Over-expression of Tgs1 in Mycobacterium marinum enhances virulence in adult zebrafish.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can persist in the host for decades without causing TB symptoms and can cause a latent infection, which is an intricate challenge of current TB control. The DosR regulon, which contains approximately 50 genes, is crucial in the non-replicating persistence of Mtb. tgs1 is one of the most powerfully induced genes in this regulon during Mtb non-replicating persistence. The gene encodes a triacyl glycerol synthase catalyzing synthesis of triacyl glycerol (TAG), which is proposed as an energy source during bacilli persistence. Here, western blotting showed that the Tgs1 protein was upregulated in clinical Mtb strains. To detect its physiological effects on mycobacterium, we constructed serial recombinant M. marinum including over-expressed Tgs1(Tgs1-H), reduced-expressed Tgs1(Tgs1-L), and wild type M. marinum strains as controls. Tgs1 over-expression did not influence M. marinum growth under aerobic shaking and in hypoxic cultures, while growth advantages were observed at an early stage under nutrient starvation. Transmission electron microscopy revealed more lipid droplets in Tgs1-H than the other two strains; the droplets filled the cytoplasm. Two-dimensional thin-layer chromatography revealed more phosphatidyl-myo-inositol mannosides in the Tgs1-H cell wall. To assess the virulence of recombinant M. marinum in the natural host, adult zebrafish were infected with Tgs1-H or wild type strains. Hypervirulence of Tgs1-H was characterized by markedly increased bacterial load and early death of adult zebrafish. Remarkably, zebrafish infected with Tgs1-H developed necrotizing granulomas much more rapidly and in higher amounts, which facilitated mycobacterial replication and dissemination among organs and eventual tissue destruction in zebrafish. RNA sequencing analysis showed Tgs1-H induced 13 genes differentially expressed under aerobiosis. Among them, PE_PGRS54 (MMAR_5307),one of the PE_PGRS family of antigens, was markedly up-regulated, while 110 coding genes were down-regulated in Tgs1-L.The 110 genes included 22 member genes of the DosR regulon. The collective results indicate an important role for the Tgs1 protein of M. marinumin progression of infection in the natural host. Tgs1 signaling may be involved in a previously unknown behavior of M. marinum under hypoxia/aerobiosis.

Recurrent gain-of-function USP8 mutations in Cushing's disease.

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

Growth differentiation factor 15, ischemia modified albumin and pregnancy-associated plasma protein A in patients with coronary artery disease.

BACKGROUND: Growth differentiation factor 15 (GDF-15), ischemia modified albumin (IMA) might aid in the early diagnosis and risk stratification of patients with coronary artery disease (CAD), while pregnancy associated plasma protein-A (PAPP-A) can serve as a useful marker of vulnerable plaques and acute coronary syndrome (ACS). We sought to determine serum levels of GDF-15, IMA, PAPP-A and evaluate their diagnostic value in different types of CAD. METHODS: We detected serum levels of GDF-15, IMA and PAPP-A in 348 patients with CAD and 205 controls. Levels of high-sensitivity C reactive protein, creatine kinase isoenzyme MB, and high-sensitivity cardiac troponin-T, which represent biomarkers of inflammation, damage or necrosis, were also evaluated. RESULTS: There were significant differences of GDF-15, IMA and PAPP-A in patients with CAD compared with the controls, where GDF-15 seemed to be associated with severity of CAD. GDF-15 demonstrated a sensitivity of 84.0% and specificity of 84.8% for diagnosis of UAP, while the negative predictive value was 87.4%. The sensitivity and specificity, negative predictive value of IMA in the diagnosis of UAP were 70.0%, 69.5%, and 70.0%, respectively. PAPP-A had the lowest sensitivity and negative predictive value compared with other markers. CONCLUSIONS: GDF-15 demonstrated a significant improvement in earlier prediction and assessment of overall patient risk of UAP comparing to IMA and PAPP-A.

[Chemical constituents from traditional Chinese medicine Siegesbeckia pubescens].

Column chromatography on silica gel was used to study the chemical constituents of traditional Chinese medicine Siegesbeckia pubescens. The chemical structures of the separated compounds were elucidated by spectroscopic data analyses. As a result, eighteen compounds were obtained and identified as 3, 4'-dimethoxy quercetin(1), 3, 3', 4'-trimethoxy quercetin(2), 3, 3'-dimethoxy quercetin(3), 7, 3', 4'-trimethoxy luteolin(4), ursolic acid(5), 2ß,19α-dihydroxyursolic acid(6), 2ß-hydroxyursolic acid (7), stigmasterol-7-one(8), 5α, 8α-epidioxy-24(R)-methyl-cholesta-6, 22-diene-3ß-ol(9), ß-sitosterol(10), 2, 6-di(3-hydroxy-4-methoxyphenyl)-3, 7-dioxacyclo [3. 3. 0] octane (11), aurantiamide acetate (12), 3-(m-hydroxyl-p-methoxy)-N-(2'-p-hydroxyl-phenethyl)-2E-acrylamide(13), p-hydroxy benzaldehyde (14), m-hydroxy-p-methoxy benzaldehyde (15), 3, 4, 5-trimethoxybenzoic acid(16), monoethyl malonate(17), and p-hydroxylcinnamic acid(18). Among them, compounds 1-9, 11-18 were isolated from this plant for the first time.

Comparing the performance of traditional non-treponemal tests on syphilis and non-syphilis serum samples.

The goal of the present study was to determine the performance of two traditional non-treponemal tests for syphilis. Syphilis sera (n = 209) included different stages of disease, and control sera (n = 247) were from patients with tumours, leprosy, systemic lupus erythematosus, hepatitis, pregnant women and healthy individuals. Treponema pallidum ELISA, Treponema pallidum particle agglutination and rapid treponema-specific tests were used as gold standards. Rapid plasma reagin or toluidine red unheated serum test had a sensitivity and specificity of over 95%. False-negative reactions of rapid plasma reagin and toluidine red unheated serum test were observed mainly in primary and latent syphilis cases, and false-positive reactions were present in systemic lupus erythematosus, hepatitis-infected patients. Overall, both non-treponemal tests had high sensitivities and specificities making the assays attractive as screening tests for syphilis. When examined on WHO reference serum samples and based on lower limits of detection, non-treponemal tests were less sensitive than treponema-specific tests.

[Screening and culture of rat spermatogonial stem cells].

AIM: To establish a system of screening and culture of rat spermatogonial stem cells (SSCs). METHODS: We prepared the rat testis cells using the improved two-step enzymatic digestion, and then isolated the rat SSCs by the differential adherence selection method. The highly enriched SSCs were cultured in the serum-free culture medium of DMEM/F12 supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GFRα1 and basic fibroblast growth factor (bFGF) and on rat embryonic fibroblast (REF)feeder layer. The activity of stem cells was examined morphologically and by RT-PCR and immunocytochemical analysis for the SSCs marker gene expressions. RESULTS: The modified two-step enzymatic digestion could effectively isolate the rat testis cells, and from the isolated testis cells, rat SSCs could be successfully purified by the improved method of differential adherence selection. After over-20-day culture of the rat SSCs in serum-free medium, big colonies of SSCs were observed, and the activity of SSCs got affirmed by the positive expressions of SSCs marker genes. CONCLUSION: The system of screening and culture of rat SSCs has been established, which provide a basis for the long-term culture of rat and other animal SSCs.

Phenazinolins A-E: novel diphenazines from a tin mine tailings-derived Streptomyces species.

Phenazinolins A-E (1-5), which possess a carbon skeleton unique to diphenazines (the azabicyclo[3.3.1]nonadienol moiety in 1-3 and the oxabicyclo[3.3.1]nonadienol moiety in 4 and 5), were isolated from tin mine tailings-derived Streptomyces diastaticus YIM DT26, with 1-3 exhibited appreciable cytotoxicity and antibiotic effects.

Ankrd7, a novel gene specifically expressed in Sertoli cells and its potential roles in Sertoli cell maturation.

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immuno-histochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.

Application of LightCycler polymerase chain reaction and melting curve analysis to the authentication of the traditional Chinese medicinal plant Cimicifuga foetida.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and fluorescence melting curve analysis of LightCycler real-time polymerase chain reaction products were exploited for their applications in the authentication of the traditional Chinese medicinal plant Cimicifuga foetida from four substitutes: C. heracleifolia, C. dahurica, C. acerina, and C. simplex. According to the melting temperature--which is a function of the GC/AT ratio, length, and nucleotide sequences of the amplified product--C. foetida was differentiated from C. heracleifolia, C. dahurica, C. acerina, and C. simplex. Melting curve analysis offers a rapid and reliable method for the authentication of the traditional Chinese medicinal plant C. foetida.

WITHDRAWN: Fem1b interacts with Ankrd37 in mouse testis and induces its degradation by ubiquitin-mediated proteolysis pathway.

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[The proliferation profile of mouse spermatoganial stem cells in three types of culture media].

AIM: To establish an in vitro long-term culture system of mouse Spermatogonial stem cells (SSCs). METHODS: Three types of serum-free culture media, namely, DMEM/F12, KSR (KnockoutM Serum Replacement) and StemPro-34 SFM, to which the same growth factors including GDNF, soluble GFRalpha1 and bFGF were added equally, and MEF(mouse embryonic fibroblast) feeder layer were used to culture mouse SSCs enriched from pup mice testes through differential adherence selection. The activity of stem cells was examined morphologically, and the marker gene expression of SSCs was detected by RT-PCR and immunocytochemical analysis. RESULTS: The activity of SSCs cultured in DMEM/F12 and KSR serum-free media was only maintained for 6-7 days. However, the StemPro-34 SFM medium could maintain the proliferation of cultured SSCs nearly one month. CONCLUSION: StemPro-34 SFM serum-free medium sustains the proliferation of mouse SSCs in vitro.

Benzimidazolium 2-(2,4-dichloro-phen-oxy)acetate monohydrate.

In the crystal of the title hydrated mol-ecular salt, C(7)H(7)N(2) (+)·C(8)H(5)Cl(2)O(3)·H(2)O, the components inter-act by way of N-H⋯O and O-H⋯O hydrogen bonds, leading to chains propagating in [100].

Evaluation of a recombinant BCG expressing antigen Ag85B and PPE protein Rv3425 from DNA segment RD11 of Mycobacterium tuberculosis in C57BL/6 mice.

Antigen 85B (Ag85B) is an important immunodominant antigen of Mycobacterium tuberculosis, and is a very promising vaccine candidate molecule. Rv3425 is a member of the subgroup 3 of the PPE family, which does not exist in all BCG strains. In this study we constructed a new rBCG which included this united gene (Ag85B-Rv3425). The level of antigen-stimulated T cells expressing IFN-gamma was significantly higher in the C57BL/6 mice vaccinated with rBCG::Ag85B-Rv3425 than with BCG. In addition, the sera from mice immunized with rBCG::Ag85B-Rv3425 revealed an increase in the specific immunoglobulin G titers than that from mice immunized with BCG. Antigen specific IgG subclass analysis showed that rBCG::Ag85B-Rv3425 tended to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-gamma. These results suggested that this rBCG::Ag85B-Rv3425 could be a strong vaccine candidate for further study.

Identification and quantification of the traditional Chinese medicinal plant Gentiana macrophylla using Taqman real-time PCR.

Gentiana macrophylla Pall. is a commonly used antirheumatic herb. There are four species of Gentiana recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as G. macrophylla, and thus the therapeutic effects of G. macrophylla are not achieved. A novel one-step methodology based on real-time polymerase chain reaction (PCR) technology has been developed for the identification of G. macrophylla. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.

[Serum-free knockout SR medium supports the short-time viability of mouse spermatogonial stem cells].

Spermatogonial stem cells (SSCs), the only stem cells that are capable of transmitting genetic information to subsequent generations in adult animals,have abilities to self-renew and differentiate into spermatozoa. Therefore, SSCs are not only the study object of stem cell biology, but also the valuable resource of in vitro spermatogenesis, gene analysis and functional genomics. In the present study, we selected the mouse SSCs from mice on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders by differential adherence selection in DMEM/F12 containing 5% FBS, and used a serum-free defined medium prepared with Knockout SR basal medium to culture SSCs. Our results showed that enriched SSCs could be maintained for a short of time and form colonies, but the proliferation of SSCs was unconspicuous, suggesting that some factors that are detrimental to the self-renewal of SSCs possibly existed in the Knockout SR basal medium. However, the use of KSR medium, which was widely used in the culture of embryonic stem cells (ESCs), in the SSC maintenance was the first time, and our results indicated that the Knockout SR basal medium don't appropriate for the long-time culture of SSCs.

Differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from adulterants by TaqMan real-time polymerase chain reaction.

DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional chinese medicinal plants euphorbia humifusa and e. maculata from three related adulterants e. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.

Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis.

To develop novel delivery system for tuberculosis (TB) subunit vaccine, biodegradable chitosan microspheres were prepared and used to deliver a fusion protein, Ag85B-MPT64(190-198)-Mtb8.4 (AMM for short), made from three Mycobacterium tuberculosis genes. AMM-loaded microspheres were first characterized for their morphology, size, zeta potential, loading efficiency, and in vitro release of AMM. C57BL/6 mice were immunized at weeks 1, 3 and 5 subcutaneously with AMM formulated in chitosan microspheres, in incomplete Freund's adjuvant (IFA), or in phosphate-buffered saline (PBS), respectively. Three weeks after the last immunization, humoral and cell-mediated immune responses were examined. It was shown that the microspheres bound AMM quite efficiently (loading efficiency: >99%). AMM-loaded chitosan microspheres were observed as aggregated shapes with the average particle size of 5.78+/-0.65 microm and zeta potential of 32.77+/-1.51 mV. In vitro release studies revealed that only small amount of antigen was released in 16 days. Following subcutaneous administration, splenocytes immunized with AMM in chitosan microspheres produced higher levels of IFN-gamma compared to administration of AMM in PBS upon stimulation with Ag85B and synthetic peptide MPT64(190-198). The levels of Ag85B-specific IgG (H+L), IgG1 and IgG2a in sera of mice immunized with AMM in chitosan microspheres were also higher than those with AMM in PBS. These results indicate that chitosan microspheres when used as a carrier for fusion protein AMM could elicit strong humoral and cell-mediated immune responses.

Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis.

As a cell-specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1-positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.

In vitro propagation of spermatogonial stem cells from KM mice.

Spermatogonial stem cells (SSCs) are a unique type of stem cells in that they transmit genetic information to the next generation by producing sperms. Studies of SSC proliferation and differentiation have been hampered by the inability of reconstructing these processes in vitro, particularly in a serum-free culture system. Several groups have reported the long term culture of SSCs during which SSCs self-renew and restore spermatogenesis when transplanted back to recipient testes. However, different protocols and mice with particular genetic background have been used by different laboratories, and the techniques have not been adopted widely. In the present study, we first established a SSC isolation and culture system composed of differential adherence selection of SSCs, serum-free medium and mouse embryonic fibroblast (MEF) feeder cells. SSCs from KM pups could be cultured on MEF feeders in StemPro-34 SFM Medium supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GDNF family receptor alpha-1 (GFRa1) and basic fibroblast growth factor (bFGF) for 1 month. These SSCs were characterized morphologically and by examining the expression of marker genes. Expression of Oct4 and Sox2, which are crucial factors in embryonic stem cell (ESC) self-renewal, were detected in our cultured SSCs, suggesting that SSCs may share with ESCs some common mechanisms in self-renewal regulation. We also found that LIF had no effect on the proliferation of cultured SSCs derived from KM mice.