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Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can lead to serious central nervous system complications. The blood-brain barrier(BBB) is essential for maintaining the environmental stability of the central nervous system. Hyperglycemia may cause blood-brain barrier dysfunction and lead to central nervous system complications, but the mechanism is not clear. To explore the molecular mechanism of BBB injury caused by high glucose (HG), we hypothesize that p66Shc damaged BBB by promoting ROS production and autophagy. Human brain microvascular endothelial cells (HBMEC) were treated with different concentrations of glucose, Mito-tempo (MT), autophagy inhibitor (3-MA), autophagy inducer rapamycin (RAP), and P66Shc siRNA. Western blot was used to detect the expression of LC3, P62, ZO-1, claudin-5, and P66Shc in HBMEC. Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence staining was used to observe the expression of ZO-1, occludin, and ROS. The autophagy level was enhanced by HG. HG decreased the expression of ZO-1 and increased the expression of P66Shc and ROS. Inhibition of ROS levels under HG reversed the high level of autophagy induced by HG and increased the expression of ZO-1. The autophagy level was negatively correlated with the ZO-1 expression in HG. In HG, inhibit the expression of P66Shc reduced the ROS and autophagy levels and increased the ZO-1 expression. HG increased the autophagy level and destroyed the integrity of the tight junctions between HBMEC. Silencing P66Shc repaired HG damaged-HBMEC by inhibiting ROS production and reducing autophagy. To explore the molecular mechanism of BBB injury caused by high glucose (HG), we hypothesize that p66Shc damaged BBB by promoting ROS production and autophagy.

OBJECTIVE: To explore the protective effect of SBi4211 (heptamidine), an inhibitor of S100B, against central nervous system injury induced by HIV-1 envelope protein gp120. METHODS: In an in vitro model, U251 glioma cells were co-cultured with SH-SY5Y cells to explore the protective effect of SBi4211 against gp120-induced central nervous system injury. In a gp120 transgenic (Tg) mouse model (8 months old) mimicking HIV-associated neurocognitive disorder (HAND), the effect of treatment with gp120 or both gp120 and SBi4211 on neuronal activity and apoptosis were assessed using Cell Counting kit-8 (CCK-8) and flow cytometry. ELISA, Western blotting and immunohistochemistry were used to determine the expression levels of S100B, RAGE, GFAP, NeuN, Syn, MAP-2 and the inflammatory factors IL-6 and TNF-α. RESULTS: In the cell co-culture system, SBi4211 treatment significantly inhibited gp120-induced expression of S100B, RAGE and GFAP in U251 cells (P < 0.001), reduced the levels of inflammatory factors iNOS, IL-6 and TNF-α (P < 0.001) and enhanced the expressions of neuron-related proteins NeuN, Syn and MAP-2 (P < 0.001). In the transgenic mouse model, SBi4211 treatment significantly reduced the expressions of S100B, RAGE and inflammation levels (P < 0.05), inhibited the activation of astrocytes in the brain, and maintained the integrity of the neurons (P < 0.05). CONCLUSION: SBi4211 can protect neurons from gp120-induced neurotoxicity possibly by inhibiting the S100B/ RAGE-mediated signaling pathway.

Methicillin-resistant Staphylococcus aureus (MRSA) carries the resistance gene mecA in the staphylococcal cassette chromosome (SCC) that disseminates among staphylococci but the cell-to-cell transmission mechanism of SCC has not been clarified for half a century1. Here, we present evidence for efficient natural transformation in Staphylococcus aureus and its relevance in SCCmec transmission. We found that growth in biofilm conditions increased the transformation efficiency in a dependent manner on two component signal transduction systems, TCS13 (AgrCA) and TCS17 (BraSR). Strikingly, we demonstrate that natural transformation mediates the transfer of SCCmec from MRSA or methicillin-resistant coagulase negative staphylococci to methicillin-sensitive S. aureus. The site-specific insertion/excision system mediated by cassette chromosome recombinases was essential for SCCmec transformation while the stability of SCCmec varied depending on SCC types and recipients. We propose that natural transformation is the key process in the emergence of MRSA.

OBJECTIVE: To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout. METHODS: Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM(+/+), VIM(-/-), VIM(+/+)/gp120 Tg and VIM(-/-)/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting. RESULTS: The results of PCR showed the presence of the target bands in VIM(+/+), VIM(-/-), VIM(+/+)/gp120 Tg and VIM(-/-)/gp120 Tg mice. In VIM(-/-)/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected. CONCLUSION: We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.

Studies have shown that exposure to environmental tobacco smoke can increase the risk of bacterial meningitis, and nicotine is the core component of environmental tobacco smoke. Autophagy is an important way for host cells to eliminate invasive pathogens and resist infection. Escherichia coli K1 strain (E. coli K1) is the most common Gram-negative bacterial pathogen that causes neonatal meningitis. The mechanism of nicotine promoting E. coli K1 to invade human brain microvascular endothelial cells (HBMECs), the main component of the blood–brain barrier, is not clear yet. Our study found that the increase of HBMEC autophagy level during E. coli K1 infection could decrease the survival of intracellular bacteria, while nicotine exposure could inhibit the HBMEC autophagic response of E. coli K1 infection by activating the NF-kappa B and PI3K/Akt/mTOR pathway. We concluded that nicotine could inhibit HBMEC autophagy upon E. coli K1 infection and decrease the scavenging effect on E. coli K1, thus promoting the occurrence and development of neonatal meningitis.

OBJECTIVE: To construct a HIV-1 gp120 transgenic mouse model (gp120(+)) with α7 nicotinic acetylcholine receptor (α7nAChR) gene knockout. METHODS: The α7nAChR gene knockout mice (α7R(-/-)) were crossed with HIV-1gp120 transgenic mice (gp120(+)) to generate F1 generation mice. We selected the F1 mice with the genotype of α7R(+/-)/gp120(+) to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and α7nAChR inhibitor, and the expressions of IL-1β and TNF-α were detected using ELISA. RESULTS: The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (α7R(-/-)/gp120(+)) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express α7 nAChR but with high gp120 protein expression. In the in vitro cell experiment, treatment with gp120 promoted the secretion of IL-1β and TNF-α in BV2 cells, while inhibition of α7nAChR significantly decreased the expression of IL-1β and TNF-α (P < 0.001). CONCLUSION: By mating gp120 Tg mice with α7R(-/-) mice, we obtained gp120 transgenic mice with α7nAChR gene deletion, which serve as a new animal model for exploring the role of α7nAChR in gp120-induced neurotoxicity.

d(GC)n repeats form Z-DNA within promoter region and repress the promoter activity in Escherichia coli Shenghe Huang1, Chuxin Wu2, Dongming Li1, Haizhou Wang3, Zechang Rao1, Qiling Shen1, Chunxiang Chen1, Yong Liu3, Xun Xu1, and Chengyu Hu3,* Fuzhou Medical College, Nanchang University, Fuzhou 344000, China, Nanchang Teachers College, Nanchang 330103, China, and Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China

The immunostimulatory effect and antiviral activity of grass carp (Ctenopharyngodon idella) recombinant interferon (rCiIFN) were investigated in the present study. The rCiIFN fusion protein (≈ 38 kD) was expressed and purified from BL21 Escherichia coli. To evaluate in vivo protective efficacy of rCiIFN against grass carp hemorrhagic virus (GCHV) infection, we administered it by intraperitoneal (ip) injection and oral feeding in our experiments. Intraperitoneal injections with three different rCiIFN dosages (0.1, 1, 10 μg/g fish body weight) at 24 h prior to GCHV infection all improved the survival rate of the fish. The relative percentage survival (RPS) from 1 μg/g fish body weight injection was higher than those from other two dosages. Fish were fed with pellets mixed with rCiIFN at 1 μg/g fish body weight once daily for 1, 2 and 5 days before GCHV infection. The results indicated that the RPS from feeding for 2 consecutive days was higher than those from feeding for just 1 d or 5 consecutive days. Additionally, we observed that several IFN-stimulated genes (ISGs) were greatly up-regulated. After stimulation with rCiIFN, the expression levels of Mx and IFN genes in grass carp kidney cells (CIK) were highly enhanced and peaked within 6 and 12 h, respectively. By comparison with gene expression levels observed in PBS-injected controls, expressions of IFN, IRF1, IRF7, PKR, Mx and STAT3 were found to be significantly up-regulated in tested tissues of rCiIFN-injected fish, and most of these ISGs reached the highest level within 12 h post-treatment and declined within 72 h. After feeding fish once daily for 1–5 days with pellets mixed with rCiIFN at 1 μg rCiIFN/g fish body weight, it was found that the expression level of IFN was significantly up-regulated and reached its peak level in tissues of those fish treated for 1 or 2 days. These results demonstrated that rCiIFN had antiviral activity and ip injection as well as oral administration of rCiIFN was effective in protecting grass carp against GCHV infection.

The prevalence of type two diabetes (T2D) has been increasing sharply worldwide. Many recent studies have been done to determine the effective strategies for better management of type two diabetes. One of these strategies was the Ketogenic Diet, which when performed on rats and human showed very impressive results. The benefits of having some ketone bodies circulating in the body have numerous benefits such as weight loss, improvement of HbA1c levels, reversal of nephropathy, cardiac benefits and treatment for dyslipidemia.

To establish a method for detecting circulating brain microvascular endothelial cells (cBMECs), a novel biomarker of blood-brain barrier (BBB) injury.Blood samples were collected from 33 patients with AIDS encephalitis and 13 healthy subjects for detection of cBMECs, cECs and EPCs using magnetic affinity isolation and immune identification technology.The numbers of cBMECs, cECs and EPCs were significantly higher in the AIDS patients than in the control subjects (t=4.298, P0.01; t=4.886, P0.01; t=4.889, P0.01). An significant association was also noted between HIV load and cBMEC number (r=0.928, P0.01).We have successfully established a method for detecting peripheral blood cBMECs, which can be of important value in non-invasive assessment of BBB injury.

Genomic DNA of Proteus mirabilis C05028 was sequenced by an Illumina HiSeq platform and was assembled to 39 scaffolds with a total length of 3.8 Mb. Next, open reading frames (ORFs) were identified and were annotated by the KEGG, COG, and NR databases. Finally, we found special virulence factors only existing in P. mirabilis C05028.

Proteus mirabilis is a common urinary tract pathogen, and may induce various inflammation symptoms. Its notorious ability to resist multiple antibiotics and to form urinary tract stones makes its treatment a long and painful process, which is further challenged by the frequent horizontal gene transferring events in P. mirabilis genomes. Three strains of P. mirabilis C02011/C04010/C04013 were isolated from a local outbreak of a food poisoning event in Shenzhen, China. Our hypothesis is that new genes may have been acquired horizontally to exert the digestion tract infection and toxicity. The functional characterization of these three genomes shows that each of them independently acquired dozens of virulent genes horizontally from the other microbial genomes. The representative strain C02011 induces the symptoms of both vomit and diarrhea, and has recently acquired a complete type IV secretion system and digestion tract toxic genes from the other bacteria.

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene.

A novel gene named TaSC was cloned from salt-tolerant wheat. Northern blot showed that the expression of TaSC in salt-tolerant wheat was up-regulated after salt stress. Real-time quantitative PCR analyses showed that TaSC expression was induced by salt and ABA in wheat. Localization analysis showed that TaSC proteins were localized to the plasma membrane in transgenic Arabidopsis thaliana. The overexpression of TaSC in Col-0 and atsc (SALK_072220) Arabidopsis strains resulted in increased salt tolerance of the transgenic plants. TaSC overexpression in Col-0 and atsc significantly up-regulated the expression of AtFRY1, AtSAD1, and AtCDPK2. AtCDPK2 overexpression in atsc rescued the salt-sensitive phenotype of atsc. The TaSC gene may improve plant salt tolerance by acting via the CDPK pathway.

Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo.To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis.After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13.We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT.Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.