Ciprofloxacin induced antibiotic resistance in Salmonella Typhimurium mutants and genome analysis.

Antibiotic resistance of Salmonella species is well reported. Ciprofloxacin is the frontline antibiotic for salmonellosis. The repeated exposure to ciprofloxacin leads to resistant strains. After 20 cycles of antibiotic exposure, resistant bacterial clones were evaluated. The colony size of the mutants was small and had an extended lag phase compared to parent strain. The whole genome sequencing showed 40,513 mutations across the genome. Small percentage (5.2%) of mutations was non-synonymous. Four-fold more transitions were observed than transversions. Ratio of < 1 transition vs transversion showed a positive selection for antibiotic resistant trait. Mutation distribution across the genome was uniform. The native plasmid was an exception and 2 mutations were observed on 90 kb plasmid. The important genes like dnaE, gyrA, iroC, metH and rpoB involved in antibiotic resistance had point mutations. The genome analysis revealed most of the metabolic pathways were affected.

DrRecQ regulates guanine quadruplex DNA structure dynamics and its impact on radioresistance in Deinococcus radiodurans.

The GC-rich genome of Deinococcus radiodurans contains a very high density of putative guanine quadruplex (G4) DNA motifs and its RecQ (drRecQ) was earlier characterized as a 3'→5' dsDNA helicase. We saw that N-Methyl mesoporphyrin IX (NMM), a G4 DNA binding drug affected normal growth as well as the gamma radiation resistance of the wild-type bacterium. Interestingly, NMM treatment and recQ deletion showed additive effect on normal growth but there was no effect of NMM on gamma radiation resistance of recQ mutant. The recombinant drRecQ showed ~400 times higher affinity to G4 DNA (Kd  = 11.74 ± 1.77 nM) as compared to dsDNA (Kd  = 4.88 ± 1.30 µM). drRecQ showed ATP independent helicase function on G4 DNA, which was higher than ATP-dependent helicase activity on dsDNA. Unlike wild-type cells that sparingly stained for G4 structure with Thioflavin T (ThT), recQ mutant showed very high-density of ThT fluorescence foci on DNA indicating an important role of drRecQ in regulation of G4 DNA structure dynamics in vivo. These results together suggested that drRecQ is an ATP independent G4 DNA helicase that plays an important role in the regulation of G4 DNA structure dynamics and its impact on radioresistance in D. radiodurans.

Analysis of translesion polymerases in colorectal cancer cells following cetuximab treatment: A network perspective.

INTRODUCTION: Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. METHODS: In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. RESULTS: Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. CONCLUSION: We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

DR2417, a hypothetical protein characterized as a novel ß-CASP family nuclease in radiation resistant bacterium, Deinococcus radiodurans.

BACKGROUND: Deinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair. SCOPE OF REVIEW: DR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance. MAJOR CONCLUSIONS: Sequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with ß-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3'→5' exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon γ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of γ radiation resistance in Deinococcus. CONCLUSION: Our results suggest that DncA was a novel nuclease of ß CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and γ radiation resistance of this bacterium. GENERAL SIGNIFICANCE: Traditionally DncA should have shown both DNase and RNase functions as other members of ß CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.

Hypothetical proteins present during recovery phase of radiation resistant bacterium Deinococcus radiodurans are under purifying selection.

Deinococcus radiodurans has an unusual capacity to recover from intense doses of ionizing radiation. The DNA repair proteins of this organism play an important role in repairing the heavily damaged DNA by employing a novel mechanism of DNA double-strand break repair. An earlier report stated that genes of many of these repair proteins are under positive selection implying that these genes have a tendency to mutate, which in turn provides selective advantage to this bacterium. Several "hypothetical proteins" are also present during the recovery phase and some of them have also been shown for their roles in radiation resistance. Therefore, we tested the selection pressure on the genes encoding these poorly characterized proteins. Our results show that a number of "hypothetical proteins" present during the repair phase have structural adaptations compared to their orthologs and the genes encoding them as well as those for the DNA repair proteins present during this phase are under purifying selection. Evidence of purifying selection in these hypothetical proteins suggests that certain novel characteristics among these proteins are conserved and seem to be under functional constraints to perform important functions during recovery process after gamma radiation damage.

Artificial insemination of pigs reared under smallholder production system in northeastern India: success rate, genetic improvement, and monetary benefit.

The study investigated the success rate, genetic improvement, and monetary benefit of artificial insemination (AI) technology in smallholder backyard pig production system. The pig production system was studied, and performance of nondescript and crossbred pigs under the traditional system was evaluated. Litter size and growth rate of crossbred pig was significantly (P < 0.05) higher compared to the nondescript pigs. Non-availability of superior germplasm to produce crossbred pigs and high mating cost were the major constraints observed in the study in addition to indiscriminate mating and non-availability of breeding boar. For genetic improvement of nondescript local pigs and to produce crossbred pigs, AI delivery mechanism was developed in participatory mode including farmers, village leaders, and key persons in 36 villages. The information system was designed in such a way that AI was carried out at the doorstep of the farmer upon request. A total of 167 estrus sow/gilts were artificially inseminated, and a farrowing rate of 78.44 % was obtained with a mean litter size of 7.86 ± 0.65 following AI, which did not differ significantly from natural service. However, the growth rate of crossbred piglets obtained through AI was significantly higher than the growth rate of piglets born out of natural service. The tribal farmers were benefited by AI in several ways: (1) timely availability of superior germplasm to produce crossbred piglets; (2) saved the mating cost of INR 1,000-1,200 and transport of cost (INR 300-400) of female to the boar premises and (3) controlled mating to prevent inbreeding. The present study clearly demonstrates the feasibility and potential benefit of AI technique to smallholder backyard pig production system in tribal rural areas. In addition to genetic improvement of nondescript local pigs, this technology can help in overcoming breeding constraints in smallholder backyard pig production for increasing productivity.

Detection of Pasteurella multocida isolates from local pigs of India by polymerase chain reaction and their antibiogram.

Pasteurella multocida has been recognized as an important veterinary pathogen for over a century. Conventional methods for diagnosis of pasteurellosis rely on the detection of the organism by microscopy and its isolation and identification. However, as far as pasteurellosis is concerned, it is not just sufficient to know the identity of the organism. To constitute effective control measures, it is important to know the serotype of the organism. A study was undertaken to characterize the Pasteurella isolates from local pigs in India with clinical respiratory disease by determination of their capsule types and presence or absence of toxin gene. Pasteurella could be isolated from 66.70% of pigs with clinical respiratory disease. All the isolates were confirmed through biochemical characterization and P. multocida-specific polymerase chain reaction. It has also been observed that all the isolates belonged to capsular type D. All the isolates were sensitive to chloramphenicol, chlortetracycline, doxycycline, and enrofloxacin, while the rest of the antibiotics were less effective. It has also been observed that all isolates were resistant to cephalexin, penicillin G, and sulphadiazine. The study revealed the detection of P. multocida serotype D from clinical respiratory diseases of local pigs of India, which could be one of the important respiratory tract pathogens responsible for mortality of local pigs in India.

Characterization of DRA0282 from Deinococcus radiodurans for its role in bacterial resistance to DNA damage.

DRA0282, a hypothetical protein, was found in a pool of nucleotide-binding proteins in Deinococcus radiodurans cells recovering from gamma radiation stress. This pool exhibited an unusual inhibition of nuclease activity by ATP. The N terminus of DRA0282 showed similarity to human Ku80 homologues, while the C terminus showed no similarities to known proteins. The recombinant protein required Mn(2+) for its interaction with DNA and protected dsDNA from exonuclease III degradation. The binding of the protein to supercoiled DNA with a K(d) of ~2.93 nM was nearly 20-fold stronger than its binding to ssDNA and nearly 67-fold stronger than its binding to linear dsDNA. Escherichia coli cells expressing DRA0282 showed a RecA-dependent enhancement of UV and gamma radiation tolerance. The ΔdrA0282 mutant of D. radiodurans showed a dose-dependent response to gamma radiation. At 14 kGy, the ΔdrA0282 mutant showed nearly 10-fold less survival, while at this dose both pprA : : catΔdrA0282 and pprA : : cat mutants were nearly 100-fold more sensitive than the wild-type. These results suggested that DRA0282 is a DNA-binding protein with a preference for superhelical DNA, and that it plays a role in bacterial resistance to DNA damage through a pathway in which PprA perhaps plays a dominant role in D. radiodurans.

Targeting RECQL5 Functions, by a Small Molecule, Selectively Kills Breast Cancer in Vitro and in Vivo.

Clinical and preclinical data reveal that RECQL5 protein overexpression in breast cancer was strongly correlated with poor prognosis, survival, and therapeutic resistance. In the current investigation, we report design, synthesis, and specificity of a small molecule, 4a, which can preferentially kill RECQL5-expressing breast cancers but not RECQL5 knockout. Our stringent analysis showed that compound 4a specifically sensitizes RECQL5-expressing cancers, while it did not have any effect on other members of DNA RECQL-helicases. Integrated approaches of organic synthesis, biochemical, in silico molecular simulation, knockouts, functional mutation, and rescue experiments showed that 4a potently inhibits RECQL5-helicase activity and stabilizes RECQL5-RAD51 physical interaction, leading to impaired HRR and preferential killing of RECQL5-expressing breast cancer. Moreover, 4a treatment led to the efficient sensitization of cisplatin-resistant breast cancers but not normal mammary epithelial cells. Pharmacologically, compound 4a was orally effective in reducing the growth of RECQL5-expressing breast tumors (human xenograft) in NUDE-mice with no appreciable toxicity to the vital organs.

Integrated resource-driven pig production systems in a mountainous area of Northeast India: production practices and pig performance.

Data on pig production system was derived through structured household interviews from a total number of 320 rural households and performance of pigs was assessed. Results revealed that the pig production system represented mixed farming based mainly on the common property resources. Majority of the pigs were reared in intensive system and fed with home made cooked feed (kitchen waste and locally available plants). The body weight of crossbred, Burmese and local pigs were 67, 65.4 and 45.6 kg, respectively at 12 months of age with average daily body weight of 184, 179 and 125 g, respectively. The overall mortality among the pigs was 17.96%. The major causes of mortality in pigs were Swine fever, Swine erysipelas, digestive disorders, nephritis and respiratory disorders. The body weight gain in pigs subjected to deworming and mineral mixture supplementation (218 g/day) was significantly (p < 0.05) higher than the control group (178 g/day). The input output ratio was 1:1.7 for both crossbred and Burmese pigs, while the corresponding ratio for local pigs was 1:1.2. It is inferred that the smallholder resource driven pig production system is economically viable and sustainable at household level and there is enough scope to improve the smallholder resource driven pig production system.

Quality and shelf life evaluation of pork nuggets incorporated with fermented bamboo shoot (Bambusa polymorpha) mince.

Pork nuggets were processed by incorporating fermented bamboo shoot mince and their physico-chemical, microbiological and sensory characteristics were evaluated during 35 day storage at refrigeration temperature (4±1 °C). Addition of fermented bamboo shoot significantly affects the pH, moisture, protein, fat, fiber, instrumental color values and texture profiles of nuggets, especially at 6% and 8% addition levels. Nuggets with 6% and 8% FBSM had significantly lower TBARS values at the end of the storage period (i.e. 0.39 and 0.33 mg malonaldehyde/kg respectively) compared to the control (0.84 mg malonaldehyde/kg). Bamboo shoot addition significantly improved the microbial and sensory qualities of the pork nuggets. Incorporation of fermented bamboo shoot at 8% level increased the shelf life of pork nuggets by at least two weeks i.e. from 21 days to 35 days at 4±1 °C compared to the control.

Association of Body Mass Index (BMI) with Lung Function Parameters in Non-asthmatics Identified by Spirometric Protocols.

INTRODUCTION: Association between obesity and asthma has been reported widely, with disparity between males and females. Epidemiological data which indicate that obesity precedes development of asthma and increases the prevalence and incidence of asthma, raises the possibility of a causal association. AIM: To find out the association, including gender differences, between Body Mass Index (BMI) and lung functions in non-asthmatics identified by spirometric protocols. METHODS: A retrospective analysis was conducted of the spirometry results obtained between October 2012 and March 2013 for six months. Participants were referred by a variety of medical specialties to the pulmonary function laboratory in Physiology department, R.G. Kar Medical College, Kolkata, India of the 590 test results recorded in the study database 424 non-asthmatic subjects were reviewed. RESULTS: Significant differences in the spirometric parameters, measured as a percentage of predicted were evident between male and female. Among obese subjects significant correlation is found between BMI and pulmonary function values, FEV1(r = -0.531, p=0.009); FEF25-75% (r= -0.653, p=0.001); FEV1/FVC (r= - 0.603, p=0.002). Significant association was found between BMI and lung function in obese female but not in obese male. CONCLUSION: Association was found between indices of spirometry and BMI in non-asthmatic obese group along with a gender disparity.

Effect of dietary sunflower oil and coconut oil on adipose tissue gene expression, fatty acid composition and serum lipid profile of grower pigs.

The present study was conducted to assess whether the partial replacement of feed energy by vegetable oils containing high medium-chain saturated fatty acids (MCFA) and n-6 polyunsaturated fatty acids (PUFA) would modify lipogenic gene expression and other parameter of fat metabolism in pigs. Eighteen pigs (17-19 kg body weight) received one of three experimental diets for 60 days (six animals per group): (i) Control diet; (ii) a diet with sunflower oil (SO) or (iii) a diet with coconut oil (CO). In diets SO and CO, 10% of the feed energy was replaced by the respective oils. The experimental treatment did not influence the performance of the pigs. In blood serum, an increased content of total cholesterol was observed for SO and CO fed animals, whereas no significant changes for total triglycerides and different lipoprotein fractions were detected. The fatty acid composition of adipose tissue was significantly modified, with an increased content of MCFA and n-6 PUFA in CO and SO fed pigs, respectively. The gene expression for fatty acid synthase was decreased for SO and CO fed pigs; for stearoyl CoA desaturase and sterol regulatory element binding protein, a depression was observed in SO but not in CO fed pigs. The results of present study suggest that the type of dietary fat can modulate the adipose tissue gene expression and fatty acid composition differentially, with minimal effect on serum lipid profile.

Mineral profiling of local pig-feeds and pigs reared under resource driven production system to reduce porcine mineral deficiency in subtropical hill ecosystem of Northeastern India.

The present study assessed the mineral status of pigs fed with local feed resources. The commonly used plants for feeding pigs and blood serum samples from Hampshire, Large White Yorkshire and indigenous pigs were analyzed for total protein, albumin and cholesterol levels. Processed plant and serum samples were also analyzed for calcium, phosphorus, magnesium, sodium, potassium, copper, cobalt, manganese, iron and zinc. The incidence and extent of mineral deficiency in pigs was quantified. No significant difference was observed in total protein and albumin levels between any two breed/types of pigs, however the Indigenous pigs showed significantly (P < 0.05) higher cholesterol level compared to other two breeds. Among different plants, Spilanthus sp had majority of macro and micro nutrients in high levels. Regarding incidence of mineral deficiency in pigs, it was observed that 90, 67.1, 61.4, 48.6, 95.7% of the pigs were deficient in calcium, phosphorus, sodium, magnesium and potassium. An interesting finding was that all the pigs (100%) utilized in the study were deficient in zinc. From this study, it was inferred that there are good numbers of potential source of mineral that might be used more economically to improve the mineral availability to pigs.

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa.

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.