Enumerating the phytic acid content in maize germplasm and formulation of reference set to enhance the breeding for low phytic acid

Phytic acid (Myoinositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is a ubiquitous compound present in plants. It is an important constituent in seed reducing the bioavailability of phosphorous and mineral nutrients when fed to monogastric animals like swine, poultry, fish etc. Hence, identification of maize germplasm with reduced phytic acid content is imperative to formulate the breeding programs to evolve low phytate lines. Towards this, three hundred and thirty-eight maize germplasm accessions available at Department of Millets, TNAU, were raised and screened for phytic acid content which varied from 2.77 to 16.70 mg/g of seed. Based on the variability present, a reference set with fifty-eight genotypes for phytic acid was formulated. The reference set was formed with random genotypes selected from the base population to follow a normal distribution (skewness; 0.17, kurtosis; 0.61 and K–S test for normality Dn = 0.70) for phytic acid. The non-significant difference between the means of the base and the reference ensured the entire representation of the base in the formulated reference for phytic acid. Among all the lines in the reference set, the lowest phytic acid content were observed in the lines UMI-113 (2.77 mg/g) followed by UMI-300-1 (3.17 mg/g), UMI-467 (5.50 mg/g) and UMI-158 (6.58 mg/g) could be used as donors for low phytic acid in breeding programs. The principal component analysis for studying the extent of variability in the reference, revealed six major principal components that exhibited 80.40% of variation with flowering traits, ear height and phytic acid as a major contributor for variability. The characters namely plant stand, germination percentage, kernel yield, ear length, ear diameter and number of kernels per row were found to be positively correlated with the phytic acid and this emphasizes the negative pleiotropic effects of low phytic acid lines in germination and seed set. Thus this formulated reference set enables the breeders to handle minimum population for further grouping the genotypes to analyse their heterotic potential combined with low phytic acid.     

Are Current UK National Institute for Health and Clinical Excellence (NICE) Obesity Risk Guidelines Useful? Cross-Sectional Associations with Cardiovascular Disease Risk Factors in a Large,Representative English Population

The National Institute for Health and Clinical Excellence (NICE) has recently released obesity guidelines for health risk. For the first time in the UK, we estimate the utility of these guidelines by relating them to the established cardiovascular disease (CVD) risk factors. Health Survey for England (HSE) 2006, a population-based cross-sectional study in England was used with a sample size of 7225 men and women aged ≥35 years (age range: 35–97 years). The following CVD risk factor outcomes were used: hypertension, diabetes, total and high density lipoprotein cholesterol, glycated haemoglobin, fibrinogen, C-reactive protein and Framingham risk score. Four NICE categories of obesity were created based on body mass index (BMI) and waist circumference (WC): no risk (up to normal BMI and low/high WC); increased risk (normal BMI & very high WC, or obese & low WC); high risk (overweight & very high WC, or obese & high WC); and very high risk (obese I & very high WC or obese II/III with any levels of WC. Men and women in the very high risk category had the highest odds ratios (OR) of having unfavourable CVD risk factors compared to those in the no risk category. For example, the OR of having hypertension for those in the very high risk category of the NICE obesity groupings was 2.57 (95% confidence interval 2.06 to 3.21) in men, and 2.15 (1.75 to 2.64) in women. Moreover, a dose-response association between the adiposity groups and most of the CVD risk factors was observed except total cholesterol in men and low HDL in women. Similar results were apparent when the Framingham risk score was the outcome of interest. In conclusion, the current NICE definitions of obesity show utility for a range of CVD risk factors and CVD risk in both men and women.     

Footprint of APOBEC3 on the Genome of Human Retroelements

Almost half of the human genome is composed of transposable elements. The genomic structures and life cycles of some of these elements suggest they are a result of waves of retroviral infection and transposition over millions of years. The reduction of retrotransposition activity in primates compared to that in nonprimates, such as mice, has been attributed to the positive selection of several antiretroviral factors, such as apolipoprotein B mRNA editing enzymes. Among these, APOBEC3G is known to mutate G to A within the context of GG in the genome of endogenous as well as several exogenous retroelements (the underlining marks the G that is mutated). On the other hand, APOBEC3F and to a lesser extent other APOBEC3 members induce G-to-A changes within the nucleotide GA. It is known that these enzymes can induce deleterious mutations in the genome of retroviral sequences, but the evolution and/or inactivation of retroelements as a result of mutation by these proteins is not clear. Here, we analyze the mutation signatures of these proteins on large populations of long interspersed nuclear element (LINE), short interspersed nuclear element (SINE), and endogenous retrovirus (ERV) families in the human genome to infer possible evolutionary pressure and/or hypermutation events. Sequence context dependency of mutation by APOBEC3 allows investigation of the changes in the genome of retroelements by inspecting the depletion of G and enrichment of A within the APOBEC3 target and product motifs, respectively. Analysis of approximately 22,000 LINE-1 (L1), 24,000 SINE Alu, and 3,000 ERV sequences showed a footprint of GG→AG mutation by APOBEC3G and GA→AA mutation by other members of the APOBEC3 family (e.g., APOBEC3F) on the genome of ERV-K and ERV-1 elements but not on those of ERV-L, LINE, or SINE.     

Carbohydrate-conjugate heterobimetallic complexes: synthesis, DNA binding studies, artificial nuclease activity and in vitro cytotoxicity

New carbohydrate-conjugated heterobimetallic complexes [C(32)H(62)N(10)O(8)NiSn(2)Cl(4)]Cl(2)(1) and [C(32)H(62)N(10)O(8)CuSn(2)Cl(4)]Cl(2) (2) were synthesized and characterized by spectroscopic (IR, (1)H, (13)C, and (119)Sn NMR, EPR, UV-vis, ESI-MS) and analytical methods. The interaction studies of 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 toward CT DNA. The extent of interaction was further confirmed by the interaction of 2 with the nucleotides viz.; 5'-AMP, 5'-CMP, 5'-GMP, and 5'-TMP, by absorption titration. (1)H, (31)P, (119)Sn NMR spectroscopy further validated the interaction mode of 2 with 5'-GMP. The electrophoresis pattern observed for 2 with supercoiled pBR322 DNA, exhibited significantly good nuclease activity following oxidative pathway. The preferential selectivity of 2 toward the major groove was observed on interaction of 2 with pBR322 DNA, in the presence of standard groove binders viz.; DAPI and methyl green. Additionally, in vitro antitumor activity of 2 was evaluated on a panel of human cancer cell lines, exhibiting remarkable cytotoxicity activity against Colo205 (colon) and MCF7 (breast) cell lines with GI(50) values <10 μg/mL.     

Resistance to Chilo infuscatellus (Lepidoptera: Pyraloidea) in transgenic lines of sugarcane expressing Bacillus thuringiensis derived Vip3A protein

Molecular Biology Reports - Sustainable agriculture requires management of insect pests through resistance development. The biological potential of Cry toxins and Vip protein, derived from Bacillus...     

Host-adaptation of Francisella tularensis alters the bacterium's surface-carbohydrates to hinder effectors of innate and adaptive immunity

Background

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium''s mammalian, extracellular phase.

Methods/Findings

SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.

Conclusion

F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.     

Designing of highly effective complementary and mismatch siRNAs for silencing a gene

In past, numerous methods have been developed for predicting efficacy of short interfering RNA (siRNA). However these methods have been developed for predicting efficacy of fully complementary siRNA against a gene. Best of author's knowledge no method has been developed for predicting efficacy of mismatch siRNA against a gene. In this study, a systematic attempt has been made to identify highly effective complementary as well as mismatch siRNAs for silencing a gene.Support vector machine (SVM) based models have been developed for predicting efficacy of siRNAs using composition, binary and hybrid pattern siRNAs. We achieved maximum correlation 0.67 between predicted and actual efficacy of siRNAs using hybrid model. All models were trained and tested on a dataset of 2182 siRNAs and performance was evaluated using five-fold cross validation techniques. The performance of our method desiRm is comparable to other well-known methods. In this study, first time attempt has been made to design mutant siRNAs (mismatch siRNAs). In this approach we mutated a given siRNA on all possible sites/positions with all possible nucleotides. Efficacy of each mutated siRNA is predicted using our method desiRm. It is well known from literature that mismatches between siRNA and target affects the silencing efficacy. Thus we have incorporated the rules derived from base mismatches experimental data to find out over all efficacy of mutated or mismatch siRNAs. Finally we developed a webserver, desiRm (http://www.imtech.res.in/raghava/desirm/) for designing highly effective siRNA for silencing a gene. This tool will be helpful to design siRNA to degrade disease isoform of heterozygous single nucleotide polymorphism gene without depleting the wild type protein.     

Catechin Hydrate Ameliorates Redox Imbalance and Limits Inflammatory Response in Focal Cerebral Ischemia

Epidemiologic studies have shown that foods rich in polyphenols, such as flavonoids, can lower the risk of ischemic disease; however, the mechanism of protection has not been clearly investigated. In this study, we hypothesized that pretreatment effect of catechin hydrate (CH) on functional outcome, neuronal damage and on secondary injuries in the ischemic brain of rats. To test this hypothesis, male Wistar rats were pretreated with CH (20 mg/kg b.wt) for 21 days and then subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. After 2 h MCAO/22 h reperfusion, neurological deficit, infarct sizes, activities of antioxidant enzymes and cytokines level were measured. Immunohistochemistry and western blot were used to analyse the expression of glial fibrillary acidic protein (GFAP), inducible nitric oxide (iNOS) and NF-kB in ischemic brain. The administration of CH showed marked reduction in infarct size, reduced the neurological deficits, suppressed neuronal loss and downregulate the iNOS, GFAP and NF-kB expression in MCAO rats. A significantly depleted activity of antioxidant enzymes and content of glutathione in MCAO group were protected significantly in MCAO group pretreated with CH. Conversely, the elevated level of thiobarbituric acid reactive species and cytokines in MCAO group was attenuated significantly in CH pretreated group when compared with MCAO group. The results indicated that CH protected the brain from damage caused by MCAO, and this effect may be through downregulation of NF-kB expression.     

Antileishmanial evaluation of clubbed bis(indolyl)-pyridine derivatives: One-pot synthesis,in vitro biological evaluations and in silico ADME prediction

A new series of bis(indolyl)-pyridine derivatives 6(a–m) were synthesized by Chichibabin reaction process and evaluated for antileishmanial and antibacterial activities to establish structure–activity relationship. The synthesis was carried out through one-pot multicomponent reaction of 3-acetylindole, aromatic aldehydes, and ammonium acetate in the presence of camphor-10-sulfonic acid as a catalyst. The compounds 6d (IC₅₀ = 102.47 μM) and 6f (IC₅₀ = 99.49 μM) had shown promising antileishmanial against L. donovani promastigotes when compared with standard sodium stibogluconate (IC₅₀ = 490.00 μM). All the synthesized compounds (MIC range = 41.35–228.69 μg/mL) had shown potent antibacterial activity than standard ampicillin (MIC range = 100.00–250.00 μg/mL) against all the tested bacterial strains. In silico ADME and metabolic site prediction studies were also held out to set an effective lead candidate for the future antileishmanial and antibacterial drug discovery initiatives.