New epitopes in ovalbumin provide insights for cancer neoepitopes.

MHC I-restricted epitopes of chicken ovalbumin (OVA) were originally identified using CD8 T cells as probes. Here, using bioinformatics tools, we identify four additional epitopes in OVA in addition to a cryptic epitope. Each new epitope is presented in vivo, as deduced from the lack of CD8 response to it in OVA-transgenic mice. In addition, CD8 responses to the known and novel epitopes are examined in C57BL/6 mice exposed to the OVA-expressing tumor E.G7 in several ways. No responses to any epitope including SIINFEKL are detected in mice with growing E.G7 or mice immunized with the tumor. Only in E.G7-bearing mice treated with an anti-CTLA4 antibody which depletes tumor-infiltrating regulatory T cells, CD8 responses to SIINFEKL and the novel epitope EKYNLTSVL are detected. Finally, all epitopes fails to treat mice with pre-existing tumors. These observations force an important re-consideration of the common assumptions about the therapeutic value of neoepitopes detected by CD8 responses in tumor-bearing hosts.

Reagent-Free and Rapid Assessment of T Cell Activation State Using Diffraction Phase Microscopy and Deep Learning.

CD8+ T cells constitute an essential compartment of the adaptive immune system. During immune responses, naïve T cells become functional, as they are primed with their cognate determinants by the antigen presenting cells. Current methods of identifying activated CD8+ T cells are laborious, time-consuming and expensive due to the extensive list of required reagents. Here, we demonstrate an optical imaging approach featuring quantitative phase imaging to distinguish activated CD8+ T cells from naïve CD8+ T cells in a rapid and reagent-free manner. We measured the dry mass of live cells and employed transport-based morphometry to better understand their differential morphological attributes. Our results reveal that, upon activation, the dry cell mass of T cells increases significantly in comparison to that of unstimulated cells. By employing deep learning formalism, we are able to accurately predict the population ratios of unknown mixed population based on the acquired quantitative phase images. We envision that, with further refinement, this label-free method of T cell phenotyping will lead to a rapid and cost-effective platform for assaying T cell responses to candidate antigens in the near future.

The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

Isolation and enzymatic properties of a nonspecific acid phosphatase from Vigna aconitifolia seeds.

Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulfate fractionation and cation-exchange chromatography [carboxymethyl (CM) cellulose]. The enzyme was 228-fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 column showed that Mr of native enzyme was 58 kDa and denaturing PAGE demonstrated that it was made up of two subunits of 24 and 27 kDa. The enzyme showed its optimum activity at pH 5.0 and 60°C. It exhibited broad substrate specificity and showed a higher specificity constant for para-nitrophenyl phosphate, Na ß-naphthyl phosphate, and adenosine monophosphate (AMP). Cu²âº, Mo6⁺, Fe³âº, phosphate, and fluoride ions were reported as strong inhibitors for the enzyme. Active site study for the enzyme demonstrated that tryptophan and aspartic acid may be important for the catalysis.

Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization.

Acid phosphatase isolated from Vigna aconitifolia seeds was immobilized onto glutaraldehyde activated chitosan beads by crosslinking method. Chitosan beads activated with 2% of glutaraldehyde have demonstrated maximum immobilization yield (∼ 83%). The immobilized enzyme showed optimum activity at pH 7.0, while soluble form was maximally active in acidic range (pH 5.0). With respect to free form, immobilized acid phosphatase showed better activity in alkaline range. On the other side, immobilization does not affect the optimum temperature range i.e., both, soluble and immobilized acid phosphatase exhibited maximum activity at 60 °C. The Km and Vmax values for the immobilized enzyme were calculated to be 0.37 mM and 13.5 U/mg. The immobilization on chitosan beads enhanced the shelf life of acid phosphatase. The immobilized enzyme retained its more than 50% hydrolytic activity for approximately two months. The immobilized acid phosphatase was reusable for more than 40 cycles of reaction.

In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus.

The Staphylococcus aureus bacterium, a nosocomial pathogen often causing untreatable and lethal infection in patients, mutated to become resistant to all the first-line drugs. The present study details the potential of clerodane diterpene 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide (CD) isolated from Polyalthia longifolia against methicillin-resistant S. aureus (MRSA) through in vitro and in vivo assays. Minimum inhibitory concentration (MIC) of CD exhibited significant anti-MRSA activity (15.625-31.25 mg/l) against reference strain and seven clinical isolates, while time kill assays at graded MICs indicated 2.78-9.59- and 2.9-6.18-fold reduction in growth of reference strain and clinical isolates of S. aureus, respectively. The combined effect of the CD and 7.5 % NaCl resulted in significant reduction in microbial count within 24 h, indicating the loss of the salt tolerance ability of S. aureus. Further, release of 260-nm absorbing material and flow cytometric analysis revealed an increased uptake of propidium iodide. These assays may indicate the membrane-damaging potential of CD. The molecule CD was found to interact synergistically with clinically used antibiotics (FICI ≤ 0.5) against all clinical isolates. In infected mice, CD significantly (P < 0.001) lowered the systemic microbial load in blood, liver, kidney, lung and spleen tissues and did not exhibit any significant toxicity at 100 mg/kg body weight.

Immobilization and applications of glucose-6-phosphate dehydrogenase: a review.

Immobilized enzymes have been used extensively in the fields of food industry, materials processing, textiles, detergents, biochemical and chemical industries, biotechnology, and pharmaceuticals. Studies on immobilization of glucose-6-phosphate dehydrogenase have been less extensive than those for other industrially applicable enzymes. Immobilization of glucose-6-phosphate dehydrogenase has been carried out for the formation of biosensors for the estimation of glucose, ATP, phosphate, and so on. The present review deals with the attempts made for immobilization of glucose-6-phosphate dehydrogenase and its applications for various purposes.

Changing epidemiology of community-acquired acute kidney injury in developing countries: analysis of 2405 cases in 26 years from eastern India.

BACKGROUND: The epidemiology of acute kidney injury (AKI) differs from country to country and varies from center to center within a country. Owing to the absence of a central registry, data on overall epidemiology of AKI are scanty from India. METHODS: This study aimed at describing changes in epidemiology of community-acquired AKI (CAAKI) over a time span of 26 years in two study periods, namely, 1983-95 and 1996-2008. RESULTS: We studied 2405 (1375 male and 1030 female) cases of AKI in the age range 1-95 (mean: 40.32) years. The incidence of CAAKI in 1983-95 and 1996-2008 was 1.95 and 4.14 per 1000 admission, respectively (P < 0.01). Obstetrical AKI has decreased because of the declining number of post-abortal AKI. Surgical AKI decreased from 13.8% in 1983-95 to 9.17% in 1996-2008(P < 0.01). Malarial AKI increased significantly from 4.7% in the first half of the study to 17% in the later period (P < 0.01). Diarrhea-associated AKI had significantly decreased from 36.83% in 1983-95 to 19% in 1996-2008 (P < 0.01). Sepsis-related AKI had increased from 1.57% in 1983-95 to 11.43% in 1996-2008 (P < 0.01). Nephrotoxic AKI showed an increasing trend in recent years (P < 0.01) and mainly caused by rifampicin and NSAIDs. Liver disease-related AKI increased from 1.73% in 1983-95 to 3.17% in 1996-2008 (P < 0.01). Myeloma-associated acute renal failure (ARF) accounted for 1.25% of the total number of ARF cases in the period 1996-2008. HIV infection contributed to 1.65% of ARF of the total number of AKI cases in the second period (1996-2008). Incidence of renal cortical necrosis (RCN) decreased significantly from 5.8% in 1983-95 to 1.3% in 1996-2008 of the total number of ARF cases (P < 0.01). However, during the same period ARF due to acute tubular necrosis, acute glomerulonephritis and acute interstitial nephritis remained unchanged. The mortality rate from AKI decreased significantly from 20% in 1983-95 to 10.98% in 1996-2008 (P < 0.01). CONCLUSIONS: The epidemiological characteristics of CAAKI have changed over the past three decades. There has been an increase in the overall incidence of ARF with the changing etiology of AKI in recent years. Incidences of obstetrical, surgical and diarrheal AKI have decreased significantly, whereas those of AKI associated with malaria, sepsis, nephrotoxic drugs and liver disease have increased. RCN has decreased significantly. In contrast to developed nations, community-acquired AKI is more common in developing countries. It often affects younger individuals and is caused by single and preventable diseases.

Rodent models and contemporary molecular techniques: notable feats yet incomplete explanations of Parkinson's disease pathogenesis.

Rodent models and molecular tools, mainly omics and RNA interference, have been rigorously used to decode the intangible etiology and pathogenesis of Parkinson's disease (PD). Although convention of contemporary molecular techniques and multiple rodent models paved imperative leads in deciphering the role of putative causative factors and sequential events leading to PD, complete and clear-cut mechanisms of pathogenesis are still hard to pin down. The current article reviews the implications and pros and cons of rodent models and molecular tools in understanding the molecular and cellular bases of PD pathogenesis based on the existing literature. Probable rationales for short of comprehensive leads and future possibilities in spite of the extensive applications of molecular tools and rodent models have also been discussed.

A molecular description of acid phosphatase.

Acid phosphatase is ubiquitous in distribution in various organisms. Although it catalyzes simple hydrolytic reactions, it is considered as an interesting enzyme in biological systems due to its involvement in different physiological activities. However, earlier reviews on acid phosphatase reveal some fragmentary information and do not give a holistic view on this enzyme. So, the present review summarizes studies on biochemical properties, structure, catalytic mechanism, and applications of acid phosphatase. Recent advancement of acid phosphatase in agricultural and clinical fields is emphasized where it is presented as potent agent for sustainable agricultural practices and diagnostic marker in bone metabolic disorders. Also, its significance in prostate cancer therapies as a therapeutic target has been discussed. At the end, current studies and prospects of immobilized acid phosphatase are included.

Allethrin-induced genotoxicity and oxidative stress in Swiss albino mice.

Allethrin (C(19)H(26)O(3)) is non-cyano-containing pyrethroid insecticide that is used extensively for controlling flies and mosquitoes. Apart from its neurotoxic effects in non-target species, allethrin is reported to be mutagenic in bacterial systems. In this study, we observed oxidative damage-mediated genotoxicity caused by allethrin in Swiss albino mice. The genotoxic potential of allethrin was evaluated using chromosome aberrations (CAs) and a micronuclei (MN) induction assay as genetic end-points. The oral intubation of allethrin (25 and 50mg/kg b.wt.) significantly induces CAs and MN in mouse bone marrow cells. The DNA-damaging potential of allethrin was estimated in mouse liver using the DNA alkaline unwinding assay (DAUA) and by measuring the levels of 8-hydroxy-2'-deoxy-guanosine (8-OH-dG). Furthermore, a dose-dependent increase in reactive oxygen species (ROS) generation and lipid peroxidation (LPO), with a concurrent decrease in superoxide dismutase (SOD) and catalase, confirm its pro-oxidant potential. The DNA-damaging potential of allethrin was found to be mediated through the modulation of p53, p21, GADD45α and MDM-2. These results confirm the genotoxic and the pro-oxidant potential of allethrin in Swiss albino mice.

Mancozeb-induced genotoxicity and apoptosis in cultured human lymphocytes.

AIMS: Mancozeb is a dithiocarbamate fungicide known to be genotoxic and induces tumors in rodents at various sites. There is no report in the literature about its genotoxicity in humans. Here, we investigated the association between mancozeb exposure and induction of genotoxic and proapoptotic changes in cultured human lymphocytes (CHLs). MAIN METHODS: Lymphocytes were isolated from peripheral blood of healthy non-smoking donors. Induction of micronuclei and chromosomal aberrations was recorded both by conventional and flow cytometric methods. Annexin-V FITC was used for the differentiation of apoptotic and necrotic cells by flow cytometry. KEY FINDINGS: Mancozeb exposure (0.5, 2 and 5 µg/ml) to CHLs leads to significant induction in the frequency of chromosomal aberrations (CAs) and micronuclei (MN), in a dose-dependent manner. Concomitantly, pro-oxidant potential of mancozeb was also recorded, by increase in the levels of reactive oxygen species (ROS) generation. Our results demonstrated that ROS plays a critical role in the initiation of mancozeb induced apoptosis in CHLs through two ways, primarily through mitochondria-mediated pathway including induction of ROS, decrease in mitochondrial membrane potential (ΔΨm), along with cytochrome c release from mitochondria, and activation of the caspase cascade. The other pathway includes increase in ROS, which resulted in activation of NF-κB, expression of FasL and triggered FasL-dependent pathway, which also involves caspase-8. Therefore, exposure to mancozeb can lead to induction of apoptosis in CHLs through both mechanisms. SIGNIFICANCE: The results of study confirm that mancozeb exposure can induce genotoxicity and apoptosis in CHLs, thus pose a potential risk to exposed human population.

Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson's disease phenotype in the mouse.

Maneb and paraquat are known to induce Parkinson's disease (PD) phenotype, however, caffeine offers neuroprotection. Nitric oxide (NO) acts an important mediator in PD phenotype and tyrosine kinase (TK), nuclear factor kappa B (NF-kB), p38 mitogen activated protein kinase (p38 MAPK) are known to regulate its production. The present study aimed to elucidate the role of caffeine in the regulation of NO production and microglial activation and their subsequent contribution in dopaminergic neuroprotection. The animals were treated with caffeine and/or maneb and paraquat along with controls. In a few sets of experiments, the animals were also treated with aminoguanidine, an inhibitor of inducible NO synthase, pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB, genistein, an inhibitor of TK or SB202190, an inhibitor of p38 MAPK. Tyrosine hydroxylase (TH)-immunoreactivity and anti-integrin αM (OX-42) staining were performed to assess the number of dopaminergic neurons and activation of microglia, respectively. NO was measured in terms of nitrite, however, the expressions of p38 MAPK, interleukin (IL)-1ß, NF-kB and TK were checked by western blot analyses. Maneb and paraquat induced the number of degenerating dopaminergic neurons, microglial cells, nitrite content, expressions of IL-1ß, p38 MAPK, NF-kB and TK and caffeine co-treatment reduced the level of such alterations. Reductions were more pronounced in the animals co-treated with aminoguanidine, PDTC, genistein or SB202190. The results obtained thus demonstrate that caffeine down-regulates NO production, neuroinflammation and microglial activation, which possibly contribute to neuroprotection.

Association of single nucleotide polymorphisms in CYP1B1 and COMT genes with breast cancer susceptibility in Indian women.

Cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) enzymes play critical roles in estrogen metabolism. Alterations in the catalytic activity of CYP1B1 and COMT enzymes have been found associated with altered breast cancer risk in postmenopausal women in many populations. The substitution of leucine (Leu) to valine (Val) at codon 432 increases the catalytic activity of CYP1B1, however, substitution of Val to methionine (Met) at codon 158 decreases the catalytic activity of COMT. The present study was performed to evaluate the associations of CYP1B1 Leu(432)Val and/or COMT Val(158)Met polymorphisms with total, premenopausal and postmenopausal breast cancer risks in Indian women. COMT and CYP1B1 polymorphisms in controls and breast cancer patients were analyzed employing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by gel electrophoresis. Although CYP1B1 and COMT genotypes did not exhibit statistically significant association with breast cancer risks when analyzed individually, COMT wild type (Val(158)Val) in combination with CYP1B1 heterozygous variant (Leu(432)Val) [OR: 0.21; 95% CI (0.05-0.82), p value; 0.021] and COMT heterozygous variant (Val(158)Met) in combination with CYP1B1 wild type (Leu(432)Leu) [OR: 0.29; 95% CI (0.08-0.96), p value; 0.042] showed significant protective association with premenopausal breast cancer risk. The results demonstrate that CYP1B1 wild type in combination with COMT heterozygous or their inverse combination offer protection against breast cancer in premenopausal Indian women.