Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044.

The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S (CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCR analysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.

Longitudinal Observation of Immune Response for 23 Months in COVID-19 Convalescent Patients After Infection and Vaccination.

To better understand dynamic changes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) immune response, a prospective, single-center, cohort study was conducted on longitudinal immune response in 34 COVID-19 convalescent patients over 23 months in Chongqing. Two blood samples from convalescent patients were collected, first sample collected during 10-13 months (M10-13) after infection (pre-SARS-CoV-2 vaccination) and second sample collected during 20-23 months (M20-23) after infection (post-SARS-CoV-2 vaccination). The SARS-CoV-2-specific humoral and cellular immunity were traced by testing total antibody (Ab), anti-nucleocapsid (NP) immunoglobulin M (IgM), anti-NP immunoglobulin G (IgG), and anti-spike (S) IgG Abs, lymphocyte subset count, and Th1 cytokines. Healthy donors (30) were also included in the study as the uninspected healthy controls. Our data showed significant change in mean titer of SARS-CoV-2-specific Ab response from M10-13 to M20-23 included, namely, SARS-CoV-2-specific total Ab as 219 AU/mL increasing to 750.9 AU/mL; anti-NP IgM as 3.5 AU/mL decreasing significantly (p < 0.001) to 0.6 AU/mL; anti-NP IgG as 7.9 AU/mL increasing to 87.1 AU/mL; and anti-S IgG as 499.0 RU/mL increasing to 1,802.3 RU/mL. Our observations suggested that one vaccine dose might have been sufficient for COVID-19 convalescent patients. Larger sample sizes are needed to compare better immune effect of protein subunit vaccine. Besides, compared to healthy donors, patients had decreased CD3+ and CD8+ T lymphocyte counts during two periods. Patients had most cytokines recovered normally within 2 years, but IL-6 level was significantly elevated; however, IL-6 was negatively correlated with IgM and positively correlated with IgG. Changes in cytokines might have been caused by SARS-CoV-2 infection or vaccination. Patients with comorbidities were associated with decreased CD3+ and CD8+ T lymphocytes and lower Ab titers following SARS-CoV-2 vaccination. Vaccination enormously increased humoral immunity beneficial in COVID-19 convalescent patients. Elderly COVID-19 convalescent patients with comorbidities needed more attention.

Casein Reactivates Dopaminergic Nerve Injury and Intestinal Inflammation with Disturbing Intestinal Microflora and Fecal Metabolites in a Convalescent Parkinson's Disease Mouse Model.

Parkinson's disease (PD) is the fastest-growing neurodegenerative disease, with pathogenic causes elusive and short of effective treatment options. Investigations have found that dairy products positively correlate with the onset of PD, but the mechanisms remain unexplored. As casein is an antigenic component in dairy products, this study assessed if casein could exacerbate PD-related symptoms by stimulating intestinal inflammation and unbalanced intestinal flora and be a risk factor for PD. Using a convalescent PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the results showed casein reduced motor coordination, caused gastrointestinal dysfunction, reduced dopamine content, and induced intestinal inflammation. Meanwhile, casein disturbed gut microbiota homeostasis by increasing the Firmicutes/Bacteroidetes ratio, decreasing α-diversity, and caused abnormal alterations in fecal metabolites. However, these adverse effects of casein attenuated much when it had hydrolyzed by acid or when antibiotics inhibited the intestinal microbiota of the mice. Therefore, our results suggested that casein could reactivate dopaminergic nerve injury and intestinal inflammation and exacerbate intestinal flora disorder and its metabolites in convalescent PD mice. These damaging effects might be related to disordered protein digestion and gut microbiota in these mice. These findings will provide new insights into the impact of milk/dairy products on PD progression and supply information on dietary options for PD patients.

Synergistic Inhibitory Effect of Honey and Lactobacillus plantarum on Pathogenic Bacteria and Their Promotion of Healing in Infected Wounds.

Prevention and control of infections have become a formidable challenge due to the increasing resistance of pathogens to antibiotics. Probiotics have been discovered to have positive effects on the host, and it is well-known that some Lactobacilli are effective in treating and preventing inflammatory and infectious diseases. In this study, we developed an antibacterial formulation consisting of honey and Lactobacillus plantarum (honey-L. plantarum). The optimal formulation of honey (10%) and L. plantarum (1 × 109 CFU/mL) was used to investigate its antimicrobial effect and mechanism in vitro, and its healing effect on wound healing of whole skin infections in rats. Biofilm crystalline violet staining and fluorescent staining results indicated that the honey-L. plantarum formulation prevented the biofilm formation in Staphylococcus aureus and Pseudomonas aeruginosa and increased the number of dead bacteria in the biofilms. Further mechanism studies revealed that the honey-L. plantarum formulation may inhibit biofilm formation by upregulating biofilm-related genes (icaA, icaR, sigB, sarA, and agrA) and downregulating quorum sensing (QS) associated genes (lasI, lasR, rhlI, rhlR, and pqsR). Furthermore, the honey-L. plantarum formulation decreased the number of bacteria in the infected wounds of rats and accelerated the formation of new connective tissue to promote wound healing. Our study suggests that the honey-L. plantarum formulation provides a promising option for the treatment of pathogenic infections and wound healing.

Exercise training has a protective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model with improved neural and intestinal pathology and modified intestinal flora.

Parkinson's disease (PD) is a common neurodegenerative disease with the exact etiology still unclear, but gut microbial disorders are thought to be related to the initiation and progression of it. Exercise training has a significant effect on the intestinal flora, so to investigate the promotion effect of exercise training on Parkinson's disease, we performed a rotarod walking training (5 times a week at 25 rpm for 20 min for 8 weeks) on a chronic mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and observed the locomotor function of mice, function of dopaminergic neurons, intestinal mucosal barrier condition, intestinal inflammation and the structure and composition of intestinal flora. The results showed in these PD mice, exercise training improved their motility, increased the dopamine (DA) content in the striatum, along with promoted the gene expression of tyrosine hydroxylase and brain-derived neurotrophic factor in the striatum, which suggests this exercise training might protect striatal dopaminergic neurons from MPTP damage; the results also showed exercise training promoted recovery from ileal pathology, reduced the gene expression of intestinal inflammatory factors, and significantly altered the composition and structure of the intestinal flora in these mice.

iTRAQ-based quantitative proteomic reveals proteomic changes in Serratia sp. CM01 and mechanism of Cr(â ¥) resistance.

OBJECTIVE: Serratia sp. CM01 is a wild strain with the resistance and reduction ability of chromium(Ⅵ). The aim of this study it to investigate the underlying mechanisms of the Cr(Ⅵ) tolerance and reduction of strain CM01, and to explore its response to environmental pollution pressure at the molecular level. METHODS: The iTRAQ technique was utilized to investigate the differentially expressed protein patterns related to the Cr(Ⅵ)-resistance in wild-type strain CM01 and domesticated CM01. RT-qPCR was used to verify the expression levels of several functional genes. The cell surface hydrophobicity and autoaggregation, the intracellular glucose content, and the total superoxide dismutase (SOD) activity were determined. RESULTS: In total, 2750 proteins were detected and identified in WT CM01 and domesticated CM01. Compared with WT CM01, the iTRAQ results of 646 proteins were found to be significantly differentially expressed in domesticated CM01. There were 343 up-regulated and 303 down-regulated proteins, which mainly related to carbohydrate metabolism, stress responses, amino acid metabolism and some other systems. RT-qPCR results showed that the expression level of seven genes in domesticated CM01 were consistent with the iTRAQ proteomic profiles. The cell surface hydrophobicity, self-aggregation, intracellular glucose content and total SOD activity of domesticated CM01 with Cr(Ⅵ) treatment were significantly higher than without Cr(Ⅵ) treatment. CONCLUSION: Domesticated CM01 displayed a complex biological network to exhibit the tolerance of Cr(Ⅵ), which may be attributed to the following aspects: (a) CM01 reduced the consumption of glucose by inhibiting the metabolism of carbohydrates, which was an energy-saving survival mode. (b) The inositol phosphate metabolism pathway played an important role. (c) Oxidative stress proteins enhanced the adaptability. (d) CM01 enhanced biosynthesis of hydrophobic amino acids to resistance to Cr(Ⅵ). (e) Several key systems and proteins, such as UvrABC system, Lon protease, porin OmpC, also may play an important role.

Effects of exogenous sulfate on the chromium(VI) metabolism of chromium(VI)-resistant engineered strains.

OBJECTIVE: To explore the effects of exogenous sulfate on the efficiency of chromium(VI) metabolism of three chromium(VI)-resistant Escherichia coli strains (eChrA / eChrB / eChrAB) by adding chromium(VI)-resistance genes chrA and/or chrB, for better understanding and further application of these Cr(VI)-resistant strains in environmental and industrial chromium removal. METHODS: Based on three engineered Cr(VI)-resistant strains exposed to different concentrations of sulfate: i) Evaluation of Cr(VI) metabolism characteristics, including the growth rate, the Cr(VI) tolerance, the removal, absorption and efflux capacity of Cr(VI); ii) Detection the expressions of Cr(VI) resistance-related genes (chrA and chrB), and sulfate channel protein-related genes (sbp, cysA, cysU and cysW genes) by RT-qPCR. RESULTS: Exogenous sulfate enhanced the Cr(VI) tolerance and the removal rate of these three engineered Cr(VI)-resistant strains, and promoted their growth rate under Cr(VI) stress, while suppressed their absorption and efflux capacity. Under a certain sulfate concentration, the Cr(VI) tolerance, removal ability and efflux capacity of these three strains were ranked as follow: eChrAB > eChrA > eChrB, while ranked as eChrB > eChrA > eChrAB for the Cr(VI) absorption rate, respectively. Opposite to the Cr(VI) treatment, exogenous sulfate suppressed the transcription levels of the Cr(VI) resistance-related genes (chrA and chrB) with gradually increased concentrations, and reduced those of sulfate channel protein related genes (sbp,cysA, cysU and cysW) under the medium and high concentrations. CONCLUSION: Sulfate can enhance the Cr(VI) tolerance and growth of Cr(VI)-resistant strains, via inhibiting the Cr(VI) absorption and efflux in a concentration-dependent manner. The underlying mode of action might be the competition of transport channels between sulfate and Cr(VI), and the suppression of sulfate channel protein related genes expressions by exogenous sulfate. Our results demonstrated an appropriate supplication of exogenous sulfate could contribute to the Cr(VI) pollution management by genes chrA/chrB related Cr(VI)-resistant strains. Additionally, the engineered E. coli strain eChrAB showed more potential for the actual Cr(VI) pollution application than strain eChrA and eChrB.

Intragastric Administration of Casein Leads to Nigrostriatal Disease Progressed Accompanied with Persistent Nigrostriatal-Intestinal Inflammation Activited and Intestinal Microbiota-Metabolic Disorders Induced in MPTP Mouse Model of Parkinson's Disease.

Gut microbial dysbiosis and alteration of gut microbiota composition in Parkinson's disease (PD) have been increasingly reported, no recognized therapies are available to halt or slow progression of PD and more evidence is still needed to illustrate its causative impact on gut microbiota and PD and mechanisms for targeted mitigation. Epidemiological evidence supported an association between milk intake and a higher incidence of Parkinson's disease (PD), questions have been raised about prospective associations between dietary factors and the incidence of PD. Here, we investigated the significance of casein in the development of PD. The mice were given casein (6.75 g/kg i.g.) for 21 days after MPTP (25 mg/kg i.p. × 5 days) treatment, the motor function, dopaminergic neurons, inflammation, gut microbiota and fecal metabolites were observed. The experimental results revealed that the mice with casein gavage after MPTP treatment showed a persisted dyskinesia, the content of dopamine in striatum and the expression of TH in midbrain and ileum were decreased, the expression of Iba-1, CD4, IL-22 in midbrain and ileum increased continuously with persisted intestinal histopathology and intestinal barrier injury. Decreased intestinal bile secretion in addition with abnormal digestion and metabolism of carbohydrate, lipids and proteins were found, whereas these pathological status for the MPTP mice without casein intake had recovered after 24 days, no significant differences were observed with regard to only treated with casein. Our study demonstrates that intestinal pathologic injury, intestinal dysbacteriosis and metabolism changes promoted by casein in MPTP mice ultimately exacerbated the lesions to dopaminergic neurons.

Measuring urinary concentrations of neonicotinoid insecticides by modified solid-phase extraction-ultrahigh performance liquid chromatography-tandem mass spectrometry: Application to human exposure and risk assessment.

Neonicotinoid insecticides are the most widely used insecticides in the world. However, some experiments in vivo and vitro have shown association between neonicotinoids exposure and adverse effects in non-target mammals. The aims of this study were to 1) develop a robust method for simultaneous quantification of urinary neonicotinoids with a wide water solubility range by modified solid-phase extraction-ultrahigh performance liquid chromatography- tandem mass spectrometry and 2) quantify the concentrations of ten neonicotinoids in 386 adolescents in Chongqing of Southwest China by using the developed method and assess health risks of exposure to neonicotinoids. The introduction of extractive crystallization effectively removed interfering inorganic salts and improved the sensitivity of the method. The mean recoveries of all the analytes were satisfactory in the range of 89.1-104.8% and the limits of detection ranged from 0.001 to 0.02 ng/mL. The developed method was sensitive, accurate and suitable for trace detection and batch analysis in biomonitoring-based studies. Of the ten examined neonicotinoids, acetamiprid had the highest geometric mean concentration (49.43 µg/g creatinine), followed by clothianidin (5.01), imidacloprid (3.80), thiamethoxam (3.24), thiacloprid (2.25), nitenpyram (1.79), dinotefuran (1.76), sulfoxaflor (1.65), imidaclothiz (1.28) and flonicamid (1.01). High detection rates of neonicotinoids (79.3-100.0%) indicated a ubiquitous adolescents' exposure to neonicotinoids in urban areas of Chongqing. Nevertheless, hazard quotient and hazard index data exhibited a low health risk caused by the individual and cumulative exposure to neonicotinoids on the basis of the reference limit values recommended by the U.S. Environmental Protection Agency and the National Food Safety Standard of China.

Chromium metabolism characteristics of coexpression of ChrA and ChrT gene.

OBJECTIVE: Serratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. METHODS: Using Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. RESULTS: ChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). CONCLUSION: ChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.

Metabolic disturbance in hippocampus and liver of mice: A primary response to imidacloprid exposure.

Imidacloprid (IMI) is one of the most frequently used neonicotinoid insecticides, but recent studies have shown adverse effects on mammals. IMI was found to be neurotoxic and hepatotoxic. In the present study, the effects of repeated oral administration of two doses of IMI (5 and 20 mg/kg/day) for 28 days on hippocampus and liver of female KM mice were studied. The histopathological and biochemical experiments indicated obvious damages to the hippocampus and liver of mice in the high-dose group (20 mg/kg/day). Using a high-throughput metabolomics platform based on ultrahigh performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS), we studied effects of IMI on metabolic profiles in the hippocampus and liver of mice. Significant differences among the control group, the low-dose group and the high-dose group were clearly presented using multivariate analysis. The changed metabolic profile in the low-dose group (5 mg/kg/day) revealed that the metabolic disturbance in the hippocampus and liver of mice had been induced by low-dose of IMI, although no significant histopathological changes were observed in the low-dose group. Six differential metabolites in the hippocampus and 10 differential metabolites in the liver were identified as the possible biomarkers to distinguish IMI exposure from the control group using the variable importance in projection (VIP) value and receiver operating characteristic (ROC) analysis. The metabolism disturbances of important biochemical pathways in the hippocampus and liver of mice in the exposed groups were elucidated, mostly concentrated in lipid metabolism, amino acid metabolism, nucleotide metabolism, carbohydrate metabolism, and energy metabolism (p < 0.05). Such investigations give out a global view of IMI-induced damages in the hippocampus and liver of mice and imply a health risk associated with early metabolic damage in mice.

Twice subacute MPTP administrations induced time-dependent dopaminergic neurodegeneration and inflammation in midbrain and ileum, as well as gut microbiota disorders in PD mice.

Parkinson's disease (PD) is a common progressive neurodegenerative disease. PD produces a pathological state in the intestine and disordered gut microbiota (GM), which may be important for the pathogenesis and progression of PD, but it is not clear. To explore the conditions and characteristics of intestinal pathology and GM disorders when PD-related injuries occur, we used twice 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) subacute administration with an interval of 3 weeks (each was an intraperitoneal injection of 25 mg/kg MPTP for 5 consecutive days). We observed the changes in intestinal and brain immune status, intestinal barrier function and GM in different injury states one day, one week, and three weeks after the first stimulus and one day and one week after the second stimulus. Our study found that two subacute administrations of MPTP induced dopaminergic (DAergic) neuron injury and inflammation in the midbrain and ileum, impaired intestinal barrier function and GM disorders closely related to administration. These changes recovered after the first administration, but after repeated administration, some indicators showed more dramatic changes than during the first administration. Our results suggest that the intestinal tract is sensitive to PD-related injury, and the GM is susceptible to disturbances caused by intestinal function, which may be concerned in local immune disorders of the intestine.

Intestinal Pathology and Gut Microbiota Alterations in a Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Mouse Model of Parkinson's Disease.

Patients with Parkinson's disease (PD) often have non-motor symptoms related to gastrointestinal (GI) dysfunction, such as constipation and delayed gastric emptying, which manifest prior to the motor symptoms of PD. Increasing evidence indicates that changes in the composition of the gut microbiota may be related to the pathogenesis of PD. However, it is unclear how GI dysfunction occurs and how gut microbial dysbiosis is caused. We investigated whether a neurotoxin model of PD induced by chronic low doses of MPTP is capable of reproducing the clinical intestinal pathology of PD, as well as whether gut microbial dysbiosis accompanies this pathology. C57BL/6 male mice were administered 18 mg/kg MPTP twice per week for 5 weeks via intraperitoneal injection. GI function was assessed by measuring the 1-h stool frequency and fecal water content; motor function was assessed by pole tests; and tyrosine hydroxylase and alpha-synuclein expression were analyzed. Furthermore, the inflammation, intestinal barrier and composition of the gut microbiota were measured. We found that MPTP caused GI dysfunction and intestinal pathology prior to motor dysfunction. The composition of the gut microbiota was changed; in particular, the change in the abundance of Lachnospiraceae, Erysipelotrichaceae, Prevotellaceae, Clostridiales, Erysipelotrichales and Proteobacteria was significant. These results indicate that a chronic low-dose MPTP model can be used to evaluate the progression of intestinal pathology and gut microbiota dysbiosis in the early stage of PD, which may provide new insights into the pathogenesis of PD.

Chromium resistance characteristics of Cr(VI) resistance genes ChrA and ChrB in Serratia sp. S2.

OBJECTIVE: To find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored. METHODS: The PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored. RESULTS: ChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN-) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05). CONCLUSION: The Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer.

Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2.

This study is to investigate the genome sequence of Serratia sp. S2. The genomic DNA of Serratia sp. S2 was extracted and the sequencing library was constructed. The sequencing was carried out by Illumina 2000 and complete genomic sequences were obtained. Gene function annotation and bioinformatics analysis were performed by comparing with the known databases. The genome size of Serratia sp. S2 was 5,604,115 bp and the G+C content was 57.61%. There were 5373 protein coding genes, and 3732, 3614, and 3942 genes were respectively annotated into the GO, KEGG, and COG databases. There were 12 genes related to chromium metabolism in the Serratia sp. S2 genome. The whole genome sequence of Serratia sp. S2 is submitted to the GenBank database with gene accession number of LNRP00000000. Our findings may provide theoretical basis for the subsequent development of new biotechnology to repair environmental chromium pollution.

Reducing capacity and enzyme activity of chromate reductase in a ChrT-engineered strain.

In order to remediate the metal-contaminated soil and water ecosystems with microorganisms, an engineered strain, which contained the chromate reductase ChrT gene from Serratia sp. S2, was studied in detail for its Cr (VI) reduction efficiency, optimal culture condition and chromate reductase activity. Results demonstrated that the engineered strain had a high Cr (VI) reduction rate of up to 40% at a concentration of 50 mg/l after being cultured for 48 h. Additionally, the optimal culture conditions were pH 7.0 and 37°C. Furthermore, the carbon sources and metal cations exhibited significant effects on the Cr (VI) reduction rate of the engineered bacterium. Sodium lactate, sodium acetate, Cu2+, Co2+ and Pb2+ were positively correlated with the reduction rate. Chromate reductase was soluble and presented in the cytoplasm. Furthermore, the enzymatic activity with nicotinamide adenine dinucleotide phosphate, which was as an electron donor, reached 14.83 U/mg.

Rotenone-induced energy stress decompensated in ventral mesocerebrum is associated with Parkinsonism progression in rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, which is characterized by the hallmark feature of loss of dopaminergic neurons in the substantia nigra. Energy metabolic disorder is associated with the pathogenesis of PD; however, the development of this disorder is yet to be elucidated. PD-like characteristics have been demonstrated in a rotenone rat model. In the present study, energy metabolism status was investigated in a rat model following intraperitoneal treatment with 1.0 mg/kg rotenone every 48 h. The behavior and tyrosine hydroxylase-positive levels in the substantia nigra of rats that were treated with rotenone for 24 weeks demonstrated that these rats developed more severe parkinsonism, as compared with that were treated for 16 weeks. Detection of ATP, lactic acid, NADH dehydrogenase 1 mRNA and lactate dehydrogenase B mRNA levels in the ventral mesocerebrum (VM) and skeletal muscle (SM) of the rats that had been treated with rotenone for 16 and 24 weeks demonstrated that the energy stress induced by rotenone progressed in both VM and SM. Notably, the energy stress detected in VM was more severe, and this energy stress was decompensated in the VM of rats that had been treated with rotenone for 24 weeks. The progression of energy stress and the incidence of energy decompensation in VM may be important for the improvement of PD pathology.

Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

Rotenone induces KATP channel opening in PC12 cells in association with the expression of tyrosine hydroxylase.

The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibitor glibenclamide were used to treat cells for 24 h, and the expression of TH was detected. Our results showed that treatment of PC12 cells with glibenclamide for 24 h can notably promote TH expression and can also enhance the expression of TH which were reduced by rotenone. These data indicate that the energy states in PC12 induced by various doses of rotenone could significantly influence the opening states of KATP channels. However long-term energy stress may raise the opening rate and opening sensitivity of this channel. In addition, our results demonstrate for the first time that activation of plasma membrane KATP channels induced by rotenone inhibits TH expression which influences DA synthesis in PC12 cells.

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro.

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50~200 µg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.