Background

The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed.

Results

We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS).

Conclusions

We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN.

Imatinib is a tyrosine kinase inhibitor and is considered the first line of non stem cell transplantation treatment for patients diagnosed with CML. We evaluated the response rates and adverse reactions to Imatinib in our patients and tried to identify factors which affected the response to Imatinib. Eighty-four patients were diagnosed on the basis of clinical and haematological variables with confirmation by FISH, detecting Philadelphia chromosome or bcr-abl translocation and were then started on oral capsule Imatinib. A complete haematological response was seen in 78.04% patients, while complete cytogenetic response (CCR) was seen in 12.2% of patients and major cytogenetic response (MCR) was seen in 64.63% of patients. It was found that that a greater total leukocyte count (TLC) on presentation had a negative correlation with cytogenetic response. Cytopenias were seen in 36 patients (43.82%). 34.9% of patients having CCR/MCR required dose reduction while 73.6% of patients not achieving CCR/MCR required dose reduction. This was a significant difference, confirmed on statistical analysis (P < 0.05; P = 0.019), establishing the negative prognostic value of dose reduction due to cytopenias.

Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene. Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST. For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker, EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1− CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and “ancestral,” were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.

Background and Aim:

Platelets are routinely isolated from whole blood and stored in plasma for 5 days. This study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C.

Materials and Methods:

The study sample included 30 blood donors of both sex in State Blood Bank, C S M Medical University, Lucknow. Random donor platelets were prepared by the platelet-rich plasma method. Whole blood (350 ml) was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators with and without additive solution.

Results:

Platelet swirling was present in all the units at 22°C on day 7 with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution while platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001) on day 7 without additive solution at 22°C.

Conclusion:

Our study infers that the platelet viability and aggregation were the best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

Background & objectives:

Leukaemia and lymphoma are common paediatric haematological malignancies acquiring human parvovirus B19 (B19) infection. In some studies anaemia has been found in children with acute lymphoblastic leukaemia (ALL) during maintenance therapy and rarely in lymphoma. We studied frequency of B19 infection and its implications in new onset acute leukaemia (mostly ALL) and lymphoma in children.

Methods:

Seventy serum samples from 35 children (age <12 yr, 29 males) newly diagnosed with haematological malignancies (on induction therapy) were collected together with 34 controls (solid tumours). Children were examined clinically and for anti-B19 IgM antibodies by quantitative ELISA and B19 DNA by PCR (VP1-VP2) and nested-PCR (VP1 unique). Bone marrow aspirates were examined histopathologically, whenever possible.

Results:

Of the 35 children, 22 had acute leukaemia while 13 had lymphoma. B19 infection was seen in six (17.1%) of 35 children (5 ALL, 1 NHL), two at diagnosis and four during follow up compared to none in the control. Among five B19 IgM positive ALL (n=18) children, two had B19 genome and two had giant pronormoblasts (lantern cells; but one lacked B19 DNA). Of the 70 serum samples tested, eight (11.4%) had anti-B19 IgM as two children had persistent B19 infection and one showed atypical maculopapular rashes (lower limbs) while 12 (34.3%) had anti-B19 IgG antibodies. B19 infected children had unexplained anaemia (80%), required more blood transfusions (6.6 ± 4.8 Units vs 3.0 ± 2.6 Units) besides induction chemotherapy was delayed (60%) and required longer duration of therapy (29.2 ± 20 vs 6.3 ± 7.8 days) (P<0.02). Five children (2 ALL, 2 AML, 1 NHL) died but none were infected with B19.

Interpretation & conclusions:

B19 infection should be considered in children with ALL as it frequently caused unexplained anaemia and delay in induction chemotherapy.

Extraintestinal pathogenic Escherichia coli (ExPEC) are of significant health concern. The emergence of drug resistant E. coli with high virulence potential is alarming. Lack of sufficient data on transmission dynamics, virulence spectrum and antimicrobial resistance of certain pathogens such as the uropathogenic E. coli (UPEC) from countries with high infection burden, such as India, hinders the infection control and management efforts. In this study, we extensively genotyped and phenotyped a collection of 150 UPEC obtained from patients belonging to a semi-urban, industrialized setting near Pune, India. The isolates representing different clinical categories were analyzed in comparison with 50 commensal E. coli isolates from India as well as 50 ExPEC strains from Germany. Virulent strains were identified based on hemolysis, haemagglutination, cell surface hydrophobicity, serum bactericidal activity as well as with the help of O serotyping. We generated antimicrobial resistance profiles for all the clinical isolates and carried out phylogenetic analysis based on repetitive extragenic palindromic (rep)-PCR. E. coli from urinary tract infection cases expressed higher percentages of type I (45%) and P fimbriae (40%) when compared to fecal isolates (25% and 8% respectively). Hemolytic group comprised of 60% of UPEC and only 2% of E. coli from feces. Additionally, we found that serum resistance and cell surface hydrophobicity were not significantly (p = 0.16/p = 0.51) associated with UPEC from clinical cases. Moreover, clinical isolates exhibited highest resistance against amoxicillin (67.3%) and least against nitrofurantoin (57.3%). We also observed that 31.3% of UPEC were extended-spectrum beta-lactamase (ESBL) producers belonging to serotype O25, of which four were also positive for O25b subgroup that is linked to B2-O25b-ST131-CTX-M-15 virulent/multiresistant type. Furthermore, isolates from India and Germany (as well as global sources) were found to be genetically distinct with no evidence to espouse expansion of E. coli from India to the west or vice-versa.

Background and Aim:

Platelets are routinely isolated from whole blood and stored in plasma for 5 days. The present study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C.

Materials and Methods:

The study sample included 30 blood donors of both sex in State Blood Bank, CSM Medical University, Lucknow. Random donor platelets were prepared by platelet rich plasma method. Whole blood (350 ml) was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators, with and without additive solution.

Results:

Platelet swirling was present in all the units at 22°C on day 7, with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution, whereas platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001) on day 7 without additive solution at 22°C.

Conclusion:

Our study infers that platelet viability and aggregation were best maintained within normal levels on day 7of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

Aim

It is often difficult and challenging task to differentiate aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (HMDS) among the patients with bone marrow aplasia. This is possibly because of the considerable clinical, cytological and histological similarities between these two disorders. As prognostic and therapeutic approach to AA and HMDS are different, it is imperative to differentiate them at the time of initial diagnosis. Various attempts have been made in the past to differentiate AA from HMDS. In the present study, we explored the value of certain new parameters i.e. S-phase fraction (SPF) and aneuploidy that could be used for this purpose.

Materials and methods

The study included 46 consecutive patients with aplastic anemia, 15 patients with HMDS along with 32 age and sex-matched control subjects. S-phase fraction and aneuploidy analysis was carried out by flow cytometry using Mod Fit-LT V3.0 software.

Results

The mean SPF value was significantly lower (p=0.02) in patients with AA and higher (p=0.01) in HMDS as compared to that of the control. Aneuploidy was present in 15.2% patients with AA and in 33.3% HMDS cases. During follow-up, 4 patients with AA developed MDS, out of these, three patients had aneuploidy as well as high SPF value at the time of diagnosis. Two patients with HMDS who had aneuploidy and high SPF, converted into AML. Eleven patients died during the study, in whom 8 had aneuploidy and high SPF value.

Conclusion

We conclude that high SPF value and presence of aneuploidy favour the diagnosis of HMDS rather than AA. SPF and aneuploidy may be important parameters in patients with AA to predict their propensity to evolve into myelodysplastic syndrome and acute myeloid leukemia. SPF value may also be useful in the early diagnosis of HMDS before morphologically evidence of dysplasia is apparent.

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling.

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

Objective

To evaluate the incidence of hereditary bleeding disorders, registered at our centre from all over Uttar Pradesh over an extended period of 10 years.

Material and methods

A complete coagulation profile of 240 cases conducted, followed by clinical history of the patient. The frequency and type of bleeding, age of onset, treatment taken, consanguinity and the blood transfusion if any, are little information collected from the patient.

Results

The complete scenario of bleeders indicated maximum number (92%) of hemophilia ‘A’ from northern UP as compared from other zones (76.5% ± 3). Contrarily, hemophilia ‘B’ registered maximum from eastern zone (26%) and minimum from the northern zone (8%). The age diversity at diagnosis ranged between 1–32 years. The reasons for difference are all speculative. Definite cause needs extensive research.

Conclusions

The study showed that elderly patients with hemophilia had significantly more impairments in physical functioning and depression than younger ones.

Chronic Eosinophilic Leukemia (CEL) is a rare type of chronic myeloproliferative disorder of unknown etiology with no available true incidence. The vaguely overlapping clinico — pathological picture of CEL with idiopathic hypereosinophilic syndrome (IHES) often adds to the diagnostic confusion. An evidence of genetic clonality of eosinophils or an increase in blast cells in the blood or bone marrow is mandatory for diagnosis of CEL while no specific diagnostic tests exist for IHES; making it an entity of exclusion. Till date, CEL is a rarely reported entity in India. We add yet another case of eosinophilic leukemia along with review of available literature.

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on three dimensional quantitative structure–activity relationship (3D-QSAR) studies were conducted on a series (78 compounds) of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as potent anticancer agents. The best prediction were obtained with a CoMFA standard model (q2 = 0.530, r2 = 0.903) and with CoMSIA combined steric, electrostatic, hydrophobic and hydrogen bond donor fields (q2 = 0.548, r2 = 0.909). Both models were validated by a test set of ten compounds producing very good predictive r2 values of 0.935 and 0.842, respectively. CoMFA and CoMSIA contour maps were then used to analyze the structural features of ligands to account for the activity in terms of positively contributing physiochemical properties such as steric, electrostatic, hydrophobic and hydrogen bond donor fields. The resulting contour maps produced by the best CoMFA and CoMSIA models were used to identify the structural features relevant to the biological activity in this series of analogs. This study suggests that the highly electropositive substituents with low steric tolerance are required at 5 position of the pteridine ring and bulky electronegatve substituents are required at the meta-position of the phenyl ring. The information obtained from CoMFA and CoMSIA 3-D contour maps can be used for the design of deazapteridine-based analogs as anticancer agents.

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r2 = 0.499) for the 73 compounds between docking score and IC50 values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.

Objective

We have previously analyzed protein profiles using Surface Enhanced Laser Desorption and Ionization Time-Of-Flight Mass Spectroscopy (SELDI-TOF-MS) [Kozak et al. 2003, Proc. Natl. Acad. Sci. U.S.A. 100:12343–8] and identified 3 differentially expressed serum proteins for the diagnosis of ovarian cancer (OC) [Kozak et al. 2005, Proteomics, 5:4589–96], namely, apolipoprotein A-I (apoA-I), transthyretin (TTR) and transferin (TF). The objective of the present study is to determine the efficacy of the three OC biomarkers for the detection of early stage (ES) OC, in direct comparison to CA125.

Methods

The levels of CA125, apoA-I, TTR and TF were measured in 392 serum samples [82 women with normal ovaries (N), 24 women with benign ovarian tumors (B), 85 women with ovarian tumors of low malignant potential (LMP), 126 women with early stage ovarian cancer (ESOC), and 75 women with late stage ovarian cancer (LSOC)], obtained through the GOG and Cooperative Human Tissue Network. Following statistical analysis, multivariate regression models were built to evaluate the utility of the three OC markers in early detection.

Results

Multiple logistic regression models (MLRM) utilizing all biomarker values (CA125, TTR, TF and apoA-I) from all histological subtypes (serous, mucinous, and endometrioid adenocarcinoma) distinguished normal samples from LMP with 91% sensitivity (specificity 92%), and normal samples from ESOC with a sensitivity of 89% (specificity 92%). MLRM, utilizing values of all four markers from only the mucinous histological subtype showed that collectively, CA125, TTR, TF and apoA-I, were able to distinguish normal samples from mucinous LMP with 90% sensitivity, and further distinguished normal samples from early stage mucinous ovarian cancer with a sensitivity of 95%. In contrast, in serum samples from patients with mucinous tumors, CA125 alone was able to distinguish normal samples from LMP and early stage ovarian cancer with a sensitivity of only 46% and 47%, respectively. Furthermore, collectively, apoA-I, TTR and TF (excluding CA-125) distinguished i) normal samples from samples representing all histopathologic subtypes of LMP, with a sensitivity of 73%, ii) normal samples from ESOC with a sensitivity of 84% and iii) normal samples from LSOC with a sensitivity of 97%. More strikingly, the sensitivity in distinguishing normal versus mucinous ESOC, utilizing apoA-I, TF and TTR (CA-125 excluded), was 95% (specificity 86%; AUC 95%).

Conclusions

These results suggest that the biomarker panel consisting of apoA-I, TTR and TF may significantly improve early detection of OC.

In an open trial, drug-resistant schizophrenics were treated with clozapine for sixteen weeks. The patients were rated on BPRS, PANSS, Side effects scale and Global Impression Scale at weeks 0. 9 and 16. A battery of base-line investigations were done, and hemogram was repeated at weekly intervals. Of the total 29 patient included. 25 completed the trial. The patients showed significant improvement on both BPRS and PANSS, although the improvement was more in initial weeks than between 9 and 16 weeks. The most common side effects observed were sedation, hypersalivation and tachycardia. Issues of response of clozapine on negative symptoms and a longer duration of the drug therapy are discussed.