Macro-, micro-, and trace element distributions in areca nut, husk, and soil of northeast India.

In areca nut and husk, 14 elements (As, Ca, Cd, Cl, Co, Cu, K, Mg, Mn, Na, Rb, Sb, and Zn) were determined, while 34 elements including rare earth elements were detected in the corresponding soil samples using instrumental neutron activation analysis and atomic absorption spectrometry methods, whereas the concentration levels of Hg in tested samples are negligible, perhaps, below the detection limits. No rare earth elements were detected in edible areca nut. The concentration levels of various essential elements and heavy elements such as As, Cd, and Cu present in areca nut are within the permissible levels, whereas Pb content is relatively higher than FAO/WHO's permissible levels. The order of bioaccumulation index for heavy metals in areca nut was Cd > Sb > Cu > Zn ≥ Mn ≥ Co > Pb ≥ As. Bioaccumulation index values are indicating that areca palm may not be able to accumulate other heavy elements in the edible areca nut, except for Cd. On the basis of pollution indices, Northeast Indian soil may be relatively unpolluted.

Amplicon sequencing and imputed metagenomic analysis of waste soil and sediment microbiome reveals unique bacterial communities and their functional attributes.

The discharge of solid and liquid waste from domestic, municipal, and hospital premises pollutes the soil and river ecosystems. However, the diversity and functions of the microbial communities present in these polluted environments are not well understood and may contain harmful microbial communities with specialized metabolic potential. In this present study, we adapted the Illumina sequencing technology to analyze microbial communities and their metabolic capabilities in polluted environments. A total of 1113884 sequences of v3-v4 hypervariable region of the 16S rRNA were obtained using Illumina sequencing and assigned to the corresponding taxonomical ranks using Greengenes databases. Proteobacteria and Bacteroidetes were dominantly present in all the four studied sites (solid waste dumping site (SWD); Chite river site (CHR), Turial river site (TUR), and Tuikual river site (TUKR)). It was found that the SWD was dominated by Firmicutes, Actinobacteria; CHR by Acidobacteria, Verrucomicrobia, Planctomycetes; TUR by Verrucomicrobia, Acidobacteria; and TUKR by Verrucomicrobia and Firmicutes, respectively. The dominant bacterial genus present in all samples was Acinetobacter, Flavobacterium, Prevotella, Corynebacterium, Comamonas, Bacteroides, Wautersiella, Cloacibacterium, Stenotrophomonas, Sphingobacterium, and Pseudomonas. Twenty-seven putative bacterial pathogens were identified from the contaminated sites belonging to Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. Functional analysis showed a high representation of genes in the KEGG pathway involved in the metabolism of amino acids and carbohydrates and identified several genes associated with antibiotic resistance and xenobiotic degradation in these environments, which can be a serious problem for human health and environment. The results from this research will provide a new understanding of the possible management practices to minimize the spread of pathogenic microorganisms in the environment.

Mitochondrial D-loop and cytochrome oxidase C subunit I polymorphisms among the breast cancer patients of Mizoram, Northeast India.

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations as it is prone to oxidative stress. The aim of the present study is to assess the novel mutations in mitochondrial genes from blood samples among the breast cancer patients from a less studied Northeast Indian population. D, B, L haplogroups were observed in the cancer samples and a total of 44 mtDNA D-loop sequence variations at 42 distinct nucleotide positions were found. All the sequence variations were transitional substitutions and 6 were heteroplasmic states, except for a cytosine copy number change (9C/8C) at np 303e309 in three samples examined. A total of 88 Cytochrome Oxidase C subunit I (COXI) sequence differences with respect to the Revised Cambridge Reference Sequence (rCRS) were identified including 20 missense variants with 100 % sample mutation frequency. All 20 missense mutations are highly conserved with a Cumulate Index of 100 %. Among 88 COXI mutations, 24 (13 were Non-Synonymous and 11 were Synonymous) were not previously reported (novel mutation) in the literature or the public mtDNA mutation databases. Analysis of three-dimensional structure of COXI open reading frame (ORF) predicted the effect of one single codon (96R > C, 217T > I, 224-225GG > EE and 227D > T) mutations located in the signal peptide binding position. Analysis of mitochondrial DNA mutations, as a viable alternative, has the advantage of being capable of detecting inherent risk factors for breast cancer development.

Larvicidal activity of Ipomoea cairica (L.) Sweet and Ageratina adenophora (Spreng.) King & H. Rob. plant extracts against arboviral and filarial vector, Culex quinquefasciatus Say (Diptera: Culicidae).

Culex quinquefasciatus Say, an arboviral and filarial vector, is one of the most widespread mosquitoes in the world, and insecticide-resistant populations have been reported worldwide. Due to the emergence of resistance in C. quinquefasciatus plant based products or plant extracts may be alternative sources in integrated vector management program. The present study was carried out to establish the larvicidal activities of crude solvent extracts prepared from flowers and leaves of Ipomoea cairica and Ageratina adenophora against third instar larvae of C. quinquefasciatus as target species. The plant extracts were prepared with petroleum ether, chloroform and methanol solvents using sequential extraction method to determine the best extractant for subsequent isolation and characterization of active ingredient. The total yield of plant extract in the Soxhlet extraction ranged between 0.79% and 19.35%. The qualitative phytochemical study of the plant extracts from different solvents showed the presence of alkaloids, flavonoids, phenols, terpenes, saponins and tannins in different combinations. I. cairica and A. adenophora plant extracts were found to be effective against third instar larvae of C. quinquefasciatus causing 77-100% mortality at 48h. Highest mortality was observed at 500ppm and the order of larvicidal action was observed to be of methanol extract of I. cairica flower>petroleum ether extract of A. adenophora leaf>chloroform extract of I. cairica leaf. High mortality (100%) with low LC50 and LT50 were observed in methanolic flower extract (LC50 - 8.43ppm; LT50 - 2.51h at 48h) of I. cairica, and petroleum ether (LC50 - 133.56ppm; LT50 - 9.45h at 48h) leaf extract of A. adenophora. Lethal concentration (LC50 and LC90) values gradually decreased with the exposure periods, lethal time (LT50 and LT90) decreased with the concentration in bioassay experiment with the crude plant extracts. There was a significant correlation (three-way factorial ANOVA) was noticed among concentration of the plant extracts, exposure time and solvent extraction in relation to larval mortality (P<0.0001), which indicates that larval mortality is concentration dependent as well as time-dependent. Further in-depth study is needed to identify and characterize the active components present in the plant solvent extracts and implement the effective arboviral and filarial mosquito vector management program.

Genetic and epigenetic instability induced by betel quid associated chemicals.

Over the years, betel quid chewing and tobacco use have attracted considerable interest as they are implicated as the most likely causative risk factors of oral and esophageal cancers. Although areca nut use and betel quid chewing may lead to apoptosis, chronic exposure to areca nut and slaked lime may promote pre-malignant and malignant transformation of oral cells. The putative mutagenic and carcinogenic mechanisms may involve endogenous nitrosation of areca and tobacco alkaloids as well as the presence of direct alkylating agents in betel quid and smokeless tobacco. Metabolic activation of carcinogenic N-nitrosamines by phase-I enzymes is required not only to elicit the genotoxicity via the reactive intermediates but also to potentiate the mutagenicity with the sporadic alkylations of nucleotide bases, resulting in the formation of diverse DNA adducts. Persistent DNA adducts provides the impetus for genetic and epigenetic lesions. The genetic and epigenetic factors cumulatively influence the development and progression of disorders such as cancer. Accumulation of numerous genetic and epigenetic aberrations due to long-term betel quid (with or without tobacco) chewing and tobacco use culminates into the development of head and neck cancers. We review recent evidence that supports putative mechanisms for mutagenicity and carcinogenicity of betel quid chewing along with tobacco (smoking and smokeless) use. The detailed molecular mechanisms of the extent of accumulation and patterns of genetic alterations, indicative of the prior exposure to carcinogens and alkylating agents because of BQ chewing and tobacco use, have not yet been elucidated.

Microwave-Assisted Synthesis of Red Emitting Copper Nanoclusters Using Trypsin as a Ligand for Sensing of Pb2+ And Hg2+ Ions in Water and Tobacco Samples.

In this work, a microwave assisted method was developed for synthesis of red fluorescent copper nanoclusters (NCs) using trypsin as a template (trypsin-Cu). The as-synthesized trypsin-Cu NCs are stable and water soluble, exhibiting fluorescence emission at 657 nm when excited at 490 nm. The as-prepared red-emitting trypsin-Cu NCs were characterized by using several analytical techniques such as ultraviolet-visible (UV-Vis) and fluorescence, fluorescence lifetime, Fourier transform infrared, and X-ray photoelectron spectroscopic techniques. Red-emitting trypsin-Cu NCs acted as a nanosensor for sensing both Pb2+ and Hg2+ ions through fluorescence quenching. Using this approach, good linearities are observed in the range of 0.1-25 and of 0.001-1 µM with the lower limit of detection of 14.63 and 56.81 nM for Pb2+ and Hg2+ ions, respectively. Trypsin-Cu NCs-based fluorescence assay was successfully applied to detect both Hg2+ and Pb2+ ions in water and tobacco samples.

Simultaneous determination of areca nut- and tobacco-specific alkaloids in saliva by LC-MS/MS: Distribution and transformation of alkaloids in oral cavity.

Areca nut and tobacco are frequently used in combination. Cigarette smoking and betel quid (BQ) chewing habits impose greater oral cancer risk than either habit alone. Saliva is a better noninvasive diagnostic material as it is in direct contact with oral mucosa and cancerous lesions. This study describes the application of isotope-dilution LC-MS/MS for simultaneous quantitation of five areca nut-specific alkaloids (ASAs) and three tobacco-specific alkaloids (TSAs) in human saliva. With this method, we demonstrate that the distribution of ASAs vary significantly in smokers who chew BQ habitually, due to the hydrolysis of ASAs and metabolic activity in the oral cavity. The alkaline condition formed due to slaked lime in BQ, plays an important role in the distribution of ASAs and TSAs, by catalyzing the hydrolysis of ester forms of ASAs to their respective carboxylic acid forms besides facilitating the TSA (i.e., nicotine) absorption in the oral cavity. Moreover, our results reveal that oral mucosa rather than saliva contributes to the metabolism of ASAs at oral cavity. Less than 2.1% of ASAs were metabolized by saliva, as determined by in vitro test. Our findings may provide a better insight into the pathobiology of oral carcinogenesis due to BQ chewing.

Determination of Multi Elements in Tobacco Plant of Northeast India by Neutron Activation Analysis and Atomic Absorption Spectrometry.

Even when cultivated in uncontaminated soils, tobacco plant has higher propensity to extract and accumulate trace elements. The concentrations (mass fractions) of essential elements (K, Ca, Mg, Na, Cl, Mn, Fe, Cu, and Zn) and 28 non-essential elements in tobacco plant (leaves, stem, and root) of Northeast India and their respective soils were quantitatively measured. Hg mass fraction in all samples analyzed were found to be < 10 mg/kg. The heavy element mass fractions of tobacco are weakly correlated to different soil parameters. The bioconcentration factor values indicated that Cd (7) is selectively absorbed and translocated in the tobacco leaves compared to Zn (1.7), Cu (1.5), Ni (0.12), and Pb (0.1). Under acidic soil conditions, tobacco plant efficiently absorbed and translocated Cl- ion with great ease, whereas it may be a very low accumulator of rare-earth elements. The mass fractions of Mn, Cu, Sb, Cs, Rb, and Pb are very similar to the "reference plant," whereas significantly higher mass fractions of Al, Sc, Ti, Zr, Hf, Ta, Th, and U are present in the roots of tobacco plant relative to the "reference plant." Principal component analysis has revealed that Northeast Indian tobacco can be clearly differentiated from other varieties of tobaccos used in different countries because of their element profiles.

15N-labelled nitrite/nitrate tracer analysis by LC-MS/MS: Urinary and fecal excretion of nitrite/nitrate following oral administration to mice.

Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed a15N-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of 15N-labelled nitrite/nitrate as 15N-2,3-naphthotriazole (15N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background 14N-NAT isotopomers to 15N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background 14N-NAT isotopomers to 15N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of 14N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background 14N-NAT isotopomers to 15N-NAT. With the proposed 15N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of 15NO2- and 15NO3-, both in urine and feces, after oral administration of 15N-labelled nitrite/nitrate are clearly demonstrated. The excretions of 15NO2- and 15NO3- were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of 15NO2- and 15NO3-, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.

Metagenomic analysis and the functional profiles of traditional fermented pork fat 'sa-um' of Northeast India.

Fermented pork fat (sa-um) is traditionally and extensively consumed in Northeast Indian region for several decades. However, no scientific reports are available regarding its nutritional value as well as its potential health risks. The objective of this work was essentially the characterization of sa-um using a polyphasic approach, viz., physicochemical, electrospray ionization-mass spectrometry (ESI+-MS) and metagenomic analysis in order to gain an understanding of the nutrient contents and microbial population diversity. On a dry weight basis, about 91% fat, 2% carbohydrate and 0.70% protein were present. ESI+-MS analysis of sa-um revealed the presence of various polar and neutral lipids corresponding to monoacylglyceride, diacylglyceride and triacylglyceride species. The dominant bacterial phyla were Firmicutes, Proteobacteria and Bacteroidetes. A total of 72 bacterial genera were identified, largely abundant with Clostridium species including C. butyricum, C. citroniae, C. methylpentosum, C. perfringens, C. saccharogumia and C. tetani. The imputed functional profiles of bacterial communities were predominantly involved in energy, carbohydrate and amino acid metabolisms. Furthermore, this study deduces the presence of pro-inflammatory molecules as well as antibiotic resistance genes associated with the bacterial families such as Bacillaceae, Bacteroidaceae, Clostridiaceae, Corynebacteriaceae and Enterobacteriaceae which might be a major health concern for the sa-um consuming population.

A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis.

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4-6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Probing the iron-substrate orientation for taurine/alpha-ketoglutarate dioxygenase using deuterium electron spin echo envelope modulation spectroscopy.

The structural relationship between substrate taurine and the non-heme Fe(II) center of taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) was measured using electron spin echo envelope modulation (ESEEM) spectroscopy. Studies were conducted on TauD samples treated with NO, cosubstrate alphaKG, and either protonated or specifically deuterated taurine. Stimulated echo ESEEM data were divided to eliminate interference from 1H and 14N modulations and accentuate modulations from 2H. For taurine that was deuterated at the C1 position (adjacent to the sulfonate group), 2H ESEEM spectra show features that arise from dipole-dipole and deuterium nuclear quadrupole interactions from a single deuteron. Parallel measurements taken for taurine deuterated at both C1 and C2 show an additional ESEEM feature at the deuterium Larmor frequency. Analysis of these data at field positions ranging from g = 4 to g = 2 have allowed us to define the orientation of substrate taurine with respect to the magnetic axes of the Fe(II)-NO, S = 3/2, paramagnetic center. These results are discussed in terms of previous X-ray crystallographic studies and the proposed catalytic mechanism for this family of enzymes.

O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD, an alpha-keto acid-dependent non-heme iron dioxygenase.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.