Pattern of hemoglobinopathy among the young tribes of West Bengal: A completely different scenario from Rest of India.

Hemoglobinopathy is a major concern among the tribal population which constitutes 8.6% of the total population, and West Bengal (WB) is the home to 5.3 million tribes. The present study was conducted on 52,880 tribal school students from all the districts of WB. Written informed consent and peripheral blood were collected for complete blood count and high-performance liquid chromatography analysis. Beta trait was 5.3%, sickle trait was 2.35%, and hemoglobin (Hb) E (HbE) trait was 1.4% in this population. About 37.8% of beta trait belonged to the Santal tribe and 21.5% belonged to Oraon. HbS is mainly found in Alipurduar and Jalpaiguri districts at the prevalence of 3.69% and 5.96%, respectively. HbE trait is found at 6.06% in Alipurduar, of which 51% of cases are from Mech tribe only found in this district. Unlike central and Western parts of India, HbS trait in WB was significantly low among the tribes. A high prevalence of consanguinity among the tribes is considered responsible for the high rate of hemoglobinopathy.

Gold Nanoparticles (AuNPs) Conjugated with Andrographolide Ameliorated Viper (Daboia russellii russellii) Venom-Induced Toxicities in Animal Model.

Andrographolide, a diterpenoid compound found in the aerial parts of Andrographis paniculata (a well known anti snake venom plant) was conjugated with gold nanoparticle (andrographolide-AuNPs) and its efficacy against Daboia russellii russellii venom (DRRV) induced local damage, organ toxicity and inflammatory response was evaluated in animal models. Ethical clearance was obtained before animal experiments. Andrographolide-AuNPs was formed by adsorption method. Physico-chemical characterization of particle was done by dynamic light scattering (DLS), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). Swiss albino male mice were divided into 5 groups: Gr. 1-Sham control, Gr. 2-DRRV control, Gr. 3-anti snake venom serum treated, Gr. 4-andrographolide treated and Gr. 4-andrographolide-AuNPs treated. 1/5th minimum lethal dose of DRRV (10 µg/s.c./20 g mice) was induced in animals of group 2, 3, 4 and 5 animals, followed by treatment with anti snake venom serum (2 mg/20 g mice, i.v.) andrographolide (50 µg/20g mice, i.p.) and andrographolide-AuNPs (50 µg/20 g mice, i.v.) in group 3, 4 and 5 animals, respectively. Blood was collected after 18 h, serum was prepared and organ toxicity markers (transaminases, phosphatases, lactate dehydrogenase, creatine phosphate, urea, creatinine, Ca2+, phosphorous), inflammatory markers (interleukin 1ß, 6, 17a, 10, tumor necrosis factor α) and local damage testings (defibrination, edema, hemorrhage) were assessed. Values were expressed as mean ± SEM (n = 4), one way analysis of variance was done, P < 0.05 was considered as statistically significant. Formed andrographolide-AuNPs were pink in color with hydrodynamic diameter 30-50 nm, polydispersity index 0.412 and zeta potential -16.21 mV. XRD data confirmed the presence of crystalline gold in andrographolide-AuNPs. TEM (20-50 nm) and FE-SEM (20-25 nm) indicated the presence of nearly spherical particle. DRRV envenomation followed by treatment with andrographolide-AuNPs provided protection against venom induced edema, hemorrhage, defibrination, organ toxicity and inflammation in animal model. Venom neutralization by andrographolide-AuNPs was > andrographolide, which confirmed the increased efficacy of andrographolide after gold nanoparticle conjugation, may be due to anti-oxidant/anti-inflammatory activity of andrographolide, showing increased efficacy after gold nanoparticle tagging. Thus, andrographolide-AuNPs may serve as a supportive therapy in snakebite (against venom induced local damage, organ toxicity and inflammatory response) subject to further detail studies.

Nanosensing of Pesticides by Zinc Oxide Quantum Dot: An Optical and Electrochemical Approach for the Detection of Pesticides in Water.

Present study reveals the low concentrations (∼4 ppm) of pesticide sensing vis-à-vis degradation of pesticides with the help of nontoxic zinc oxide quantum dots (QD). In our study, we have taken four different pesticides viz., aldrin, tetradifon, glyphosate, and atrazine, which are widely used in agriculture and have structural dissimilarities/diversity. By using optical sensing techniques such as steady state and time-resolved fluorescence, we have analyzed the detailed exciton dynamics of QD in the presence of different pesticides. It has been found that the pesticide containing good leaving groups (-Cl) can interact with QD promptly and has high binding affinity (∼107 M-1). The different binding signatures of QD with different pesticides enable us to differentiate between the pesticides. Time resolved fluorescence spectroscopy provides significant variance (∼150-300 ns) for different pesticides. Furthermore, a large variation (105 Ω to 7 × 104 Ω) in the resistance of QD in the presence of different pesticides was revealed by electrochemical sensing technique. Moreover, during the interaction with pesticides, QD can also act as a photocatalyst to degrade pesticides. Present investigation explored the fact that the rate of degradation is positively affected by the binding affinity, i.e., the greater the binding, the greater is the degradation. What is more, both optical and electrochemical measurements of QD, in tandem, as described in our study could be utilized as the pattern recognition sensor for detection of several pesticides.

Protection against osteoarthritis in experimental animals by nanogold conjugated snake venom protein toxin gold nanoparticle-Naja kaouthia cytotoxin 1.

BACKGROUND & OBJECTIVES: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment led to the search for alternative therapies. It was previously reported that snake venom protein toxin Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin NKCT1 (GNP-NKCT1) against experimental OA. METHODS: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin). Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline, deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-17, vascular endothelial growth factor (VEGF)] and matrix metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface were also done. RESULTS: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage degradation in osteoarthritic rats. INTERPRETATION & CONCLUSIONS: In this study restoration of the arthritic markers and bone degradation by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies need to be done to confirm these findings.

Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0 nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

Nano gold conjugation, anti-arthritic potential and toxicity studies of snake Naja kaouthia (Lesson, 1831) venom protein toxin NKCT1 in male albino rats and mice.

Nanoscience and Nanotechnology have found their way in the fields of pharmacology and medicine. The conjugation of drug to nanoparticles combines the properties of both. In this study, gold nanoparticle (GNP) was conjugated with NKCT1, a cytotoxic protein toxin from Indian cobra venom for evaluation of anti-arthritic activity and toxicity in experimental animal models. GNP conjugated NKCT1 (GNP-NKCT1) synthesized by NaBH4 reduction method was stable at room temperature (25 +/- 2 degrees C), pH 7.2. Hydrodynamic size of GNP-NKCT1 was 68-122 nm. Arthritis was developed by Freund's complete adjuvant induction in male albino rats and treatment was done with NKCT1/GNP-NKCT1/standard drug. The paw/ankle swelling, urinary markers, serum markers and cytokines were changed significantly in arthritic control rats which were restored after GNP-NKCT1 treatment. Acute toxicity study revealed that GNP conjugation increased the minimum lethal dose value of NKCT1 and partially reduced the NKCT1 induced increase of the serum biochemical tissue injury markers. Histopathological study showed partial restoration of toxic effect in kidney tissue after GNP conjugation. Normal lymphocyte count in culture was in the order of GNP-NKCT1 > NKCT1 > Indomethacine treatment. The present study confirmed that GNP conjugation increased the antiarthritic activity and decreased toxicity profile of NKCT1.

A new approach for the delivery of artemisinin: formulation, characterization, and ex-vivo antileishmanial studies.

HYPOTHESIS: Artemisinin, a potential antileishmanial compound with poor bioavailability and stability has limited efficacy in visceral leishmaniasis. Encapsulating artemisinin into poly lactic-co glycolic nanoparticles may improve its effectiveness and reduce toxicity. EXPERIMENTS: Artemisinin-loaded nanoparticles were prepared, optimized (using Box-Behnken design) and characterized by dynamic light scattering technique, Atomic force microscopy (AFM), Transmission electron microscopy (TEM) and Fourier Transform-Infra Red spectroscopy. Release kinetics of artemisinin from optimized nanoformulation was studied by dialysis method at pH 7.4 and 5.5. Cytotoxicity and antileishmanial activity of these nanoparticles was tested on murine macrophages by MTT assay and macrophage-infested Leishmania donovani amastigotes ex vivo, respectively. FINDINGS: Artemisinin-loaded nanoparticles were 221±14nm in diameter, with polydispersity index, zeta potential, drug loading and entrapment efficiency of 0.1±0.015, -9.07±0.69mV, 28.03±1.14 and 68.48±1.97, respectively. AFM and TEM studies indicated that the particles were spherical in shape. These colloidal particles showed a sustained release pattern in vitro. Treatment with artemisinin-loaded nanoparticles significantly reduced the number of amastigotes per macrophage and percent infected macrophages ex vivo compared to free artemisinin. These nanoparticles were also non-toxic to macrophages compared to artemisinin alone.

Influence of gold nanoparticle tagged snake venom protein toxin NKCT1 on Ehrlich ascites carcinoma (EAC) and EAC induced solid tumor bearing male albino mice.

Earlier the conjugation of gold nanoparticle (GNP) and snake venom protein toxin NKCT1 was reported and primary characterization was performed. In the present communication, further characterizations of GNP-NKCT1 were done with SEM, EDS, XRD and Raman spectra for its physio-chemical nature and bonding. SEM showed the formation of gold nanoparticles, whereas EDS and XRD confirmed 60-90% gold nanoparticles in the solution. Raman shift corresponding to (C=O), (N-H), (C-N) confirmed the proper conjugation of GNP with NKCT1. GNP-NKCT1 showed anticancer effect both in vivo and in vitro in EAC cell and antitumor effect in EAC induced mice. In in vivo studies, GNPNKCT1 increased MST 108.30% and decreased viable EAC cell count 51.39%. Fluorescent micrograph showed signs of apoptosis (membrane blebbing, membrane disruption). Decreased level of IL-10 and low incorporation of BrdU showed decreased proliferation of EAC induced by GNP-NKCT1. With upregulation of Bax, down regulation of Bcl2 and increased expression of caspase 3/9, it was confirmed that GNP-NKCT1 induced caspase dependent apoptosis pathway in EAC cell. In in vitro studies, GNP-NKCT1 increased the late apoptotic stage of cell and arrested cell cycle division at G0/G1 state. GNP-NKCT1 also decreased the tumor volume and tumor weight in EAC induced tumor in male albino mice. It inhibited angiogenesis, which was confirmed by lower percentage of expression of VEGF. This study indicated the capability of gold nanoparticles which enhanced the tumor uptake of NKCT1 and also suggested that GNP-NKCT1 might be a good source for anti-carcinoma and anti-tumor agents.

In vivo and in vitro toxicity of nanogold conjugated snake venom protein toxin GNP-NKCT1.

Research on nanoparticles has created interest among the biomedical scientists. Nanoparticle conjugation aims to target drug delivery, increase drug efficacy and imaging for better diagnosis. Toxicity profile of the nanoconjugated molecules has not been studied well. In this communication, the toxicity profile of snake venom cytotoxin (NKCT1), an antileukemic protein toxin, was evaluated after its conjugation with gold nanoparticle (GNP-NKCT1). Gold nanoparticle conjugation with NKCT1 was done with NaBH4 reduction method. The conjugated product GNP-NKCT1 was found less toxic than NKCT1 on isolated rat lymphocyte, mice peritoneal macrophage, in culture, which was evident from the MTT/Trypan blue assay. Peritoneal mast cell degranulation was in the order of NKCT1 > GNP-NKCT1. The in vitro cardiotoxicity and neurotoxicity were increased in case of NKCT1 than GNP-NKCT1. On isolated kidney tissue, NKCT1 released significant amount of ALP and γ-GT than GNP-NKCT1. Gold nanoconjugation with NKCT1 also reduced the lethal activity in mice. In vivo acute/sub-chronic toxicity studies in mice showed significant increase in molecular markers due to NKCT1 treatment, which was reduced by gold nanoconjugation. Histopathology study showed decreased toxic effect of NKCT1 in kidney tissue after GNP conjugation. The present study confirmed that GNP conjugation significantly decreased the toxicity profile of NKCT1. Further studies are in progress to establish the molecular mechanism of GNP induced toxicity reduction.

Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis.

The effect of gold nanoparticles (AuNPs) on the polymerization of tubulin has not been examined till now. We report that interaction of weakly protected AuNPs with microtubules (MTs) could cause inhibition of polymerization and aggregation in the cell free system. We estimate that single citrate capped AuNPs could cause aggregation of ∼10(5) tubulin heterodimers. Investigation of the nature of inhibition of polymerization and aggregation by Raman and Fourier transform-infrared (FTIR) spectroscopies indicated partial conformational changes of tubulin and microtubules, thus revealing that AuNP-induced conformational change is the driving force behind the observed phenomenon. Cell culture experiments were carried out to check whether this can happen inside a cell. Dark field microscopy (DFM) combined with hyperspectral imaging (HSI) along with flow cytometric (FC) and confocal laser scanning microscopic (CLSM) analyses suggested that AuNPs entered the cell, caused aggregation of the MTs of A549 cells, leading to cell cycle arrest at the G0/G1 phase and concomitant apoptosis. Further, Western blot analysis indicated the upregulation of mitochondrial apoptosis proteins such as Bax and p53, down regulation of Bcl-2 and cleavage of poly(ADP-ribose) polymerase (PARP) confirming mitochondrial apoptosis. Western blot run after cold-depolymerization revealed an increase in the aggregated insoluble intracellular tubulin while the control and actin did not aggregate, suggesting microtubule damage induced cell cycle arrest and apoptosis. The observed polymerization inhibition and cytotoxic effects were dependent on the size and concentration of the AuNPs used and also on the incubation time. As microtubules are important cellular structures and target for anti-cancer drugs, this first observation of nanoparticles-induced protein's conformational change-based aggregation of the tubulin-MT system is of high importance, and would be useful in the understanding of cancer therapeutics and safety of nanomaterials.

Thermostability, pH stability and dye degrading activity of a bacterial laccase are enhanced in the presence of Cu2O nanoparticles.

The present study relates to a nanotechnology enabled method in which purified laccase from Escherichia coli AKL2 was supplemented with 100 µM copper oxide nanoparticles (Cu(2)O) (NP-laccase). The activity, half life and stability of NP-laccase were enhanced by 4, 42 and 36-fold respectively at high temperature (80 °C) and also over a wide range of pH (4-12) than laccase (in the presence of 0.18 mM CuSO(4)). Thermodynamic analysis of the nanoparticle-induced enzyme stability revealed an enhanced entropy-enthalpy compensation at 80 °C, which reflected the maintenance of its native structure. This was further supported by CD studies. The enhanced activity and thermostability of NP-laccase can be utilized for efficient decolorisation of dyes (both phenolic and azo).

Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles.

The activity and half-life of pectate lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90 °C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1mM CaCl(2). Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy-enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy-entropy change to 90 °C was observed. Without nanoparticles, the linearity range was 70 °C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme.

Exploring geometric properties of gold nanoparticles using TEM images to explain their chaperone like activity for citrate synthase.

Study on geometric properties of nanoparticles and their relation with biomolecular activities, especially protein is quite a new field to explore. This work was carried out towards this direction where images of gold nanoparticles obtained from transmission electron microscopy were processed to extract their size and area profile at different experimental conditions including and excluding a protein, citrate synthase. Since the images were ill-posed, texture of a context-window for each pixel was used as input to a back-propagation network architecture to obtain decision on its membership as nanoparticle. The segmented images were further analysed by k-means clustering to derive geometric properties of individual nanoparticles even from their assembled form. The extracted geometric information was found to be crucial to give a model featuring porous cage like configuration of nanoparticle assembly using which the chaperone like activity of gold nanoparticles can be explained.

Vitamin C prevents cigarette smoke induced atherosclerosis in guinea pig model.

AIM: Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechanism is poorly understood. This paucity of knowledge is largely attributed to the lack of an animal model; therefore, our efforts were targeted towards establishing cigarette smoke (CS)-induced atherosclerosis in guinea pig. To understand the mechanism, we investigated apoptosis, an event implicated in atherosclerosis, in the aorta of CS-exposed animals. Since a major deleterious effect of CS is oxidative stress, we also examined the effect of vitamin C, an antioxidant, on CS- induced atherosclerosis. METHODS AND RESULTS: Guinea pigs on a diet with or without vitamin C supplement were exposed to CS for different time periods. Aortal sections from these animals were examined for atherosclerotic changes by staining with H&E and Oil red O. Atherogenic changes were observed in sections obtained from CS-exposed guinea pigs only. TUNEL assay showed the occurrence of apoptosis in CS-exposed guinea pig aorta. Our results revealed that CS-induced apoptosis could contribute to the progression but not to the initiation of the disease. Immunohistochemical analysis documents that CS-induced apoptosis in aortal sections is mediated at least in part by an increased Bax/Bcl2 ratio. In contrast, CS-exposed guinea pigs fed with vitamin C-supplemented diet exhibit little or no atherogenic changes. This anti-atherosclerotic activity of vitamin C can be attributed partly to its ability to inhibit CS-induced apoptosis and platelet activation. CONCLUSION: Exposure of guinea pigs to cigarette smoke causes the development of atherosclerosis, which can be prevented by vitamin C supplement.

Anti-glycation activity of gold nanoparticles.

Anti-glycation activity of gold nanoparticles (GNPs) has been reported for the first time. Nonenzymatic glycation of alpha-crystallin leads to formation of cataract, or opaque aggregate of proteins. In this article we report prevention of glycation of alpha-crystallin by conjugation with GNPs. Formation of advanced glycosylic end products is prevented even if a strong glycating agent such as fructose is used. In addition, the nanoconjugation can provide some important information on the structural distribution of this dynamic chaperone protein. Because GNPs are biocompatible, their reported anti-glycation activity may have ophthalmological implications.

Controllable self-assembly from fibrinogen-gold (fibrinogen-Au) and thrombin-silver (thrombin-Ag) nanoparticle interaction.

Fibrinogen conjugated gold nanoparticles (fibrinogen-Au) and thrombin conjugated silver nanoparticles (thrombin-Ag) were synthesized by heating (90 degrees C) the proteins (50 microg protein/ml) with 1mM AgNO(3) or AuCl(3). The resultant particles were harvested and examined by flow cytometry, scanning electron microscopy (SEM), transmission emission microscopy (TEM), optical microscopy and dynamic light scattering. SEM and TEM images revealed that the fibrinogen-Au and thrombin-Ag particles interacted. The emergent bio-nanoconjugate population could be controlled by addition of thrombin-Ag. The method may be exploited in parametrizing coagulation factors and other clinically important protein-protein interactions.

Characterization of non-planar peptide groups in protein crystal structures.

Peptide groups are generally assumed to be planar in protein structure, due to 'rigid' partial double bond character of peptide bonds, thus the value of peptide torsion angle omega should be restricted to 180 degrees for the usual trans form of peptide unit. However, on analyzing the ultra-high resolution protein crystal database, we find that in some cases, omega deviates >10 degrees from its usual value of 180 degrees, indicating significant non-planarity of peptide groups. Moreover, the non-planarity for most of the amino acids is found to be 'biased' towards values of omega smaller than 180 degrees. Similar trend for to is confirmed by the neutron diffraction data for proteins. The neutron diffraction database also reveals that non-planar peptide groups are generally correlated to 'pyramidal' structure of the peptide-nitrogen bonds. Consequently, the hydrogen atom of peptide group deviates from its planar position, as measured by the 'improper' torsion angle theta. Thus, we find that both the angles omega and theta point towards a significant amount of non-planarity of peptide groups, which cannot be ignored. The role of peptide nonplanarity in protein function is, however, not yet clear.