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Five different concentrations (100, 250, 500, 1000 and 2000 μg/L of aflatoxin B1 were found to be inhibitory to seed germination and seedling growth (root and shoot lengths) of mustard seeds (variety Pusa bold). These also lowered the levels of chlorophyll and carotenoids in the emerging leaves during seedling growth. The inhibitory effect was correlated with the concentration of applied toxin. [ABSTRACT FROM AUTHOR]

Pioneer transcription factors have the ability to access DNA in compacted chromatin 1 . Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming 2-4 . However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming., (© 2023. The Author(s).)

Tumour illness and its resistance against existing anticancer therapies pose a serious health concern globally despite the progressive advancement of therapeutic options. The prevailing treatment of HCC using numerous antitumor agents has inflated long-lived complete remissions, but a percentage of individuals still die due to disease recurrence, indicating a need for further exploration of possible anti-tumour regimes. We aim to boost the effectiveness of the HCC treatment by conducting current investigations evaluating the effect of arsenic trioxide (ATO) with different herbal compounds like quercetin and aloe-emodin against liver tumour via inhibition of telomerase, a pro-cancer enzyme. The anticancer activity of ATO with herbal compounds was investigated in human control liver cell line (Wrl-68) and cancer liver cell line (HepG2) at different time intervals. Viability and cytotoxicity in response to combinatorial drugs were assessed in vitro by trypan blue dye exclusion assay and MTT and WST assay. Apoptosis was analysed by annexin V/PI assay, and the expression of telomerase and apoptosis-regulating proteins was evaluated by immunoblotting and qRT-PCR. Arsenic trioxide in combination with quercetin and aloe-emodin reduced cell viability in cancerous cells compared to normal cells by inducing apoptosis, downregulating telomerase and Bcl-2 (anti-apoptotic protein) and upregulating the expression of Bax (pro-apoptotic protein). ATO exhibited significant anticancer effects due to the synergistic effects of quercetin and aloe-emodin in liver tumour cells. The current study data collectively suggest that a successful inhibition of cancer growth by the combination of ATO and tested herbal medicines against liver tumour growth is via the inhibition of telomerase activity., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The outbreak of waterborne diseases such as cholera and non-cholera (vibriosis) is continuously increasing in the environment due to fecal and sewage discharge in water sources. Cholera and vibriosis are caused by different species of Vibrio genus which are responsible for acute diarrheal disease and soft tissue damage. Although incidences of cholera and vibriosis have been reported from the Vaishali district of Bihar, India, clinical or environmental strains have not been characterized in this region. Out of fifty environmental water samples, twelve different biochemical test results confirmed the presence of twenty Vibrio isolates. The isolates were found to belong to five different Vibrio species, namely V. proteolyticus , V. campbellii , V. nereis , V. cincinnatiensis , and V. harveyi . From the identified isolates, 65% and 45% isolates were found to be resistant to ampicillin and cephalexin, respectively. Additionally, two isolates were found to be resistant against six and four separately selected antibiotics. Furthermore, virulent hlyA and ompW genes were detected by PCR in two different isolates. Additionally, phage induction was also noticed in two different isolates which carry lysogenic phage in their genome. Overall, the results reported the identification of five different Vibrio species in environmental water samples. The isolates showed multiple antibacterial resistance, phage induction, and virulence gene profile in their genome.

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p = 0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p = 0.035) and additive models (p = 0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p = 0.042) as well as additive models (p = 0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have developed a new series of simple biaryl piperidine derivatives (11-19) based on biaryl naphthylisoquinoline alkaloid Ealamine-A. The target compounds were synthesized, analyzed by spectral data, and evaluated for antileishmanial activity against Leishmania donovani strain Ag83 by MTT assay. The compounds have shown the best to moderate antileishmanial activity. The 5'-fluoro-2'-methoxyphenyl derivative 14 and 3',5'-difluorophenyl derivative 16 have inhibited the promastigotes by 86 % and 85 % after 24 h and 92 % and 91 % after 48 h incubation, respectively, at 400 μM concentration. The % inhibition was lower with the lowering of the concentration and increased with the incubation time. Compounds 12, 15, and 18 have solubility issues and proved to be less active than the rest of the compounds. Molecular docking studies were performed on selective active compounds and the results indicate that these compounds may act by binding to the Leishmanolysin and the docking scores are in good correlation with the antileishmanial activity. These results provide an initial insight into the design of new therapeutics for neglected tropical diseases., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

The visceral leishmaniasis is caused by L. donovani, a neglected tropical disease with an estimated number of 500,000 cases worldwide. Apart from the absence of effective vaccine, the available drugs have limitations like toxic side effects and emergence of drug resistance. The genome of Leishmania is remarkably challenged by the oxidative stress present inside the human macrophage. To maintain genomic integrity, a number of specialized DNA repair pathways assist in the recognition and repair of damaged DNA. In general, Base Excision Repair (BER) plays an essential role in the maintenance of genomic stability. We demonstrate here that the treatment of L. donovani with oxidative agents causes DNA damage and upregulation of Polβ. On the other hand, parasite overexpressing Polβ shows more resistance against Amp B, H 2 O 2 and menadione as compared to wild type cells. We also observed a higher infectivity in the parasites that overexpress Polβ. The upregulation of Polβ was also found in stationary phase and axenic amastigote of L. donovani. Overall, we propose that Polβ is crucial for infectivity and survival of the parasite. Discovery of specific inhibitors against Polβ could offer an attractive strategy against leishmaniasis., (Copyright © 2018. Published by Elsevier B.V.)

In the year 2016, European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines provided recommendations on dyslipidemia management. The recommendation from these guidelines are restricted to European subcontinent. To adapt the updated recommendations for Indian subset of dyslipidemia, a panel of experts in management of dyslipidemia provided their expert opinions. This document provides expert consensus on adapting 2016 ESC dyslipidemia guidelines recommendations in Indian setting. The document also discussed India-specific relevant literature to support the consensus opinions provided in management of dyslipidemia., (Copyright © 2018 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.)

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.

The occurrence of 7,8-dihydro-8-oxo-2'deoxyguanosine (8-oxodG), thymine glycol:guanine (Tg:G) mismatch and abasic site DNA damage lesions in close proximity induce repair refractive multicomponent clustered DNA damage. Herein, the influence of abasic sites in the processing of 8-oxodG lesion and Tg:G mismatch bistranded cluster is evaluated. Abasic sites are found to impart conformational destabilization that appreciably hinders the repair activity of the other lesions whenever present in a cluster combination. The repair process reduces the formation of double strand breaks (DSBs) and renders this three-lesion combination a non-DSB forming cluster. The stability of the DNA duplex harbouring these three lesions is highly compromised due to altered base helicity and base stacking phenomena leading to impaired repair., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1β (IL-1β) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-β. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

Leishmania donovani is an intracellular protozoan parasite that causes endemic tropical disease visceral leishmaniasis (VL). Present drugs used against this fatal disease are facing resistance and toxicity issues. Survival of leishmania inside the host cells depends on the parasite's capacity to cope up with highly oxidative environment. Base excision repair (BER) pathway in L. donovani remains unexplored. We studied uracil DNA glycosylase (UNG), the key enzyme involved in BER pathway, and found that the glycosylase activity of recombinant LdUNG ( Leishmania donovani UNG) expressed in E. coli is in sync with the activity of the parasite lysate under different reaction conditions. Overexpression of UNG in the parasite enhances its tolerance towards various agents which produce reactive oxygen species (ROS) and shows a higher infectivity in macrophages. Surprisingly, exposure of parasite to amphotericin B and sodium antimony gluconate upregulates the expression of UNG. Further, we found that the drug resistant parasites isolated from VL patients show higher expression of UNG. Mechanisms of action of some currently used drugs include accumulation of ROS. Our findings strongly suggest that targeting LdUNG would be an attractive therapeutic strategy as well as potential measure to tackle the problem of drug resistance in the treatment of leishmaniasis.

Currently, there is no approved vaccine for visceral leishmaniasis (VL) caused by L. donovani. The ability to manipulate Leishmania genome by eliminating or introducing genes necessary for parasites' survival considered as the powerful strategy to generate the live attenuated vaccine. In the present study fructose-1,6-bisphosphatase (LdFBPase) gene deleted L. donovani (Δfbpase) was generated using homologous gene replacement strategy. Though LdFBPase gene deletion (Δfbpase) does not affect the growth of parasite in the promastigote form but axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Though Δfbpase L. donovani parasite persisted in BALB/c mice up to 12 weeks but was unable to cause infection, we tested its ability to protect against a virulent L. donovani challenge. Notably, intraperitoneal immunisation with live Δfbpase parasites displayed the reduction of parasites load in mice spleen and liver post challenge. Moreover, immunised BALB/c mice showed a reversal of T cell anergy and high levels of NO production that result in the killing of the parasite. A significant, correlation was found between parasite clearance and elevated IFNγ, IL12, and IFNγ/IL10 ratio compared to IL10 and TGFβ in immunised and challenged mice. Results suggested the generation of protective Th1 type immune response which induced significant parasite clearance at 12-week, as well as 16 weeks post, challenged immunised mice, signifying sustained immunity. Therefore, we propose that Δfbpase L. donovani parasites can be a live attenuated vaccine candidate for VL and a good model to understand the correlatives of protection in visceral leishmaniasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (K m , V max , K cat ) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DNA derived well-controlled arrangement of porphyrins has emerged as promising hybrid nanostructures. Exceptional biocompatibility and DNA-directed surface addressability coupled with rich symmetry features of the porphyrin have made these hybrid nanostructures attractive candidates for potential biomedical and biotechnological applications. However, the noteworthy photophysical properties of porphyrin and related molecules when present in DNA based nanostructures are yet to be explored fully and should be a matter of intense research that may unearth a plethora of interesting applications of these nanostructures. Herein, we demonstrate the construction of novel self-assembled DNA-porphyrin hybrid nanonetworks that utilize the porphyrin core for antibacterial applications. Porphyrin derivative with four pendant NH 2 groups was synthesized and conjugated with the 5'-PO 4 of ss-DNA by solution phase phosphoramidation coupling reaction. The conjugation was followed by DNA hybridization mediated self-assembly to form DNA-porphyrin hybrid nanonetwork. The enhanced antimicrobial property of the nanonetwork was envisioned following light irradiation at relevant wavelength. In line with this, comparative antimicrobial activities against gram-negative (Escherichia coli BL-21) and gram-positive bacteria (Staphylococcus aureus) have been studied. Interestingly, DNA-porphyrin nanonetwork afforded highly efficient and coherent photoinduced reactive oxygen species (ROS) generation to display antimicrobial activity against Staphylococcus aureus. The escalated and coherent ROS generation from the nanonetworks was attributed to the ordered placement of the porphyrins that inhibits self-quenching. Our work points out to a good alternative for antibiotic free strategies for preservation of biological materials and other applications., (Copyright © 2017. Published by Elsevier B.V.)

Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.

Plasma gelsolin (pGSN) is a multifunctional protein involved mainly in severing and clearing of actin filaments. Its level correlates with inflammation and several diseases making it a potential biomarker of diagnostic and prognostic values. The pGSN level in groups of treated and untreated HIV-1-infected Indian patients is investigated in this study. This study aims at investigating the levels of pGSN in HIV-1-infected patients across different age, sex, severity of disease, and treatment status. Blood samples of 213 patients were analyzed for CD4 counts by flow cytometry and pGSN was quantified by enzyme-linked immunosorbent assay (ELISA). The level of pGSN is significantly increased in HIV-1 infected patients (227.2 ± 54.3 μg/ml) compared to healthy volunteers (167.9 ± 61.8 μg/ml). The level correlates with CD4 cell counts as patients with lower CD4 counts showed higher pGSN levels and vice versa. Gender does not affect pGSN level; however, antiretroviral (ARV) treatment reduces pGSN toward normal. Within low CD4 cell count group, the untreated patients have 52% higher pGSN than healthy volunteers, whereas with treatment, the difference reduces to 24%. Similarly, high CD4 cell count (>350 cells/mm 3 ) group of patients showed 44% increase in pGSN in untreated patients compared to 21% increase in treated patients. There is an upregulation of pGSN in HIV-1 infection and it is inversely correlated with CD4 cell counts. Treatment with ARV drugs decreases pGSN levels toward normal. The monitoring of pGSN level in HIV-1-infected patients could be an important indicator of severity of disease and recovery during treatment.

Adenosine 5'-triphosphate (ATP)-dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet, how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Using methyl transverse relaxation-optimized nuclear magnetic resonance spectroscopy on a 450-kilodalton complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes the dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h while promoting octamer eviction by the SWI-SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling., (Copyright © 2017, American Association for the Advancement of Science.)

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

l-Asparaginases belong to a family of amidohydrolases that catalyze the conversion of l-asparagine into l-aspartic acid and ammonia. Although bacterial l-asparaginases have been used extensively as anti-leukemic agents, their possible role as potential drug targets for pathogenic organisms has not been explored. The presence of genes coding for putative l-asparaginase enzymes in the Leishmania donovani genome hinted towards the specific role of these enzymes in extending survival benefit to the organism. To investigate whether this enzyme can serve as a potential drug target against the Leishmania pathogen, we obtained structural models of one of the putative Leishmania l-asparaginase I (LdAI). Using an integrated computational approach involving molecular modelling, docking and molecular dynamics simulations, we found crucial differences between catalytic residues of LdAI as compared to bacterial l-asparaginases. The deviation from the canonical acid-base pair at triad I, along with the structural reorganization of a β-hairpin loop in the presence of a substrate, indicated an altogether new mechanism of action of the LdAI enzyme. Moreover, the finding of compositional and functional differences between LdAI and human asparaginase was used as a criterion to identify specific small molecule inhibitors. Through virtual screening of a library of 11 438 compounds, we report five compounds that showed favorable interactions with the active pocket of LdAI, without adversely affecting human asparaginase. One of these compounds when tested on cultured Leishmania promastigotes displayed a promising leishmanicidal effect. Overall, our work not only provides first hand mechanistic insights of LdAI but also proposes five strongly active compounds which may prove as effective anti-leishmaniasis molecules.

Phagocytic cells produce reactive oxygen and nitrogen species (ROS & RNS) as the most common arsenal to kill intracellular pathogens. Leishmania, an obligate intracellular pathogen also confronts this antimicrobial assault during the early phase of infection but nevertheless is able to survive these attacks and proliferate in macrophage. Adaptation of Leishmania to the toxic effects of ROS and RNS, involves a rapid change in the parasite proteome to combat the host defense response that macrophage mount in combating pathogen. To understand the events associated with combating ROS and RNS species, we performed a proteomic analysis of L. donovani promastigotes treated with sub-lethal doses of menadione (ROS), S-nitroso-N-acetylpenicillamine (RNS) or combination of both compounds. Proteomic changes triggered by these reagents were evaluated by iTRAQ labeling and subsequent LC-MALDI-TOF/TOF-MS analysis. Across the 3 stress conditions, the quantitative analysis identified changes in the proteins which encompass ~20% of the parasite proteome. Major changes were observed in enzymatic machinery of pathways involved in maintaining redox homeostasis, trypanothione metabolism, oxidative phosphorylation, superoxide metabolism, mitochondrial respiration process and other essential metabolic pathways. These observations shed light on how Leishmania promastigotes counter ROS and RNS effects during the initial stage of infection. This article is part of a Special Issue entitled: From protein structures to clinical applications., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Adult granulosa cell tumor is often a hormonally active stromal cell neoplasm of the ovary with malignant potential. Intra-operative pathological assessment is a valuable tool in guiding optimal surgical treatment in patients. Of the various intra-operative cytological diagnostic modalities, scrape smear cytology is an effective, economical, simple, fast and reliable method with results comparable with frozen section diagnosis. We describe a case of adult granulosa cell tumor in a 30-years-old lady diagnosed on intra-operative scrape cytology, and further reconfirmed on frozen section and histopathology.

This article reviews a systematic approach to the design and support of pediatric cardiac surgery programs in the developing world with the guidance and strategies of Children's HeartLink, an experienced non-government organization for more than 40 years. An algorithm with criteria for the selection of a partner site is outlined. A comprehensive education strategy from the physician to the allied health care provider is the mainstay for successful program development. In a partner program, the road to successful advancement and change depends on many factors, such as government support, hospital administration support, medical staff leadership, and a committed and motivated faculty with requisite skills, incentives, and resources. In addition to these factors, it is essential that the development effort includes considerations of environment (eg, governmental support, regulatory environment, and social structure) and health system (elements related to affordability, access, and awareness of care) that impact success. Partner programs should be willing to initiate a clinical database with the intent to analyze and critique their results to optimize quality assurance and improve outcomes., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38-55 and 51-69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein (U) to unfolded protein in refolding conditions (U(C)). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate (I(E)). FRET efficiency and distance change differently for the different donor-acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U(C) and I(E) does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U(C) and to I(E) are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I(E), possesses specific structure.

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor-induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment.

RUNX1 (AML1, CBFalpha2, PEBP2alphaB) is a transcription factor essential for the establishment of the hematopoietic stem cell. It is generally thought that RUNX1 exists as a monomer that regulates hematopoietic differentiation by interacting with tissue-specific factors and its DNA consensus through its N terminus. RUNX1 is frequently altered in human leukemia by gene fusions or point mutations. In general, these alterations do not affect the N terminus of the protein, and it is unclear how they consistently lead to hematopoietic transformation and leukemia. Here we report that RUNX1 homodimerizes through a mechanism involving C terminus-C terminus interaction. This RUNX1-RUNX1 interaction regulates the activity of the protein in reporter gene assays and modulates its ability to induce hematopoietic differentiation of hematopoietic cell lines. The promoters of genes regulated by RUNX1 often contain multiple RUNX1 binding sites. This arrangement suggests that RUNX1 could homodimerize to bring and hold together distant chromatin sites and factors and that if the dimerization region is removed by gene fusions or is altered by point mutations, as observed in leukemia, the ability of RUNX1 to regulate differentiation could be impaired.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

In a multicenter study conducted by the Indian Council of Medical Research, 1,646 samples of wheat grain collected from rural and urban areas of 10 states representing different geographical regions of India were analyzed for aflatoxin B1 (AFB1). AFB1 concentrations of > or = 5 microg kg(-1) were recorded in 40.3% of the samples, and concentrations above the Indian permissible regulatory limit of 30 microg kg(-1) were found in 16% of the samples. The proportion of samples with AFB1 concentrations above the Indian regulatory limit ranged from 1.7 to 55.8% in different states, with the minimum in Haryana and the maximum in Orissa. The variation in wheat contamination among states seems to be mainly the result of unsatisfactory storage conditions. Median AFB1 concentrations of 11, 18, and 32 microg kg(-1) were observed in samples from Uttar Pradesh, Assam, and Orissa, respectively; concentrations in other states were <5 microg kg(-1). The maximum AFB1 concentration of 606 microg kg(-1) was observed in a sample from the state of Uttar Pradesh. The calculated probable daily intakes of AFB1 through consumption of contaminated wheat for the population in some states were much higher than the suggested provisional maximum tolerable daily intake. Human health hazards associated with such AFB1 exposure over time cannot be ruled out.

Converging studies from many investigators indicate that RUNX1 has a critical role in the correct maintenance of essential cellular functions during embryonic development and after birth. The discovery that this gene is also frequently mutated in human leukemia has increased the interest in the role that RUNX1 plays in both normal and transforming pathways. Here, we provide an overview of the many roles of RUNX1 in hematopoietic self-renewal and differentiation and summarize the information that is currently available on the many mechanisms of RUNX1 deregulation in human leukemia., (Copyright 2005 Wiley-Liss, Inc.)

Under a multi-centre study conducted by the Indian Council of Medical Research, 1,511 samples of parboiled rice were collected from rural and urban areas of 11 states representing different geographical regions of India. These samples were analysed for contamination with aflatoxin B(1.) The presence of aflatoxin B(1) at levels=5 microg g(-1) was found in 38.5% of the total number of samples of the parboiled rice. About 17% of the total samples showed the presence of aflatoxin B(1) above the Indian regulatory limit of 30 microg kg(-1). No statistically significant difference in percentage of samples contaminated with >30 microg kg(-1) was observed between pooled rural (19.4%) and urban (14.5%) data. A median value of 15 microg kg(-1) of aflatoxin B(1) was observed in samples from Assam, Bihar and Tripura. In all other states surveyed the median value was <5 microg?kg(-1).

Two-site fluorescence resonance energy transfer (FRET) measurements have been made to determine how two intra-molecular distances contract in the sub-millisecond collapse reaction that occurs initially during the refolding of the small protein barstar. FRET measurements were made on two, single-Cys and single-Trp-containing mutant forms of barstar, Cys25 and Cys62, in each of which a thionitrobenzoate (TNB) adduct was attached to the cysteine thiol. In each protein, the core tryptophan, Trp53, acted as the FRET donor, and the TNB adduct, located either at C25 or at C62, acted as the FRET acceptor. The stabilities as well as observable folding kinetics of the Cys25 and Cys62 mutant proteins were found to be identical. The presence of the TNB adduct on the cysteine did not alter the stability or folding kinetics of either protein. Thus, the FRET-monitored changes in the two labeled mutant proteins, Cys25-TNB and Cys62-TNB, could be compared directly. Refolding was commenced from unfolded protein in 8M urea, and both the Trp53 to C25-TNB distance and the Trp53 to C62-TNB distance were found to contract upon dilution of urea. The extent of contraction of each distance, which was measured at a few milliseconds of refolding, was dependent continuously on the concentration of urea present during refolding, and was different for the two distances. For either FRET pair, the gradual contraction of distance with a decrease in the concentration of urea in which refolding occurs, was continuous with the contraction of the polypeptide chain that is seen with a decrease in the concentration of urea in the range in which the protein remains completely unfolded. It therefore appears that the products of the initial sub-millisecond refolding reaction of barstar are collapsed forms, whose dimensions do not change cooperatively in an all-or-none manner, but instead, change gradually with a change in concentration of urea. Thus, the sub-millisecond polypeptide chain collapse reaction of barstar upon denaturant dilution, appears to be a continuous structural transition.

Transcription repression in eukaryotes is mediated by a wide variety of transcription factors that usually recruit corepressors and form corepressor complexes at the specific promoter sites. One of these corepressors is the C-terminal-binding protein (CtBP) which was first identified as a protein that binds to the C-terminal region of the adenovirus E1A protein. CtBP has a strong role in both development and oncogenesis. Till date, the mechanism of transcription repression by CtBP is unknown. Here, we report that CtBP physically interacts in vivo with HAT enzymes from different families. The vast majority of the HAT enzymes have a potential consensus site for CtBP binding within the bromodomain but we show that additional site(s) exists for CBP. The interaction between CtBP and CBP is functionally important and leads to impairment of histone H3 acetylation by CBP at specific lysine residues (Lys9, Lys14, and Lys18) in a dose-dependent and NADH-dependent manner. Based on these results, we propose that CtBP1 mediates repression by blocking histone acetylation by HAT complexes.

Friedreich ataxia (FRDA), the most common type of ataxia worldwide, is an autosomal recessive disease. Homozygous expansion of GAA repeats in the first intron of the frataxin gene constitute the major type of mutation that causes the disease. The prevalence of FRDA in diverse ethnic populations of India has not been widely studied. We have studied the distribution of polymorphic GAA repeats in the frataxin gene among 6 clinically diagnosed patients and 160 ethnically matched normal individuals, to gather information on the prevalence of FRDA in the eastern part of India. Homozygous expansion in the range of 250-730 GAA repeats was detected among the patients. Among normal individuals, we observed a unimodal distribution of GAA repeats, consisting of 10 different alleles ranging from 7 to 16 GAA repeats, where the 9 repeat allele had maximal frequency. Only 5.9% of all chromosomes were found to harbour >12 GAA repeats. Haplotype analysis using closely linked four bi-allelic markers in and around the frataxin gene indicated that 66.7% of the expanded alleles harbour the ATCC haplotype that has been reported worldwide. This haplotype was present in 53.3% of the chromosomes with >12 GAA repeats, and accounted for only 3.8% of chromosomes with 7 to 12 GAA repeats. We found one novel haplotype, ACCT, among the expanded alleles as well as among normal individuals, though at low frequency; this haplotype may be characteristic of Indian populations.

Objective: Spinocerebellar ataxia type 2 (SCA2) has been reported as the commonest dominant hereditary ataxia in India. However, India is an ethnically and religiously diverse population. Previous studies have not clearly indicated exact ethnic and religious origins, and must therefore be interpreted with caution. The purpose of this study was to determine the prevalence of different SCA mutations in a relatively homogeneous population from eastern India., Methods: We identified 28 families with autosomal dominant cerebellar ataxia from eastern India. Each underwent full clinical evaluation and were analysed for the presence of SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, and SCA17 mutations. In addition, haplotype analysis was carried out in seven of the 16 families with SCA2., Results: Seven patients from four (14%) families were positive for an expansion in SCA1 and 26 patients from 16 (57%) families were positive for an expansion in SCA2. No mutations were detected in the remaining eight families (29%). Most of the SCA1 and SCA2 families were Hindu from the state of Bihar. Five out of 26 SCA2 patients in this study did not have slow saccades. In addition, four of seven SCA1 patients had slow saccades. We found an association between the SCA2 CAG repeat expansion and the 285 base pair (bp) allele of microsatellite marker D12S1672, and also data supportive of the association between the expansion and the 225 bp allele of D12S1333, which has been previously described., Conclusions: We conclude that (1) although slow ocular saccades are highly suggestive of SCA2, that they are not universal, nor are they exclusive to this disorder and (2) SCA2 is likely to be the commonest dominant ataxia in eastern India, with further evidence for a founder effect.

Objectives: MJD1/SCA3 is the most common type of spinocerebellar ataxia (SCA) worldwide. To explain the low prevalence of the disease among SCA patients from eastern India, we analysed CAG repeats and two bi-allelic intragenic markers at SCA3 locus among 412 normal individuals and 10 patients., Materials and Methods: For CAG repeat analysis, PCR amplified fragments were run on polyacrylamide gel, transferred to a membrane, probed with (CAG)10 and detected on an autoradiograph. Bi-allelic markers were analysed using allele specific PCR amplification., Results: Large normal alleles (>33 CAG repeats) were 0.015 in pooled populations. All the patients had the common haplotype C-A as observed worldwide. Frequency of C-A haplotype among large normal alleles was 0.75., Conclusions: Observed low prevalence of SCA3 could be because of the low prevalence of large normal alleles that might act as the reservoir for the expanded alleles. SCA3 mutation in Indian populations had the same origin as found worldwide.

Acute myeloid leukemia 1 (AML1) belongs to a family of DNA-binding proteins highly conserved through evolution. AML1 regulates the expression of several hematopoietic genes and is essential for murine fetal liver hematopoiesis. We report here that the histone methyltransferase SUV39H1, a mammalian ortholog of the Drosophila melanogaster SU(VAR) 3-9, forms complex with AML1. SUV39H1 methylates lysine 9 of the histone protein H3 leading to the formation of the high-affinity binding site on chromatin for proteins of the heterochromatin protein 1 family (HP1). The interaction of AML1 with SUV39H1 requires the N-terminus of AML1 where the Runt domain is located. Binding of AML1 to SUV39H1 abrogates the transactivating and DNA-binding properties of AML1 and dissociates the net-like nuclear structure of AML1. It has been reported that AML1 is capable of interaction with histone acetyl transferases (CBP, p300, and MOZ) and with component of the histone deacetylase complex (Sin3), and that the interaction with these coregulators affects the strength of AML1 in promoter regulation. Our data suggest that other enzymes are also involved in gene regulation by AML1 activity by modulating the affinity of AML1 for DNA.

Lysine acetyltransferases modulate the activity of many genes by modifying the lysine residues of both core histones and transcription-related factors. These modifications are tightly controlled in the cell because they are involved in vital processes such as cell cycle progression, differentiation, and apoptosis. Therefore, any deregulation of acetylation/deacetylation equilibrium or inappropriate modifications could lead to different diseases. Since previous studies have shown that some oncoproteins also undergo this modification, acetylation could be involved in the processes of cell transformation and oncogenesis. Here, we report that AML1/MDS1/EVI1 (AME), a repressor produced by the t(3;21) associated with human leukemia, physically interacts with the acetyltransferases P/CAF and GCN5. Our data suggest that AME has at least two binding sites for these acetyltransferases, one of which is in the Runt domain. Both P/CAF and GCN5 efficiently acetylate AME in vivo in the central region. AME acetylation has no effect on its interaction with the co-repressor CtBP1. Finally, we demonstrate that the co-expression of AME and either P/CAF or GCN5 abrogates the repression of an AML1-dependent reporter gene.

To identify the genetic modifier(s) that might alter the age at onset in Huntington's disease (HD) we have analyzed variations in GluR6 kainate receptor (GluR6), CA150 gene, Delta2642 and polymorphic CCG repeat variation in huntingtin (htt) gene in 77 HD patients and normal individuals. In addition, variation in the RAI1 gene was analyzed in 30 spinocerebellar ataxia (SCA2) patients and normal individuals to show the possible influence on the age at onset. Multiple regression analysis indicated that variation in GluR6 and CCG repeat genotype might explain 6.2% and 3.1%, respectively, of the variability in the age at onset in HD. Similar analysis with SCA2 patients indicated that RAI1 might explain about 13% of the variability in the age at onset. Specific alleles in GluR6 and CA150 locus were only observed in HD patients.

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.