Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.     

Detailed Analysis of Gene Polymorphisms Associated with Ischemic Stroke in South Asians

The burden of stroke is disproportionately high in the South Asian subcontinent with South Asian ethnicity conferring a greater risk of ischemic stroke than European ancestry regardless of country inhabited. While genes associated with stroke in European populations have been investigated, they remain largely unknown in South Asians. We conducted a comprehensive meta-analysis of known genetic polymorphisms associated with South Asian ischemic stroke, and compared effect size of the MTHFR C677T-stroke association with effect sizes predicted from homocysteine-stroke association. Electronic databases were searched up to August 2012 for published case control studies investigating genetic polymorphisms associated with ischemic stroke in South Asians. Pooled odds ratios (OR) for each gene-disease association were calculated using a random-effects model. We identified 26 studies (approximately 2529 stroke cases and 2881 controls) interrogating 33 independent genetic polymorphisms in 22 genes. Ten studies described MTHFR C677T (108 with TT genotype and 2018 with CC genotype) -homocysteine relationship and six studies (735 stroke cases and 713 controls) described homocysteine-ischemic stroke relationship. Risk association ORs were calculated for ACE I/D (OR 5.00; 95% CI, 1.17–21.37; p = 0.03), PDE4D SNP 83 (OR 2.20; 95% CI 1.21–3.99; p = 0.01), PDE4D SNP 32 (OR 1.57; 95% CI 1.01–2.45, p = 0.045) and IL10 G1082A (OR 1.44; 95% CI, 1.09–1.91, p = 0.01). Significant association was observed between elevated plasma homocysteine levels and MTHFR/677 TT genotypes in healthy South Asians (Mean difference (ΔX) 5.18 µmol/L; 95% CI 2.03–8.34: p = 0.001). Our results demonstrate that the genetic etiology of ischemic stroke in South Asians is broadly similar to the risk conferred in Europeans, although the dataset is considerably smaller and warrants the same clinical considerations for risk profiling.     

Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients

Molecular and Cellular Biochemistry - Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their...     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.     

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Paradoxical effects of maternal stress on fetal steroids and postnatal reproductive traits in female mice from different intrauterine positions

We examined effects of maternal stress on prenatal serum concentrations of testosterone and estradiol and on postnatal reproductive traits in female mice from different intrauterine positions. On Day 18 of fetal life, control females positioned in utero between two male fetuses (2M females) had higher concentrations of testosterone and lower concentrations of estradiol in serum than control female fetuses located between two females (0M females). Control females positioned between a male and a female fetus (1M females) had intermediate levels of both hormones. Prior intrauterine position in control females accounted for differences in genital morphology (length of the anogenital separation) at birth and length of estrous cycles during adulthood. Maternal stress eliminated these postnatal differences due to prior intrauterine position: all 0M, 1M, and 2M female offspring of stressed mothers exhibited postnatal traits that were indistinguishable from those of control 2M females. Maternal stress resulted in an increase of over 1 ng/ml in serum testosterone in all female fetuses; the magnitude of the increase was similar for 0M, 1M, and 2M females. There was no effect of maternal stress on serum concentrations of estradiol in 0M and 2M female fetuses. Maternal stress resulted in a dramatic change in the postnatal traits of 0M females, whereas 2M females showed no change. Since the effect of maternal stress on sex steroids was similar among fetuses from different intrauterine positions but postnatal response to maternal stress varied by intrauterine position, other components of the endocrine system may mediate effects of maternal stress on these postnatal characteristics.     

The behavioural thermal preference of the common triplefin (Forsterygion lapillum) tracks aerobic scope optima at the upper thermal limit of its distribution

Ocean temperatures are rising and fish are redistributing themselves poleward and into deeper waters to retain a favourable thermal environment (11 and 30). To investigate whether biogeographical shifts might occur through behavioural redistribution into optimal environments, we examined whether a common triplefin species (Forsterygion lapillum) would behaviourally select (i.e. track) a temperature that matches its physiological optimum under laboratory conditions. F. lapillum were acclimated to 15, 18 or 21 °C for at least 4 weeks, after which various rates of oxygen consumption (MO₂) were measured using automated respirometry and their behavioural thermal preferenda assessed using an electronic shuttle choice tank. Aerobic metabolic scope (resolved as the difference between maximal and maintenance MO₂) did not differ across all thermal treatments (i.e. specimens acclimated to 15, 18 or 21 °C) revealing that F. lapillum is a eurythermal species with a range of optimal physiological performance that closely matches the environmental conditions they are exposed to. A comparably wide range of behavioural preference would perhaps be expected but all three acclimation groups showed a surprisingly narrow behavioural preference range of 20–21 °C. The results therefore suggest that, irrespective of acclimation, eurythermal species may have a tendency to select optimal temperatures at the upper limit of their thermal distribution range. The results are discussed in the context of the ecology and the expected response of F. lapillum to future thermal change.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

Effects of exogenously applied salicylic acid and putrescine alone and in combination with rhizobacteria on the phytoremediation of heavy metals and chickpea growth in sandy soil

The present attempt was made to study the role of exogenously applied salicylic acid (SA) and putrescine (Put) on the phytoremediation of heavy metals and on the growth parameters of chickpea grown in sandy soil. The SA and Put were applied alone as well as in combination with plant growth promoting rhizobacteria (PGPR). The PGPRs, isolated from the rhizosphere of chickpea, were characterized on the basis of colony morphology and biochemical traits through gram staining, catalase and oxidase tests, and identified by 16S-rRNA gene sequencing as Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium. The chickpea seeds were soaked in 24 h old fresh cultures of isolates for 2–3 h prior to sowing. The growth regulators (PGRs), SA and Put (150 mg/L), were applied to the seedlings as foliar spray at three-leaf stage. The result revealed that plants treated with SA and Put alone or in combination with PGPRs, significantly enhanced the accumulation of heavy metals in plant shoot. PGPR induces Ni accumulation in sensitive variety and Pb in both the varieties, the PGR in combination augment the bioremediation effects of PGPR and both sensitive and tolerant variety showed significant accumulation of Ni, Cd, and Pb. SA was more effective in accumulating Ni and Cd whereas, significant accumulation of Pb was recorded in Put. PGPRs further augmented the PGRs induced accumulation of heavy metals and macronutrients in chickpea shoot and in rhizosphere. SA increased the proline content of tolerant variety while decreasing the lipid peroxidation and proline content of the sensitive variety but decreased the stimulating effect of PGPR in proline production. Interactive effects of PGPR and PGRs are recommended for inducing phytoremediation in chickpea plants under drought stress.