Bioremediation of tannery wastewater using UV-treated bacteria in the free and immobilized form

Aims@#Heavy metals are significant environmental pollutants and toxic to life and chromium (Cr) (VI) is one of them being discharged in the environment due to many human activities. The leather industry uses Cr(VI) salt in the tanning process, which is discharged untreated and becomes a source of many diseases. The use of microbes to remove metals is a cost-effective and clean method. The present study aims to isolate local and native microbes for Cr(VI) removal from tannery wastewater and enhance their capacity to bioremediate the tannery wastewater. Further, efficiency in free and immobilized forms was also checked.@*Methodology and results@#Microbes were isolated from a local tannery wastewater outlet and after many rounds of minimum inhibitory concentration, and concentration of 500 µg/mL was found to be that concentration at which microbes could survive, above which they died. The sequencing of 16S rRNA and its analysis showed that it was closely related to Staphylococcus saprophyticus and in the given study, it was named B6. At 37 °C, pH 7.5 and 120 h of incubation, it removed 77% Cr(VI) from the reaction mixture. B6 was exposed to UV to obtain mutant. Exposure of 15 min to a UV lamp gave mutant MB6, which showed a removal capacity of 77% after 72 h only. Cr(VI) removal capacity of the mutant was then analyzed in the free and attached form where coal and sodium alginate were used as solid surfaces. Mutants immobilized on coal showed 91% Cr(VI) removal after 96 h, while sodium alginate showed 58% Cr(VI) removal in 120 h, thus showing coal as a more effective surface for adsorption.@*Conclusion, significance and impact of study@#Our present study shows the use of cheap and environmentally friendly methods to remediate tannery wastewater, which is a big problem in a country like Pakistan. Pakistan is the second largest producer of leather but lacks a wastewater treatment facility. So, this method offers in-situ wastewater treatment, which can be further enhanced in different ways.

Molecular detection of drug-resistant Plasmodium falciparum mutants in Assam

Background & objectives: The spread of drug-resistant Plasmodium falciparum ( Pf) poses a serious threat to the control and elimination of malaria. The objective of this study was to detect the molecular biomarkers of antimalarial drug resistance in Pf in patients visiting a tertiary care hospital in Assam. Methods: Malaria was first detected in fever cases using microscopy and a rapid diagnostic test (RDT), and then confirmed using PCR. Pf chloroquine resistance transporter (Pfcrt), Pf multidrug resistance-1 (Pfmdr-1), and single-nucleotide polymorphisms linked to delayed parasite clearance after treatment with artemisinin MAL 10-688956 and MAL 13-1718319 and Kelch-13 propeller (PfK-13) genes were evaluated by PCR-restriction fragment length polymorphism (RFLP). Results: Sixty nine cases of malaria were found among 300 cases of fever. Of these, 54 were positive for Pf, 47 of which were confirmed by PCR. Pfcrt-K76T mutation was seen in 96.6 per cent and Pfmdr1-N86Y mutation in 84.2 per cent of cases. Mutation was not detected in MAL10 and MAL13 genes. Sequence analysis of Kelch-13 gene showed the presence of a novel mutation at amino acid position 675. Statistically, no significant association was found between the molecular biomarkers and demographic profile, clinical presentation and outcome of the cases. Interpretation & conclusions: Molecular surveillance is essential to assess the therapeutic efficacy of the drugs against circulating Pf isolates in Assam which are found to be highly resistant to CQ. The role of the new mutation found in the Kelch-13 gene in the development of artemisinin resistance in Assam needs to be thoroughly monitored in future research.

Prokaryotic expression and functional identification of androgen receptor functional domains / 安徽医科大学学报

Objective@#To construct the full-length prokaryotic expression plasmid of the wild type of androgen receptor (AR) and the truncated body of four functional domains，and to identify the fusion protein by Western blot and electrophoretic mobility shift assay ( EMSA) ．@*Methods@#Based on the pGEX-4T-1 vector ，the recombinant plasmids were constructed to express the full-length and functional domains of AR. IPTG was used to induce the expression of the recombinant proteins，which were isolated and purified by glutathione sepharose 4B beads under the optimized condition．The specific protein expression in the bacterial lysate and the purified protein isolated with glutathione sepharose 4B beads was identified by Western blot with AR antibody and GST labeled antibody．The purified protein was incubated with a fluorescent probe of the virus，and the complex was detected by electrophoresis in a non-denaturing gel. @*Results @# The prokaryotic recombinant plasmids of full length and three functional domain truncated AR were successfully constructed．The recombinant clones were identified by using bacterial culture as a template，and further verified by double enzyme digestion．It showed that there were identical bands in the same sizes as the inserted fragments．The nucleotide and the amino acid sequences were aligned to the reference sequence in NCBI GenBank．The GST fusion protein，GST-AR-NTD + DBD (96 ku) and GST-AR-NTD (86 ku) were successfully induced and verified． The purified protein could be directly combined with the viral genome DNA．@*Conclusion@#The prokaryotic expression conditions of truncated AR plasmid from the same gene sequence are different．The purified AR protein can be used to understand the direct interaction mechanism between functional domains of AR and other molecules.

The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Clinical characteristics of the 2019 novel coronavirus Omicron variant infected cases / 中华传染病杂志

Objective:To investigate the clinical characteristics and prognostic factors of 2019 novel coronavirus (2019-nCoV) Omicron variant infected cases.Methods:A total of 987 coronavirus disease 2019 (COVID-19) adult imported cases admitted to Shanghai Public Health Clinical Center, Fudan University from July 1, 2021 to January 6, 2022 were recruited. The cases were divided into Omicron group (193 cases) and non-Omicron group (794 cases) according to the genotype of the virus. The clinical data, imaging examination and laboratory results of two groups were collected and compared. Chi-square test and Mann-Whitney U test were used as statistical methods. Multiple linear regression analysis was used for multiple linear regression analysis. Results:The majority of patients in Omicron group were 18 to 30 years old, accounting for 51.3%(99/193), which was higher than 31.4%(249/794) in non-Omicron group. The difference was statistically significant ( χ2=52.75, P<0.001). The proportion of mild cases in Omicron group was 88.6%(171/193), which was higher than 81.6%(648/794) in non-Omicron group. The difference was statistically significant ( χ2=5.37, P=0.021). Cases with symptoms were more common in Omicron group than those in non-Omicron group (60.1%(116/193) vs 29.1%(231/794)), and the difference was statistically significant ( χ2=65.49, P<0.001), with the main clinical manifestations of sore/itchy throat, fever and cough/expectoration. The proportion of cases with pulmonary computed tomography (CT) imaging manifestations at admission in Omicron group was 13.0%(25/193), which was lower than that in non-Omicron group (215/794, 27.1%). The difference was statistically significant ( χ2=16.83, P<0.001). The proportion of cases with 2019-nCoV IgG positive at admission was 47.7%(92/193) in Omicron group, which was lower than 61.1%(485/794) in non-Omicron group, and the difference was statistically significant ( χ2=11.51, P<0.001). The hospitalization time of Omicron group was 20.0 (16.0, 23.0) d, which was longer than that of non-Omicron group (14.0 (10.0, 22.0) d), and the difference was statistically significant ( Z=-7.42, P<0.001). Multiple linear regression analysis showed that the time of hospitalization of cases with 2019-nCoV IgG positive at admission was shorter, while that of the cases with fever in Omicron group was longer (both P<0.050). Conclusions:The main clinical characteristics of cases with Omicron variant are fever and upper respiratory symptoms. Their pulmonary CT imaging manifestations are less, and the time of hospitalization is slightly longer. The time of hospitalization and the virus clearance time in Omicron variant infected cases with 2019-nCoV IgG positive at admission and not presented with fever are both shorter.

Different invasion efficiencies of Brucella abortus wild-type and mutantsin RAW 264.7 and THP-1 phagocytic cells and HeLa non-phagocytic cells / 대한수의학회지

Brucellosis is one of the common zoonoses caused by Brucella abortus (B. abortus). However, little has been reported on factors affecting invasion of B. abortus into host cells. To investigate cell-type dependent invasion of B. abortus, phagocytic RAW 264.7 and THP-1 cells and non-phagocytic HeLa cells were infected with wild-type and mutant B. abortus, and their invasion efficiencies were compared. The invasion efficiencies of the strains were cell-type dependent. Wild-type B. abortus invasion efficiency was greater in phagocytic cells than in epithelial cells. The results also indicated that there are different factors involved in the invasion of B. abortus into phagocytic cells.

Analysis of Lamivudine-resistant variants mutation patterns in patients with chronic hepatitis disease / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression.@*Methods@#Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed.@*Results@#Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*).@*Conclusions@#Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Effect of soybean ureases on seed germination and plant development

Abstract Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The ammonia (nitrogen (N) product of urease activity) is incorporated into organic compounds. Thus, urease is involved in N remobilization, as well as in primary N assimilation. Two urease isoforms have been described for soybean: the embryo-specific, encoded by the Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding gene was recently identified, designated Eu5, which encodes the putative protein product SBU-III. The present study aimed to evaluate the contribution of soybean ureases to seed germination and plant development. Analyses were performed using Eu1/Eu4/Eu5-co-suppressed transgenic plants and mutants of the Eu1 and Eu4 urease structural genes, as well as a urease-null mutant (eu3-a) that activates neither the ubiquitous nor embryo-specific ureases. The co-suppressed plants presented a developmental delay during the first month after germination; shoots and roots were significantly smaller and lighter. Slower development was observed for the double eu1-a/eu4-a mutant and the eu3-a single mutant. The N content in transgenic plants was significantly lower than in non-transgenic plants. Among the mutants, eu3-a presented the lowest and eu1-a the highest N content. Altogether, these results indicate that increased ureolytic activity plays an important role in plant development.

G1986A and G1899A in the pre-C region of HBV promote the serological conversion of e antigen / 实用医学杂志

Objective To investigate the correlation of the 1896 and 1899 mutations of hepatitis B virus (HBV)with the conversion of e antigen in serum and the progression of the disease. Methods 238 serum samples from the patients with HBsAg positive for over six months and HBV-DNA copy number > 5.0 × 102 IU/mL were collected,and the sequence analysis was used to analyze the nucleotide sequences of the 1896 and 1899 sites in the pre-C region of HBV. At the same time,the relevant clinical data and the expressions of HBeAg were collected,followed by Spearman correlation analysis and chi square test with SPSS 20.0. Results Both 1896 and 1899 sites in the pre-C region of HBV were mutated,and the base G was A,which was closely related to the expression of e antigen(P<0.05). Both G1896A and G1899A promoted the e antigen serological conversion ,and the e antigen serological conversion of G1899A was higher than that of G1896A. G1899A was associated with HBV related disease progression (correlation coefficient 0.280,P < 0.05),especially with the incurrence of HCC. Conclusions G1896A and G1899A in the pre-C region of HBV can promote the serological conversion of e antigen.

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief / 生物工程学报

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.

Crucial Residues Modulating Interface of hBcl-B - hBaxBH3 Heterodimer as Probed by Computational Methods

ABSTRACT Cancerous cells develop resistance to cell death by over expression of anti-apoptotic proteins, which are specific to interact with pro-apoptotic and BH3-only proteins of Bcl-2 family. Delineating crucial residues mediating the heterodimer complexes (anti-apoptotic proteins - pro-apoptotic/BH3-only proteins) is indispensable to develop specific antagonists to anti-apoptotic proteins. In these backgrounds, we have herein reported crucial residues of hBaxBH3 and hBcl-B (an anti-apoptotic protein specifically interacts with human Bax but does not interact with human Bak) for hetero dimerization of the polypeptides and as well validated the structural determinants of the polypeptides through variety of virtual 'alanine mutants' and 'switch mutants' by using an array of computational methods. Residues such as D53, S60, E61, K64, E69 and D71 of hBaxBH3 and R45, H50, F53, F54, Y57, M71, S74, V75, R86, V88, T89, F93 and F159 of hBcl-B were found to be crucial residues of the polypeptides for intermolecular interaction leading hetero dimerization. Moreover, 'pharmacophoric residues' for the hBaxBH3 and hBcl-B have also been figured out and rationalized.

Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

Morphological and SSR Assessment of Putative Drought Tolerant M3 and F3 Families of Wheat (Triticum aestivum L.).

In an attempt to develop drought tolerant genotypes of bread wheat, two procedures, i.e., mutation and hybridization were used to induce new genetic variation. Selection for high grain yield/plant (GYPP) and other desirable traits was practiced in the M2 populations of 7 gamma irradiated genotypes and F2 populations of 15 diallel crosses among 6 genotypes of wheat under well watering (WW) and water stress (WS) conditions. Progenies of these selections (53 M3 and 109 F3 families) and their seven parents were evaluated in the field under WW and WS. Significant yield superiority of twelve families (7 M3 ’s and 5 F2 ’s) over their original and better parents, respectively under WS reached 74.71% (SF9). These putative drought tolerant families were assessed on the DNA level using SSR analysis. Fifteen SSR primers were used for PCR amplification of the genomic DNA of these 12 selections and their parents. The SSR analysis proved that the 12 families are genetically different from their 7 parents, with an average polymorphism of 86.67%. The genetic similarities (Gs) ranged from 30% to 88%. Both mutants SF3 and SF4 exhibited very low Gs (42 and 40%, respectively) with their common parent (Giza-168), indicating that gamma rays were very effective in changing the genetic background of Giza-168 towards high GYPP under WS conditions. SSR assay permitted the identification of seven unique bands (5 positive and 2 negative) for three drought tolerant wheat genotypes (SF3, SF4 and Aseel-5). These bands might be considered useful as markers associated with drought tolerance in bread wheat breeding programs.

Rhodotorula glutinis and Its Two Mutants Ameliorate Hepato-Renal Dysfunction Induced by Ochratoxin A on Rats.

Aim: This study was designed to investigate the possible curative effect of Rhodotorula glutinis (R. glutinis) and its two mutants (Col-1R1 and Col-1R3) against hepatorenal toxicity induced by ochratoxin (OA) in rat. Methods: The strains of yeast Col- 1R1 and Col- 1R3 have been genetically improved and isolated from R. glutinis after colchicines treatment. OA was produced and determined from Aspergillus ochracus isolate from Egyptian corn. Experimental design: Five groups of rats were treated as follows: group 1, was the control group orally given 4 ml / Kg 0.1 M NaCOH3; group 2 treated with OA (1.7 mg /Kg).Groups 3, 4 and 5 orally administered the R. glutinis and its two mutants (50 X106 colony forming unit (cfu) / 10 ml saline / kg body weight) prior 1hr of OA -treatment for 15 successive days. Results: The studied autoploidy strains showed significant increase in caratenoids level, protease, β-1, 3-glucanase and chitinase activities when compared with the parental strain. Biochemical results revealed that OA significantly decreased serum total antioxidant capacity (TAC) and it caused elevation inserum transaminases (AST, ALT), creatinine, uric acid, nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and carcinoembryonic antigen (CEA) (P <0.05) as compared with the control group. The three tested yeasts significantly decreased the elevated values toward the normal levels and improved the pathological feature in liver and kidney tissues. Moreover, R. glutinis and the two mutants significantly reduced hepatorenal damaged arias, increased optical density of DNA and alleviated ochratoxin A-induced caspase-3 activation. The resultant effect of the two mutant strains had more powerful effect more than the wild strainto ameliorate hepatorenal dysfunction in ochratoxicosis-rat. Conclusion: Col-1R3 was more effective than Col-1R1 may be due to its higher contents of carotenoids, glucane and chitine, which act as antioxidants.

Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv.

Proteins are targets for photodegradation due to absorption of incident light by endogenous chromophores, e.g aromatic side chains. In this work we study the role of Trp-disulfide triads in the light induced loss of immunoglobulin activity. Study Design: We investigated a single chain variable fragment (scFv) of the Trp-disulfide triad containing monoclonal antibody 82D6A3. The scFv binds to von Willebrand factor (VWF) and upon illumination with near UV-B-light the scFv partially loses its binding capacity to VWF. In order to relate this observed degeneration to the specific Trp-disulfide triads, we mutated W35(VL) and W36(VH) which are in direct contact with the disulfide

Induction of resistant mutants of Salmonella enterica serotype Typhi under ciprofloxacin selective pressure.

Background & objectives: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. Methods: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. Results: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. Interpretation & conclusions: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 μg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective: To develop attenuated strains of Salmonella enterica serovar Typhi (. S. typhi) for the candidate vaccine by osmolar stress. Methods: S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1-0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models. Results: The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wild-type and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD

Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated. Methods: We examined the in vitro expression of HBsAg, ratio of cure and ‘e’ antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection. Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and ‘e’ antigen secretion. Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.