Neuron-Derived Exosomes Promote the Recovery of Spinal Cord Injury by Modulating Nerve Cells in the Cellular Microenvironment of the Lesion Area.

During spinal cord injury (SCI), the homeostasis of the cellular microenvironment in the injured area is seriously disrupted, which makes it extremely difficult for injured neurons with regenerative ability to repair, emphasizing the importance of restoring the cellular microenvironment at the injury site. Neurons interact closely with other nerve cells in the central nervous system (CNS) and regulate these cells. However, the specific mechanisms by which neurons modulate the cellular microenvironment remain unclear. Exosomes were isolated from the primary neurons, and their effects on astrocytes, microglia, oligodendrocyte progenitor cells (OPCs), neurons, and neural stem cells were investigated by quantifying the expression of related proteins and mRNA. A mouse SCI model was established, and neuron-derived exosomes were injected into the mice by the caudal vein to observe the recovery of motor function in mice and the changes in the nerve cells in the lesion area. Neuron-derived exosomes could reverse the activation of microglia and astrocytes and promote the maturation of OPCs in vivo and in vitro. In addition, neuron-derived exosomes promoted neurite outgrowth of neurons and the differentiation of neural stem cells into neurons. Moreover, our experiments showed that neuron-derived exosomes enhanced motor function recovery and nerve regeneration in mice with SCI. Our findings highlight that neuron-derived exosomes could promote the repair of the injured spinal cord by regulating the cellular microenvironment of neurons and could be a promising treatment for spinal cord injury.

High-Performance Evolution of Ni2P@Hierarchical HZSM-5 as the Guaiacol Hydrodeoxygenation Catalyst.

Hydrodeoxygenation (HDO) is one of the effective methods to upgrade biomass pyrolysis oil, but the development of a low-cost, high-performance HDO catalyst still faces enormous challenges. In this work, a facile and eco-friendly approach is developed to synthesize the Ni2P@hierarchical HZSM-5 catalyst. Structure and acidity of the catalyst can be controlled by simply adjusting the proportions of ammonia solution and the silicon to aluminum ratio, which are closely related to the performance of the catalyst. Experimental results reveal that the Ni2P@hierarchical HZSM-5 catalyst with Si/Al = 85 under NH3·H2O/Ni = 14 exhibits the highest activity of 98% guaiacol conversion, along with a 78.8% yield of cyclohexane (reaction conditions: 300 °C, 3 MPa H2). In addition, the guaiacol reaction network is provided.

Mycobacterial 3-hydroxyacyl-l-thioester dehydratase Y derived from Mycobacterium tuberculosis induces COX-2 expression in mouse macrophages through MAPK-NF-κB pathway.

Tuberculosis (TB) is a leading cause of global mortality due to infectious diseases. Expression of cyclooxygenase-2 (COX-2) acts as an important influencing factor favoring bacillary survival during TB infection. In this study, we investigated the Mycobacterium tuberculosis proteins recognized by sera from TB patient collected before and after anti-TB therapy by dynamic immunoproteomics and identified a novel immune-regulating protein 3-hydroxyacyl-l-thioester dehydratase Y (HtdY), which could induce COX-2 expression in mouse macrophages. Signaling perturbation data showed that the activation of p38, ERK 1/2 and JNK 1/2 MAPK as well as NF-κB played critical role in this immune response. Taken together, our findings indicated that mycobacterial HtdY might contribute to the persistence of the TB infection by inducing COX-2 expression through MAPK-NF-κB signaling pathway.

The Wag31 protein interacts with AccA3 and coordinates cell wall lipid permeability and lipophilic drug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis, especially drug resistant tuberculosis, is a serious threat to global human health. Compared with other bacterial pathogens, M. tuberculosis gains stronger natural drug resistance from its unusually lipid-rich cell wall. As a DivIVA homolog, Wag31 has been demonstrated to be closely involved in peptidoglycan synthesis, cell growth and cell division. Previous research rarely investigated the role of Wag31 in drug resistance. In this study, we found Wag31 knock-down in Mycobacterium smegmatis resulted in a co-decrease of the resistance to four lipophilic drugs (rifampicin, novobiocin, erythromycin and clofazimine) and an increase in the cell permeability to lipophilic molecules. Six proteins (AccA3, AccD4 and AccD5, Fas, InhA and MmpL3) that are involved in fatty acid and mycolic acid synthesis were identified in the Wag31 interactome through Co-Immunoprecipitation. The Wag31-AccA3 interaction was confirmed by the pull-down assay. AccA3 overexpression resulted in a decrease in lipid permeability and an increase in the resistance of rifampicin and novobiocin. It confirmed the close relationship of lipophilic drug resistance, lipid permeability and the Wag31-AccA3 interaction. These results demonstrated that Wag31 maintained the resistance to lipophilic drugs and that Wag31 could play a role in controlling the lipid permeability of the cell wall through the Wag31-AccA3 interaction.

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated from south-central in China.

Rifampicin (RIF) and isoniazid (INH) Mycobacterium tuberculosis isolates were characterized from south-central China and transmission patterns within the Beijing genotype were detected in multidrug-resistant isolates. Six genetic regions, including rpoB for RIF, and katG, inhA, ahpC, mabA-inhA promoter and oxyR-ahpC intergenic region for INH were analyzed by DNA sequencing in 60 multidrug-resistant isolates, including 7 extensively drug-resistant isolates. The genomic deletion RD105 was characterized by genotyping. The results showed that 91.7% of MDR isolates carried mutations in the rpoB gene and 85.0% of the MDR isolates had at least one mutation in the INH resistance-associated loci detected. In total, these six genetic regions are responsible for 95.0% of MDR isolates. Mutations in the XDR isolates were focused on rpoB 531 or rpoB 526, and katG 315, correlating to a higher frequency level of resistance to RIF MIC ⩾8 µg ml⁻¹ and INH MIC ⩾4 µg m⁻¹. Three novel katG mutants (G273S, I266T and P232S) and three new alleles (E458A, S509R and P535S) in the rpoB gene were identified. Among the 85 clinical isolates, 78 are Beijing genotypes and the other 7 are non-Beijing genotypes. The results present the identification of genetic markers in M. tuberculosis isolates, some of which may be unique to this particular geographic niche. An understanding of the mutations in these drug-resistant strains may aid in choosing the appropriate chemotherapy regimens on the pharmacogenetic properties of the mutations for the prevention and control of tuberculosis.

Effects of Se deficiency on serum histamine concentration and the expression of histamine H2 receptor in the jejunum of chickens.

UNLABELLED: The present study was designed to investigate the influence of Se deficiency on serum histamine concentration and the expression of histamine receptor in the jejunum of chickens. Forty neonatal chickens were randomly divided into two groups. Experimental chickens were fed a low-Se diet (0.034 mg/kg), whereas chickens in the control group were fed a diet with a Se level of 0.229 mg/kg. Ten chickens were sacrificed on days 30, 45, 60 and 75. Blood and jejunum samples were collected. Histamine concentration in the jejunum was measured by ELISA, the jejunal mast cell (MC) ultrastructure was studied by transmission electron microscopy, and the expression level of histamine H2 receptor (H2R) mRNA in the jejunum was examined using real-time PCR. RESULTS: The jejunal histamine concentration in chickens fed the low-Se diet was significantly higher than that in the control group (P < 0.01). Se deficiency induced degranulation of MC in the jejunum of chickens in the low-Se diet group; their cytoplasm was filled with fused granules and vacuoles. The expression level of jejunal H2R mRNA in chickens fed the low-Se diet was also significantly higher than that in the control group (P < 0.01). The results obtained suggest that Se deficiency stimulates MC degranulation and release of histamine, binding H2R promotes both regulation of digestion and cell proliferation while protects the jejunum from injury induced by Se deficiency.

Rapid detection and monitoring therapeutic efficacy of Mycobacterium tuberculosis complex using a novel real-time assay.

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

Evaluation of a novel fusion protein antigen for rapid serodiagnosis of tuberculosis.

The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been a rapid and important diagnostic aid for tuberculosis (TB) control and prevention. However, any single antigen is not enough to be used to cover the antibody profiles of all TB patients. In this study, a novel fusion protein was constructed using gene splicing by overlap extension (SOEing), and then the antibody level against it in 171 TB patients and 86 controls was evaluated by enzyme-linked immunosorbent assay. Compared with the three individual antigen (16 kDa: sensitivity 19.9%, specificity 96.5%; MPT64: sensitivity 75.4%, specificity 34.9%; 38 kDa: sensitivity 33.3%, specificity 83.7%), the fusion protein antigen (sensitivity 42.1%, specificity 89.5%) gave the best diagnostic performance with the largest receiver operating characteristic curve area 0.656 (95% confidence interval [CI], 0.590-0.721; P<0.01). These results suggested that the novel fusion protein antigen successfully constructed by gene SOEing provided the improved diagnostic performance for TB, and other mycobacterial multiepitope fusion proteins may also be worthy of investigation for further enhancing the detection sensitivity.

Complete genome sequence of a novel clinical isolate, the nontuberculous Mycobacterium strain JDM601.

Mycobacteriosis is on the increase. Nontuberculous mycobacteria (NTM) are resistant to most antituberculosis drugs naturally. We determined the complete genome sequence of a novel NTM strain, JDM601, of the Mycobacterium terrae complex, which was isolated from a patient with tuberculosis-like disease and with various antibiotic resistances.

[Advances in the study of Mycobacterium tuberculosis protein phosphatase and its inhibitors].

Reversible protein phosphorylation regulates multiple biochemical events. Mycobacterium tuberculosis phosphatases play important roles in regulating the pathogen physiology and interference of host signaling. They are also involved in the evasion of host immune response and blockage of the phagosome-lysosome fusion. Selective inhibition of phosphatase represents an ideal new avenue of anti-tuberculosis drug design. In this paper, we update the progresses about the regulation network of Mycobacterium tuberculosis phosphatases including MptpA, MptpB, MstP, SapM and their inhibitors. These serve as the basis for further antituberculosis drug target.

[Dimo-thylidioctyl ammonium bromide-BCG polysaccharide nucleic acid adjuvant enhanced the immunogenicity of a Mycobacterium tuberculosis subunit vaccine].

OBJECTIVE: To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). METHODS: BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively. RESULTS: With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone. CONCLUSION: Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis.

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.

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.

[Adult-to-adult living donor liver transplantation using right lobe including middle hepatic vein].

OBJECTIVE: To investigate the feasibility and the clinical efficacy of right lobe including middle hepatic vein in adult-to-adult living donor liver transplantation. METHOD: We retrospectively analyzed the clinical data of 30 adult-to-adult living donor liver transplantation using right lobe including middle hepatic vein performed in our hospital from Feb. 2007 to Nov. 2007. RESULTS: The right lobes weighed 540-1058 g (median 708 g). The remnant liver volumes were over 30% of the total liver volume in all donors. No perioperative death was noted for among donors and recipients. Complications were recorded and cured in 4 donors (13.3%) and 7 recipients (23.3%). All the donors and the recipients were followed up for 2-8 months (median 5 months), during which no donor died and 1 recipient died from aspergillus infection 4 months after operation. CONCLUSION: Adult-to-adult living donor liver transplantation using right lobe graft including middle hepatic vein is a safe and effective technique.

[Advances in the study of the microbial efflux pumps and its inhibitors development].

Drug resistant bacteria is an increasingly urgent challenge to public health. Bacteria adaptation and extensive abuse of antibiotics contribute to this dilemma. Active efflux of antibiotics is employed by the bacteria to survive the antibiotic pressure. Efflux pump is one of the hot spots of current drug related studies and ideal targets for the improvement of treatment. The efflux pumps and related mechanisms of action, regulation of expression and methodologies were summarized. Comparative genomics analyses were employed to elucidate the underlying mechanisms of action and evolution of efflux pump as exemplified by the Mycobacterium in our lab, which is a crucial re-emerging threat to global public health. The pathway and state-of-art drug development of efflux pump related drugs are included too.

[The heterologous expression and purification of membrane protein from Mycobacterium tuberculosis].

Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis, whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

[Comparison of the proteomes of isoniazid-resistant Mycobacterium tuberculosis strains and isoniazid-susceptible strains].

OBJECTIVE: To determine whether there is a major difference in protein levels between isoniazid-resistant and susceptible M. tuberculosis strains. METHODS: The proteins extracted from 9 isoniazid-resistant M. tuberculosis strains and 7 isoniazid-susceptible M. tuberculosis strains were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Protein spots remarkly increased in isoniazid-resistant M. tuberculosis strains were digested in gel by enzyme and the mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in protein database search. These genes of all strains were sequenced. RESULT: In all protein spot differences, 5 protein spots were upregulated in isoniazid-resistant strains and identified as Rv1446c, Rv3028c, Rv0491, Rv2971, and Rv2145 by (MALDI-TOF-MS). CONCLUSION: These results suggest that the differentially expressed proteins from isoniazid-resistant strains might be used as potential immuno-diagnostic antigens and candidate novel drug targets against drug resistant tuberculosis.