Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown on arsenic-contaminated soils of West Bengal

The exposure of paddy fields to arsenic (As) through groundwater irrigation is a serious concern that may not only lead to As accumulation to unacceptable levels but also interfere with mineral nutrients in rice grains. In the present field study, profiling of the mineral nutrients (iron (Fe), phosphorous, zinc, and selenium (Se)) was done in various rice genotypes with respect to As accumulation. A significant genotypic variation was observed in elemental retention on root Fe plaque and their accumulation in various plant parts including grains, specific As uptake (29–167 mg kg^?1 dw), as well as As transfer factor (4–45%). Grains retained the least level of As (0.7–3%) with inorganic As species being the dominant forms, while organic As species, viz., dimethylarsinic acid and monomethylarsonic acid, were non-detectable. In all tested varieties, the level of Se was low (0.05–0.12 mg kg^?1 dw), whereas that of As was high (0.4–1.68 mg kg^?1 dw), considering their safe/recommended daily intake limits, which may not warrant their human consumption. Hence, their utilization may increase the risk of arsenicosis, when grown in As-contaminated areas.     

An antibiotic,heavy metal resistant and halotolerant <Emphasis Type="Italic">Bacillus cereus</Emphasis> SIU1 and its thermoalkaline protease

Background  

Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min.     

Development and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Galactosylated Low Molecular Weight Chitosan Nanoparticles Bearing Doxorubicin

The aim of the present research was to evaluate the potential of galactosylated low molecular weight chitosan (Gal-LMWC) nanoparticles bearing positively charged anticancer, doxorubicin (DOX) for hepatocyte targeting. The chitosan from crab shell was depolymerized, and the lactobionic acid was coupled with LMWC using carbodiimide chemistry. The depolymerized and galactosylated polymers were characterized. Two types of Gal-LMWC(s) with variable degree of substitution were employed to prepare the nanoparticles using ionotropic gelation with pentasodium tripolyphosphate anions. Factors affecting nanoparticles formation were discussed. The nanoparticles were characterized by transmission electron microscopy and photon correlation spectroscopy and found to be spherical in the size range 106–320 nm. Relatively higher percent DOX entrapment was obtained for Gal-LMWC(s) nanoparticles than for LMWC nanoparticles. A further increase in drug entrapment was found with nanoparticles prepared by Gal-LMWC with higher degree of substitution. A hypothesis which correlates the ionic concentration of DOX in nanoparticles preparation medium and percent DOX entrapment in cationic polymer has been proposed to explain the enhanced DOX entrapment. In-vitro drug release study demonstrated an initial burst release followed by a sustained release. The targeting potential of the prepared nanoparticles was assessed by in vitro cytotoxicity study using the human hepatocellular carcinoma cell line (HepG₂) expressing the ASGP receptors on their surfaces. The enthusiastic results showed the feasibility of Gal-LMWC(s) to entrap the cationic DOX and targeting potential of developed Gal-LMWC(s) nanoparticles to HepG₂ cell line.     

Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC),¹ which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to identify informative molecular markers that can be used to replace it.Recently, small cell-derived vesicles termed exosomes that are present in body fluids (1–5) have been proposed as a potential source of diagnostic markers (2, 6–8). These nanometer-sized vesicles, which are secreted by most cell types, originate from multivesicular bodies of the endocytic tract and reflect a subproteome of the cell. Exosomes are enriched in membrane and cytosolic proteins, and this molecular repertoire appears to be of particular functional importance to the immune system (9). Exosomes also comprise an array of lipids, mRNA, and microRNA, which are likely involved in conveying intercellular communication processes (10). Importantly, many exosomal components are simply not present as free soluble molecules in body fluids, such as certain microRNA species, which are encapsulated within the exosome lumen (6, 10). Therefore, the ability to isolate exosomes from urine (2), plasma (1), saliva (11), or other physiological sources (3) holds significant potential for obtaining novel and complex sets of biomarkers in a non-invasive manner. Exosome analysis may therefore be of value in disease diagnosis and monitoring in a variety of settings (6, 7, 12–14).Exosomes as indicators of pathology were first documented in the context of renal injury where a differential proteomics approach revealed changes in urinary exosome phenotype following renal injury (7). The researchers identified exosomally expressed Fetuin-A as a marker that became elevated 50-fold within hours following nephrotoxin exposure in rodents. Exosomal Fetuin-A elevation was also apparent in patients with acute renal injury before changes in urinary creatinine were observed (7). Clinical exosome analysis may also prove useful for solid cancers, such as ovarian or lung cancer, where the quantity of epithelial cell adhesion molecule-positive serum exosomes may correlate with tumor stage/grade. Such disease-associated exosomes express microRNA species not detected in healthy subjects (6, 12), although in this respect, there is little correlation between microRNA and disease bulk (6, 12). Other recent examples include studies of urinary exosomes in prostate cancer with exosomes expressing protein markers 5T4 (15), prostate cancer gene 3 (PCA-3) (8), or mRNA (TMPRSS2-ERG) (8, 16) associated with prostate cancer. To our knowledge, exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients (17). In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. However, given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed.Identification of the principal and most relevant molecular markers in these and other clinical scenarios remains a major challenge. In part, this is because exosomes present within complex body fluids originate from heterogeneous cell types. For example, plasma exosomes may be derived from platelets, lymphocytes, or endothelial cells (1), and a proportion may arise from well perfused organs such as the liver (18) and likely other organs as well (16). Similarly, exosomes present in urine arise from urothelial cells of the kidney and downstream of the renal tract (2, 8, 15).Importantly, all proteomics studies of exosomes isolated from body fluids are unavoidably complicated by the presence of high abundance non-exosomal proteins contaminating the preparations. Examples include albumin, immunoglobulin, and complement components present in exosomes prepared from malignant effusions (5) and Tamm-Horsfall protein present in exosomes purified from urine (2). As such, great care must be taken in the interpretation of the large data sets produced by proteomics studies, requiring careful validation of the proteins of interest. The protein composition of exosomes using a single homogenous cell type is one approach that may be used to uncover the protein components of exosomes produced by various cell types.There remain two major issues in the realm of exosome proteomics that complicate our interpretation of lists of identified proteins. Foremost are the diverse methods chosen for exosome purification that in some studies have involved attempts to remove contaminants through a key biophysical property of the vesicles, i.e. their capacity to float on sucrose (19, 20) or other dense media (21). Not all published studies, however, have taken such steps, preferring a far simpler pellet (or pellet and wash) approach. These latter preparations may be significantly contaminated by components of the cellular secretome, cell fragments, and other components. All of these factors could lead to false positive identifications of exosome proteins. The second key issue centers on the MS approaches utilized in various exosome proteomics studies. Many early examples relied only on a peptide mass fingerprinting approach, lacking robust peptide sequence data (22, 23), and more recently, search criteria that are generally recommended for MS-derived sequence data have not been specified in all studies. In this study, we have listed only those proteins identified by good quality MS/MS data for two or more peptides. Variability in the robustness and bias in bioinformatics analysis of data sets and in the steps taken to validate identified proteins is an additional factor that impacts the confidence in the identification lists produced.In this study, we aimed to perform the first proteomics analysis of human bladder cancer exosomes. We took extensive steps to produce high purity and quality-assured exosome preparations prior to beginning proteomics workflows. Solubilizing the sample with SDS and a reducing agent (DTT) was a critical step that allowed for global protein identification using nanoscale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. In this study, we present the identification of a significant number of exosomally expressed proteins (353 in total) of unrivaled quality. Critical manual examination of these identifications revealed issues with multiple (physiologically impossible) MHC Class I identifications that were attributed to a misdesignation of nomenclature by MASCOT due to peptide (and target protein) homology. The data were subjected to unbiased overrepresentation analysis (examining ExoCarta and Gene Ontology databases) to reveal a proteome consistent with exosomes, particularly of carcinoma origin. Validation of several identified proteins, by combining ultracentrifugation on a linear sucrose gradient with Western blotting and/or analysis of exosome-coated latex beads, demonstrated correct surface orientation of several MS-identified membrane proteins at densities consistent with exosomes.The robust approaches taken emphasize our confidence in the validity of the identifications generated and highlight that 72 (of 353) proteins have not been previously shown to be exosomally expressed by other human proteomics studies. The data will be useful for future studies in this underinvestigated disease and will form a platform not only for future clinical validation of some of these putative markers but also to aid further investigations into novel aspects of exosome function and manufacture.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Plants at high altitude exhibit higher component of alternative respiration

Total respiration, capacities of cytochrome (CytR) and alternative respiration (AR) were studied in two varieties of barley (Horedum vulgare) and wheat (Triticum aestivum) each and one variety of pea (Pisum sativum) at low (Palampur; 1300 m) and high altitudes (Kibber; 4200 m). Similar studies were carried out in naturally growing Rumex nepalensis and Trifoilum repenses at Palampur, Palchan (2250 m) and Marhi (3250 m). All the plants species exhibited lower CytR but significantly higher AR capacity at high altitude (HA) (72-1117% higher) as compared to those at low altitude (LA). Glycolytic product, pyruvate and tricarboxylic acid cycle intermediate, citrate increased with increase in altitude. While the role of these metabolites in relation to HA biology is discussed, significantly higher AR at HA is proposed to be an adaptive mechanism against the metabolic perturbations wherein it might act to lower reactive oxygen species and also provides metabolic homeostasis to plants under the environment of HA.     

Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle

The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)).