jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol.

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.

Study of dihydropteroate synthase (DHPS) gene mutations among isolates of Pneumocystis jiroveci.

BACKGROUND & OBJECTIVES: Pneumocystis jiroveci (also known as P. carinii) causes fatal pneumonia in patients with AIDS and other immunocompromised patients. Co-trimoxazole (trimethoprim + sulphamethoxazole, TMP-SMZ) is the drug of choice for treatment and prophylaxis. Widespread use of sulpha medication has raised the possible selection of resistant P. jiroveci strains worldwide. Non-synonymous polymorphisms associated with sulpha resistance have been observed in P. jiroveci dihydropteroate synthase (DHPS) gene at codons 55 and 57. In view of this, we investigated mutation at DHPS locus amongst P. jiroveci isolates obtained at a tertiary care hospital in north India. METHODS: Microscopic examination of P. jiroveci in 69 clinical samples obtained from patients suspected to have P. carinii pneumonia (PCP), was performed by Grocott's Gomori methenamine silver and direct fluorescent antibody staining. Molecular studies were carried out by polymerase chain reaction (PCR) using major surface glycoprotein (MSG) as the target gene. Investigations for DHPS mutations were carried at specific 55th and 57th codon using PCR-RFLP (restriction fragment length polymorphism) assay. RESULTS: Microscopic examination detected P. jiroveci in four cases and MSG gene was amplified in five cases. Further, amplification of DHPS gene was successful in four of the five cases positive by MSG gene PCR. No point mutation was observed and all four isolates presented wild-type sequences at DHPS gene by RFLP analysis. INTERPRETATION & CONCLUSION: Although our findings suggest that in Indian subpopulation, point mutations in DHPS gene of P. jiroveci are not as common as in other parts of the developed world, further studies are needed.

Nutrient dynamics and decomposition rates during composting of sulphitation pressmud by different methods.

Decomposition rates and nutrient contents of sulphitation pressmud (SPM), either alone or by mixing with cow dung and rice straw, under the aerobic (heap and NADEP), anaerobic (pit) and vermicomposting methods were compared. Loss in Organic C and C/N ratios of the decomposing materials indicated the heap and NADEP methods faster for the composting of SPM alone and SPM + cow dung and vermicomposting method for SPM + rice straw registering significant reductions in Organic C ranging from 45.0 to 61.0% from the respective decomposing materials at termination time (119 days). The heap and NADEP methods recorded C/N ratio of SPM alone as low as 11 and 12 and SPM + cow dung as 12.0 and 13.9 at termination stage. Vermicomposting method brought the C/N ratio of SPM + rice straw to 16.4 as compared to 18.6 to 29.8 with the other methods. Irrespective of the composting methods, CO2 evolution from the decomposing materials was higher in early period (up to 28 days). Among methods, NADEP and heap methods recorded more CO2 evolution in early period while vermicomposting method showed more CO2 evolution at the later intervals in comparison to the other methods. Nutrient content in the decomposing materials at different intervals depended on their initial contents. Total N in the composts were statistically comparable, however, the composts of SPM alone recorded relatively more N than SPM + cow dung and SPM + rice-straw prepared by the respective methods. Total P and K contents of the composts increased with time irrespective of the composting methods. Composts of SPM alone recorded significantly more total P than SPM + cow dung and SPM + rice straw at 62, 91 and 119 days while SPM + rice straw recorded more total K than the composts of other materials. Total P in the composts did not differ with the composting methods, but total K in composts of SPM alone was relatively more with heap and NADEP methods, in SPM + cow dung with pit method and in SPM + rice straw by vermicomposting method. Total S in the composts decreased with time and both the aerobic methods showed more reductions than pit and vermicomposting methods.

Utilization of composted sugar industry waste (pressmud) to improve properties of sodic soil for rice cultivation.

Sulphitation pressmud (SPM) and its composts were prepared by heap, pit, NADEP and vermicomposting methods and their effects were compared with soil properties and growth, yield and nutrient uptake by rice in a sodic soil under pot conditions. Application of 15 t ha(-1) SPM and its different composts significantly increased the plant height and dry matter accumulation at different intervals, grain and straw yields and N, P and K uptake by the crop over the control. NADEP compost of SPM alone recorded the maximum and significant plant height by 8.5 to 19.3% and plant dry matter by 14.6 to 32.8% over the raw SPM at different intervals. NADEP composts of SPM alone and SPM + rice straw were also found to be superior than raw SPM by recording 34.8 and 27.8% more grain yield respectively. The SPM composts prepared by NADEP and SPM by vermicomposting methods significantly accumulated higher N and K in rice grains and straw, while NADEP compost of SPM and SPM + rice straw recorded more P in grains and straw than raw SPM. Application of SPM and its composts reduced the pH, EC and bulk density of the soil after rice harvesting, though the reductions were not significant in comparison to the control. However, these treatments increased the soil organic C by 33.33 to 69.0%, available N by 41.4 to 74.8%, available P by 47.1 to 97.8%, available K by 11.8 to 59.2% and available S by 10.3 to 90.7% over the control. NADEP composts, in general, were found to be superior than the raw SPM and other composts in residual soil nutrient content after rice crop.

Tuberculous osteitis of clivus.

Tuberculous osteitis of clivus is rare. Its diagnosis is difficult because of the rarity of the site and the non-specific nature of the disease. Management consists of confirmation of diagnosis by biopsy and chemotherapy with anti-tubercular drugs. Meningitis may complicate the clinical course, increase morbidity and mortality.

Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice.

The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH. The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier. Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e. rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots. It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed. Transgenic rice has also been produced with a petH promoter and gus reporter gene construct. Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.

Non-operative treatment of fractures of the humeral shaft: a comparative study.

Forty cases of diaphyseal fracture of humerus were treated by functional cast bracing (FCB). Results were compared with a series of 25 patients treated by U cast method. Average time taken for union was 7.5 weeks with a range of 5-14 weeks by FCB method. Fractures treated by U cast method took on an average 10 weeks to unite. Stiffness at shoulder and elbow was uniformly found in patients treated by U cast. More than 50% patients had varus angulation less than 5 degrees after treatment by FCB. Only 16% had varus angulation less than 5 degrees by U cast method while 32% had varus angulation more than 15 degrees. FCB is distinctly superior method of treatment for diaphyseal fracture of humerus.