43-kD Protein Research Articles

A novel gene, TARDBP, and the protein it encodes can predict glioma patient prognosis and establish a prediction model

BackgroundTDP-43 (43-kD transactive response DNA-binding protein) is a DNA-/RNA-binding protein that plays an important role in several nervous system diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Whether it plays an important role in glioma patients is unknown.MethodsDatasets were downloaded from the Chinese Glioma Genome Atlas (CGGA) website (http://www.cgga.org.cn/). Cox survival analysis was performed to determine the relationship between TARDBP gene expression and the overall survival of glioma patients. GO analyses were performed to determine the biological functions of the TARDBP gene. Finally, we used PRS type, age, grade, IDH mutation status, 1p/19q codeletion status, and expression value of the TARDBP gene to construct a prediction model. With this model, we can predict patients’ 1-, 2-, 3-, 5-, and 10-year survival rates.ResultsThe TARDBP gene plays an important role in glioma patients. The expression of the TARDBP gene has a significant correlation with glioma patient survival. We also constructed an ideal prediction model.ConclusionOur findings suggest that the TARDBP gene and the protein it encodes play important roles in glioma patients. The expression of the TARDBP gene has a significant correlation with the overall survival of glioma patients.

Discovery of novel benzimidazole derivatives as potent p300 bromodomain inhibitors with anti-proliferative activity in multiple cancer cells

Adenovirus E1A-associated 300-kD protein (p300) bromodomain, which regulates gene expression by recognizing acetylated lysine (KAc) of histone, is a promising target for the treatment of cancer. Herein, a series of potent p300 bromodomain inhibitors with novel CBP30-based scaffolds was discovered through bioisosterism and conformational restriction strategies. The most promising compound 1u showed more potent inhibitory activity (IC50 = 49 nM) against p300 bromodomain and anti-proliferative activity in various cancer cell lines compared to CBP30. Moreover, 1u suppressed the expression of c-Myc and induced G1/G0 phase arrest and apoptosis in OPM-2 cells more potently than CBP30. This study provides new lead compounds for further research on the biological functions of p300.

Thrombopoietin Induces Association of Crkl With STAT5 But Not STAT3 in Human Platelets

AbstractCrkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a β-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.

Characterization of antibody response against 16kD and 38kD of M. tuberculosis in the assisted diagnosis of active pulmonary tuberculosis.

BackgroundIn view of the inability of traditional etiological methods to diagnose pulmonary tuberculosis rapidly and effectively, the antibody responses against 38kD and 16kD-antigens of Mycobacterium tuberculosis (M. tuberculosis) were both detected in order to obtain a better serological detection method for M. tuberculosis.MethodsM. tuberculosis-secreted protein 38kD and membrane protein 16kD were prokaryotically expressed and purified, and then used as detection antigens. A novel evolved immunoglobulin-binding molecule (NEIBM)-ELISA method was used to detect antibody levels against 38kD and 16kD in active tuberculosis patients (confirmed tuberculosis cases and clinically diagnosed cases), to explore the significance of these two antigens in serological detection of M. tuberculosis, and to study the diagnostic value of the combined detection of the two antigens in active pulmonary tuberculosis.ResultsThe results showed that the positive detection rates of the 16kD antigen and 38kD antigen of M. tuberculosis were higher (about 44%) in the confirmed cases of tuberculosis, and there was no significant difference in the positive detection rates of the two antigens (P=0.786). The combined detection of these two antigens showed that the positive detection rate could be increased to 61.5%, which was significantly better than the detection effect of the two antigens alone. The positive detection rates of 16kD and 38kD antigens were 26–30% in clinically diagnosed tuberculosis cases, which were lower than those in confirmed tuberculosis cases, and there was no significant difference in the positive detection rates of the two antigens (P=0.242). The detection effect of the two combined antigens was better than that of the 16kD and 38kD antigens alone, but the detection rate was still lower than that of the confirmed tuberculosis cases.ConclusionsThis study found that the detection effect of 16kD and 38kD antigens was similar in confirmed cases and clinically diagnosed cases of pulmonary tuberculosis, and that the detection effect needs to be further improved. The combined detection of the two antigens showed a significantly better detection effect than the two antigens alone, suggesting that the combined detection of multiple antigens can be used for serological diagnosis of M. tuberculosis infection in clinic.

Pneumocystis carinii Major Surface Glycoprotein Dampens Macrophage Inflammatory Responses to Fungal β-Glucan.

Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) β-glucans. Major surface glycoprotein was shown to greatly reduce β-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc β-glucan. The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis β-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory β -glucan components of the organisms.

Cre Recombinase: a genomic manipulation tool

Comprehending human biology necessitates an understanding of molecular mechanisms that contribute to human physiology. For example, to determine why drug users relapse after stress, researchers must identify the neurological pathways involved in the addiction, and understand how certain stimuli trigger relapse. When using Cre recombinase and the LoxP DNA sites (from P1 bacteriophages), no limitations exist for knocking genes in or out and any genetic disease can be researched. By using the Cre‐LoxP system, researchers can manipulate gene expression in specific tissues by excising, inverting, inserting, and translocating sections of DNA. Cre, a 38kD protein with 341 amino acids, has 14 alpha helices and five strands of beta sheets. Cre has two domains, the N‐terminus (amino acids 20–129) and the C‐terminus (amino acids 132–341). The active site of Cre contains the residues of Arg173, His289, Arg292, Trp315, and Tyr324 which catalyzes the interactions with LoxP. This interaction facilitates the genome change and affects whether or not certain genes are expressed. The Brown Deer High School SMART (Students Modeling A Research Topic) Team has designed a model of Cre Recombinase using 3D printing technology to investigate structure‐function relationships. The Cre‐LoxP system has become a routine lab practice. For example, in animal models the interface of stress triggers and relapse is being researched using CRE‐delivery approaches to express genes in pathways that may be critical to stress and relapse. Using the Cre‐LoxP system in drug addiction studies is only one example of the system’s utility in understanding human physiology and disease mechanisms.

Autoantibodies as biomarkers for interstitial lung disease in idiopathic inflammatory myositis and systemic sclerosis: The case of anti-eIF2B antibodies

ObjectivesSerum autoantibodies are pivotal for the early detection of systemic autoimmune rheumatic diseases such as Systemic Sclerosis (SSc) and Poly/Dermatomyositis (PM/DM), and in some cases are associated with organ complications such as interstitial lung disease (ILD). A paradigmatic example is provided by the autoantibody against the Eukaryotic Initiation Factor 2B (eIF2B) that has been recently detected in SSc. MethodsSera from 118 patients with SSc, 8 Poly/Dermatomyositis, 2 overlap SSc/Polymyositis, 4 undifferentiated connective tissue disease-UCTD and 3 healthy controls were tested first by indirect immunofluorescence for anti-nuclear antibodies-ANA pattern. Further, we employed protein-radioimmunoprecipitation (IP) and IP- Western Blot for the detection and confirmation of anti-eIF2B antibodies. Serum findings were further correlated with the clinical features of patients. ResultsWe identified 3 SSc cases (2.5%) positive for anti-eIF2B antibodies while this autoantibody was not detected in control sera. Using protein-IP all three patients manifested the 38kD protein which is the antigenic target of anti-eIF2B antibodies, and this was associated with a cytoplasmic pattern at indirect immunofluorescence. The presence of anti-eIF2B was associated with ILD and a diffuse SSc variant, in one case in association with anti-Scl70/topoI. ConclusionsOur data confirm that a small subgroup (2.5%) of patients with SSc have detectable anti-eIF2B with cytoplasmic-positive staining at immunofluorescence and this reactivity is associated with ILD.

Chitooligosaccharides and Thermostable Chitinase Against Vulvovaginal Candidiasis and Saprophyte Fungi: LC Mass Studies of Shrimp Shell Fermentation by Bacillus altitudinis.

Some patients with candidiasis seek alternatives drug to treat vaginal yeast infection like herbal preparations and probiotics. However, the effectiveness of such treatments has not received much study. In this research, the unique chitinotrophic Bacillus was isolated on shrimp shell from salt lakes and identified as Bacillus altitudinis by 16SRNA sequencing. This strain produced a novel chitin-oligosaccharide material and thermostable chitinase (5.1units/ml) during 4days incubation on shrimp shell medium; nevertheless, its growth on nutrient agar was negative. The zymogram showed less than 50kD protein responsible for chitinase activities. The LC/MS detection of concentrate fermented products showed the production of oligosaccharide during chitin fermentation. As results of shrimp shell degradation, 65.6mg/l protein, 73.4mg/l N-acetyl glucose amine, and oligosaccharide were produced. Synergism activities of chitooligosaccharide and chitinase from this strain against fungi and pathogen candida (staining with methylene blue showed that almost 50% of 106 cells were died during 6h) are promising for new anti-fungal drug with no side effect.

The Association of CEP135 rs4865047 and NPY2R rs1902491 Single Nucleotide Polymorphisms (SNPs) with Rapid Progression of Proliferative Diabetic Retinopathy in Patients with Type 1 Diabetes Mellitus

BackgroundDiabetic retinopathy has a varied prevalence, severity, and rate of progression. The aim of this study was to determine whether the single nucleotide polymorphisms (SNPs) of the gene encoding a 135-kD centrosomal protein CEP135 rs4865047 and the gene encoding the type 2 NPY protein NPY2R rs1902491 were associated with the development of rapidly progressive proliferative diabetic retinopathy in patients with type 1 diabetes mellitus.Material/MethodsPatients with rapidly progressive proliferative diabetic retinopathy (n=48) were included in the study group. The control group (n=84) consisted of diabetes mellitus patients who had no proliferative diabetic retinopathy up to 15 years of diabetes duration. The reference group (n=90) included non-diabetic individuals who matched the study group by age and gender. The SNPs in the three groups were analyzed using real-time polymerase chain reaction (PCR) amplification.ResultsThe analysis of the distribution of genotypes in CEP135 rs4865047 and NPY2R rs1902491 detected significant differences only in the single nucleotide polymorphism rs4865047 genotype between the case and control group in comparison to the reference group. The co-dominant model showed that CEP135 rs4865047 was significantly associated with patients with rapidly progressive proliferative diabetic retinopathy (OR 7.2, 95% CI, 2.28–22.74, p=0.001). No significant association was found for the NPY2R SNP rs1902491 genotype.ConclusionsOur study reports a significant association of the CEP135 single nucleotide polymorphism rs4865047 genotype with rapidly progressive proliferative diabetic retinopathy and the control group. No significant association was found of the NPY2R single nucleotide polymorphism rs1902491 genotype.

Mass spectrometry identification of potential biomarker proteins in the 150-kD electrophoretic band in patients with schizophrenia.

Background:Diagnosing schizophrenia is primarily based on the presentation of defined signs and symptoms, none of which is pathognomonic for this group of syndromes. However, few significant genome-wide associations between schizophrenia and individual have detected. Protein profiling of candidate serum biomarkers in schizophrenia is therefore an area of great interest.Methods:In the present study, we used a combination of 7% polyethylene glycol (PEG) enrichment of immune complexes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate abnormal band, then analyse the band with liquid chromatography mass spectrometry (LC-MS).Results:There is a special 150-kD electrophoretic band in patients with schizophrenia, bipolar disorder, or depression relative to healthy controls (each 30 samples). Analysis of the band using LC-MS resulted in the identification of 11 serum proteins whose abundance was altered between patients and controls. Among them, 8 proteins (CFH, CFB, cDNA FLJ75416, zinc finger protein 729, isoform 2 of nidogen-1, diaphanous-1, cDNA FLJ77762, and cDNA FLJ58411) were up regulated, while one protein (isoform 1 of collagen alpha-1 (II) was down regulated in patients with schizophrenia, but only zinc finger protein 729 has statistics significance (P < .05). No differences were noted with regard to thrombospondin-1 or collagen alpha-2 (I) among the 3 groups. These proteins take part in several biological functions such as focal adhesion, complement cascades, ECM-receptor interaction, and Staphylococcus aureus infection.Conclusions:The 150-kD electrophoretic band or zinc finger protein 729 may become biomarkers in patients with schizophrenia. In the future increasing sample size and function research of zinc finger protein 729 should be executed continuously.

Lysosomal dysfunction in TMEM106B hypomyelinating leukodystrophy.

Transmembrane protein 106B ( TMEM106B ; NM_001134232) was recently identified as a gene responsible for a form of hypomyelinating leukodystrophy (HLD).1,2 All 5 cases identified to date carry the identical c.754 G > A, (p.Asp252Asn) mutation.1,2 Although the exact function is unknown,3 studies of TMEM106B in the context of frontotemporal lobar degeneration with 43-kD TAR DNA-binding protein (TDP-43) pathology (FTLD-TDP) indicate that TMEM106B likely acts as a lysosomal regulator and can modify risk for FTLD-TDP.4 However, the molecular effects of the (p.Asp252Asn) substitution have not yet been reported for TMEM106B -associated HLD. The HLDs are heterogeneous conditions, with the known disease genes playing roles in myelin sheath structure (e.g., PLP1 ) and other cellular functions that are not oligodendrocyte specific, including protein translation, molecular chaperoning, and cytoskeletal regulation.5 We set out to assess if this recurrent TMEM106B substitution was affecting lysosome biology or had an alternate role underlying the HLD pathogenesis. Implication of lysosome biology in HLD provides exciting new advances in our understanding of the molecular underpinnings of this condition and the complexities of neurodevelopment. The authors would like to thank the patient and family; without whom this work would not be possible. The authors also wish to acknowledge Dr. Wendy Mears for her tissue culture expertise. This work was supported by the Care4Rare Canada Consortium funded by Genome Canada, the Canadian Institutes of Health Research, the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec, and the Children's Hospital of Eastern Ontario Foundation.

The identification of autoantigens in mucous membrane pemphigoid using immortalized oral mucosal keratinocytes.

Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/β4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.

Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines.

Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.

SETD2 indicates favourable prognosis in gastric cancer and suppresses cancer cell proliferation, migration, and invasion

SET domain containing protein 2 (SETD2, also known as HYPB) is a 230-kD protein which is located at cytogenetic band p21.31 of chromosome 3. SETD2 is usually transformed or eradicated in multiple forms of tumours in humans. However, its primary function in gastric cancer (GC) remains unclear. In the current study, we investigated the mRNA and protein expression levels of SETD2 using immunohistochemistry, qPCR, RT-PCR, and immunoblotting. The function of SETD2 in GC cells was investigated by MTT and transwell assays. Our results revealed remarkably lower levels of SETD2 mRNA and protein in the tumour samples than in tumour-adjacent tissues. Decreased expression of SETD2 mRNA was observed in 122 (79.7%) of 153 primary tumour tissue samples. On the basis of the overall survival analysis, we could interpret that a low expression of SETD2 was correlated with a poor prognosis (P < 0.001, log-rank test). Multivariate survival analysis indicated that SETD2 was an obvious prognostic factor in patients with GC. SETD2 overexpression in GC cell lines significantly inhibited cell proliferation, migration, and invasion. Altogether, the investigation demonstrated the clinical significance of SETD2 expression, supporting the fundamental principle that a decrease in its level is an unfavourable event in the progression and prognosis of GC. Therefore, down-regulated SETD2 can presumably be a potential negative prognostic and progression marker for GC.

Evaluation of the Relation Between Omentin-1 and Vitamin D in Postmenopausal Women With or Without Osteoporosis.

Crosstalk between bone and adipose tissues is implicated in several pathologic conditions related to bone metabolism. Omentin-1, a 34-kD protein, is released from omental adipose tissue. A few studies indicated the effect of omentin-1 on bone health and bone mineral density (BMD) and the interaction of omentin-1 with vitamin D. Therefore, this study aimed to investigate the relationship between omentin-1, vitamin D, and BMD in postmenopausal women with osteoporosis compared with non-osteoporotic counterparts. Forty postmenopausal women with osteoporosis (OP), 40 counterparts without OP, and 30 premenopausal women were enrolled. Dual-energy X-ray Absorptiometry results, body mass index, and some demographic and biochemical data were recorded. Vitamin D (25-hydroxyvitamin D3) levels were measured using liquid chromatography-tandem mass spectrometry. Serum omentin-1 was determined using an enzyme-linked immunosorbent assay. Omentin-1 levels tended to increase in both postmenopausal women groups compared with the control group, but this increase was significant only in women with osteoporosis. Vitamin D levels were not different between the groups. When women were categorized according to vitamin D levels, women with normal vitamin D levels had significantly higher omentin-1 levels. A positive correlation was found between omentin-1 and vitamin D levels in all groups (r=0.197, p=0.041, n=110). The tendency to an increase in omentin-1 levels in postmenopausal women with osteoporosis may be due to a physiologic compensation against bone loss after menopause. The linear relationship between omentin-1 and vitamin D suggests that adipose tissue is one of the target tissues for the vitamin D effect.

Quercetin from Polygonum capitatum Protects against Gastric Inflammation and Apoptosis Associated with Helicobacter pylori Infection by Affecting the Levels of p38MAPK, BCL-2 and BAX.

Helicobacter pylori-associated gastritis is a major threat to public health and Polygonum capitatum (PC) may have beneficial effects on the disease. However, the molecular mechanism remains unknown. Quercetin was isolated from PC and found to be a main bioactive compound. The effects of quercetin on human gastric cancer cells GES-1 were determined by xCELLigence. H. pylori-infected mouse models were established. All mice were divided into three groups: control (CG, healthy mice), model (MG, H. pylori infection) and quercetin (QG, mouse model treated by quercetin) groups. IL-8 (interleukin-8) levels were detected via enzyme-linked immunosorbent assay (ELISA). Cell cycle and apoptosis were measured by flow cytometry (FCM). Quantitative reverse transcription PCR (qRT-PCR) and Western Blot were used to detect the levels of p38MAPK (38-kD tyrosine phosphorylated protein kinase), apoptosis regulator BCL-2-associated protein X (BAX) and B cell lymphoma gene 2 (BCL-2). The levels of IL-8 were increased by 8.1-fold in a MG group and 4.3-fold in a QG group when compared with a CG group. In a MG group, G0–G1(phases of the cell cycle)% ratio was higher than a CG group while S phase fraction was lower in a model group than in a control group (p < 0.01). After quercetin treatment, G0–G1% ratio was lower in a QG group than a MG group while S phase fraction was higher than a MG group (p < 0.01). Quercetin treatment reduced the levels of p38MAPK and BAX, and increased the levels of BCL-2 when compared with a MG group (p < 0.05). Quercetin regulates the balance of gastric cell proliferation and apoptosis to protect against gastritis. Quercetin protects against gastric inflammation and apoptosis associated with H. pylori infection by affecting the levels of p38MAPK, BCL-2 and BAX.

The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation

Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

The virulence of Streptococcus pneumoniae partially depends on dprA

Streptococcus pneumoniae is one of the most frequent opportunistic pathogens worldwide. DNA processing protein A (DprA) is an important factor involved in bacterial uptake and DNA integration into bacterial genome, but its role in S. pneumoniae virulence remains unclear. The aim of this study was to characterize the effects of the pneumococcal dprA gene on the pathogenesis of S. pneumoniae. To construct a dprA-deficient pneumococcal strain, the dprA gene of the S. pneumoniae strain D39 was inactivated. The virulence of this dprA-deficient strain, designated ΔD39, was compared with that of the wild-type strain by evaluating their respective capabilities to adhere to human pulmonary epithelial cells (PEC-A549) and by analyzing their choline-binding protein expression levels. In addition, the expression profiles of genes associated with virulence and host survival assays were also conducted with the mutant and the wild-type strain. Our results indicate that the capability of ΔD39 to adhere to the PEC-A549 airway cells was significantly lower (p < 0.01) compared with D39. Additionally, the 100-KD choline-binding protein was not detected in ΔD39. The addition of competence-stimulating peptide (CSP) lead to a significantly reduction of psaA mRNA expression in the dprA-deficient mutant and an increased level of psaA transcripts in the wild-type strain (p < 0.01). The median survival time of mice intraperitoneally infected with ΔD39 was significantly higher (p < 0.01) than that of mice infected with D39. The results of this study suggest that DprA has a significant effect on virulence characteristics of S. pneumoniae by influencing the expression of choline-binding protein and PsaA.

Characterization of Outer Membrane Proteome of Akkermansia muciniphila Reveals Sets of Novel Proteins Exposed to the Human Intestine.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.

Expression of 58-kD Microspherule Protein (MSP58) is Highly Correlated with PET Imaging of Tumor Malignancy and Cell Proliferation in Glioma Patients

Background/Aims: The nucleolar 58-kDa microspherule protein (MSP58) has important transcriptional regulation functions and plays a crucial role in the tumorigenesis and progression of cancers. 3'-deoxy-3'-[¹⁸F]fluorothymidine (FLT) has emerged as a promising positron emission tomography (PET) tracer for evaluating tumor malignancy and cell proliferation. Methods: In the present study, the expression of MSP58 was evaluated by immunohistochemistry and the corresponding PET image was examined using FLT-PET in 55 patients with various grades of gliomas. Results: The immunoreactivity score (IRS) of MSP58 increased with tumor grade with grade IV gliomas exhibiting the highest expression and showed a highly significant positive correlation with the Ki-67 index (r = 0.65, P < 0.001). The IRS of MSP58 in the tumor showed a highly significant positive correlation with corresponding FLT uptake value (r = 0.61, P < 0.001). The correlation between MSP58 expression and glioma malignancy was also confirmed by immunofluorescence, RT-PCR and western blot analysis. FLT uptake value also exhibited a highly significant positive correlation with the Ki-67 index (r = 0.59, P < 0.001). Kaplan-Meier analysis revealed that MSP58 expression has a significant prognostic ability for the overall survival time similar to that found in the uptake value of FLT-PET. Conclusion: These results indicate that MSP58 plays an important role in cell proliferation and will be one of the potential candidates of molecular therapy targeting proliferation. FLT-PET might be used as an early measure of treatment response in the proliferation-targeted therapy.