of rs4986791 and rs11536889 with cervical cancer risk. GMDR analysis suggested a significant two-locus model (p = 0.0107) involving rs352140 and HPV infection. Subjects with HPV infection and rs352140-GA + AA genotype within TLR9 gene have the highest cervical cancer risk, compared to no HPV infection participants with rs352140-GG genotype, OR (95 %CI) = 3.22 (1.68-4.81). Pairwise LD analysis did not find any significant haplotype combination associated with cervical cancer risk. The minor alleles of TLR2-rs3775290, TLR4-rs7873784, and TLR9-rs352140, and interaction between rs352140 and HPV infection were all associated with increased cervical cancer risk.

Design and construction of a bifunctional magnetically recyclable 3D CoMn2O4/CF hybrid as an adsorptive photocatalyst for the effective removal of contaminants.

Herein, a magnetic microsphere CoMn2O4 (MS-CoMn2O4) with a 3D architecture was constructed directly on cellulose fiber (CF) substrates from wastepaper by a solvothermal synthesis method with further calcination treatment. The designed hybrid shows excellent dual functions including rapid catalytic oxidation of tetracycline (TC)/methylene blue (MB) and a high adsorption capacity. What's more, the hybrid is easily recycled using an external magnetic field. In comparison with that of pure MS-CoMn2O4, the enhanced adsorption ability and photocatalytic activity of MS-CoMn2O4/CFs can mainly be attributed to the introduced cellulose fiber supporter in the hybrid system. MS-CoMn2O4 incorporated CFs can improve the efficient separation of photogenerated electron-hole pairs and the transport pathway of electrons. More importantly, introduction of CFs can help to enrich and further improve the degradation efficiency of organic contaminants. The possible mechanism for the enhancement of the photocatalytic activity has been elucidated in detail. The reusability analysis revealed that the MS-CoMn2O4/CF hybrid exhibited superb cycling stability after 5 cycles. This study provides novel insights into the design and construction of high capacity sorbents as strongly adsorptive photocatalysts to perform catalytic degradation of organic contaminants.

A novel tuberculosis antigen identified from human tuberculosis granulomas.

Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis (Mtb). Although novel Mtb biomarkers from both the pathogen and host have been studied, more breakthroughs are still needed to meet different clinic requirements. In an effort to identify Mtb antigens, chaperone-peptide complexes were purified from TB infected lungs using free-solution isoelectric focusing combined with high resolution LTQ Orbitrap Velos mass spectrometry. Antigen specific cellular immune responses in vitro were then examined. Those efforts led to the identification of six Mtb peptides only identified in Tuberculosis lung samples and that were not found in the control samples. Additionally, antigen-specific IFN-γ secretion, T-cell proliferation, cytokine expression, and a cytotoxic assay were also evaluated. Among the peptides isolated, we identified a 34 amino acid peptide named PKAp belonging to a serine/threonine-protein kinase, as being able to generate Mtb-specific cellular immune responses as noted by elevated antigen-specific cytokine secretion levels, increased CD8(+) T-cell proliferation and a strong cytotoxic lymphocyte (CTL) response. Moreover, the immune stimulating abilities of PKAp were further validated in vivo, with target peptide immunized mice showing an increased cellular IFN-γ in both the lungs and spleen without causing immunopathogenesis. In conclusion, we identified novel functional Mtb antigens directly from the granulomatous lesions of Tuberculosis patients, inducing not only significant antigen-specific IFN-γ secretion but also a marked cytotoxic lymphocyte functional response. These findings indicated that PKAp has potential as a novel antigen biomarker for vaccine development.

Lysine-Specific Demethylase 1 (LSD1) Inhibitor S2101 Induces Autophagy via the AKT/mTOR Pathway in SKOV3 Ovarian Cancer Cells.

BACKGROUND S2101 is one of the most potent LSD1 inhibitors, which can inhibit ovarian cancer cells viability. This study aimed to detect the mechanism behind the anticancer properties of S2101 in SKOV3 ovarian cells. MATERIAL AND METHODS Cell viability was tested by Cell Counting Kit-8 (CCK-8) assay. Cellular apoptosis and autophagy were evaluated by flow cytometric analysis using Annexin-V/PI staining methods and Green fluorescent protein (GFP)-fused-LC3 (GFP-LC3), respectively. Western blotting was performed for analyzing the Bax, Bcl-2, mTOR, p- mTOR, p62, LC3-I, LC3-II, AKT, and p-AKT protein expression. RESULTS Our results show that the proportion of early apoptotic and late apoptotic cells increased significantly for cells treated with S2101 at a concentration of 100 µM for 48 h. Treatment of S2101 in SKOV3 cells resulted in upregulation of Bax and downregulation of Bcl-2 in a time-dependent manner, indicating that S2101 can induce apoptosis in SKOV3. There was a downward trend in the expression of p62 when the SKOV3cells were treated with 100 µm S2101 for 12 h, 24 h and 48 h. The conversion of LC3-I to LC3-II was increased significantly at 24 h and 48 h. Autophagy was induced by S2101 in SKOV3 cells, evidenced by an increase in punctuate localization of GFP-LC3 and a change in expression of autophagy-related proteins. CONCLUSIONS S2101 treatment decreased the levels of phosphorylated AKT and mTOR. S2101 inhibits SKOV3 cells viability and induces apoptosis and autophagy. The AKT/mTOR signaling pathway was found to be affected by S2101.

Exploration of novel cellular and serological antigen biomarkers in the ORFeome of Mycobacterium tuberculosis.

Increasing evidence demonstrates that antigen-specific cellular and humoral immunity plays an indispensable role in protection against Mycobacterium tuberculosis infection. Antigen is a key element in the development of a successful diagnostic method and vaccine. However, few antigens are available, and a systemic study on M. tuberculosis ORFeome-based antigen screening is still lacking. In the current study, a genome-wide examination was conducted on high-throughput M. tuberculosis encoding proteins and novel antigens were identified via a comprehensive investigation of serological and antigen-specific cellular responses. The serological immunoglobulin G level of each protein was detected in pooled sera from 200 pulmonary tuberculosis patients by means of semi-quantitative Western blot. Of the 1,250 detected proteins, 29 were present at a higher level relative to the commercialized 38-kDa protein. Furthermore, the top 12 of the 29 proteins had not been previously reported, and their antigenicity was validated in serum from each individual patient. Results confirmed that the 12 proteins displayed nearly identical immunoglobulin G antibody levels in patients with pulmonary and extrapulmonary tuberculosis. Antigen-specific cellular interferon-γ secretion was also evaluated using a cell-based ELISPOT assay. Thirty-four of the proteins were able to induce positive interferon-γ production by peripheral blood mononuclear cells from pulmonary tuberculosis patients as judged by positive (commercial ESAT-6 antigen) and negative controls. The top 4 candidates out of the 34 proteins displayed good accuracy ranging from 50% to 80% compared with the commercial ESAT-6 antigen. Subsequent epitope examination confirmed that a pool of peptides, including a 25aa peptide from Rv1198, demonstrated significant tuberculosis-specific cellular interferon-γ production. Overall, the current study draws significant attention to novel M. tuberculosis antigens, many of which have not been previously reported. This discovery provides a large amount of useful information for the diagnosis of tuberculosis and the development of vaccines to provide protection against tuberculosis.

Proteogenomic Analysis of Trichophyton rubrum Aided by RNA Sequencing.

Infections caused by dermatophytes, Trichophyton rubrum in particular, are among the most common diseases in humans. In this study, we present a proteogenomic analysis of T. rubrum based on whole-genome proteomics and RNA-Seq studies. We confirmed 4291 expressed proteins in T. rubrum and validated their annotated gene structures based on 35 874 supporting peptides. In addition, we identified 323 novel peptides (not present in the current annotated protein database of T. rubrum) that can be used to enhance current T. rubrum annotations. A total of 104 predicted genes supported by novel peptides were identified, and 127 gene models suggested by the novel peptides that conflicted with existing annotations were manually assigned based on transcriptomic evidence. RNA-Seq confirmed the validity of 95% of the total peptides. Our study provides evidence that confirms and improves the genome annotation of T. rubrum and represents the first survey of T. rubrum genome annotations based on experimental evidence. Additionally, our integrated proteomics and multisourced transcriptomics approach provides stronger evidence for annotation refinement than proteomic data alone, which helps to address the dilemma of one-hit wonders (uncertainties supported by only one peptide).

MicroRNA-9 promotes tumorigenesis and mediates sensitivity to cisplatin in primary epithelial ovarian cancer cells.

MicroRNAs play critical roles in regulating tumor occurrence and drug sensitivity in ovarian cancers. This study aimed to investigate the key members of MicroRNAs (miRNAs) involved in modulating tumor initiation and drug resistance in primary ovarian cancer cells. An in vitro assay based on tumor clonal formation was established to evaluate tumorigenicity and cisplatin sensitivity. By performing real-time polymerase chain reaction, we examined the expression of nine microRNAs associated with the pathology of ovarian cancers in primary ovarian tumor cells, which were surgically resected from 46 patients with distinct sensitivity to platinum-based chemotherapy. MiR-9, miR-145, and miR-429 were expressed significantly higher in drug-sensitive patients (n = 26) than in drug-resistant ones (n = 20), while higher miR-26a expression was found in resistant patients (p < 0.05). In addition, tumor cells from drug sensitive patients were more tumorigenic than those of drug resistance (p = 0.0013). Cisplatin treatment led to a sharp decrease of clonal formation of drug-sensitive cells but showed slight effects on drug resistant cells. Specific anti-miRs were then employed to downregulate the expression of microRNAs in primary tumor cells. Inhibition of miR-9 resulted in decreased clonal formation and sensitivity to cisplatin, while the knockdown of other three microRNAs did not show any influence in tumorigenesis and drug sensitivity. In conclusion, this study showed that in primary ovarian tumor cells, high expression of miR-9 was associated with enhanced tumorigenesis and increased sensitivity of the tumor cells to cisplatin treatment.