Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

First fossil evidence of Palaeocarya (Engelhardioideae: Juglandaceae) from India and its biogeographical implications

Even though presently indigenous to eastern Himalaya in India, no Engelhardioideae have been reported from the Cenozoic sediments of India till date. Here, we report the first Indian occurrence of a characteristic engelhardioid winged samaroid fruit having a tri-lobed wing (oblong-ovate median lobe and two lateral lobes) and a globose nut from the latest Neogene (Pliocene: Rajdanda Formation) sediments of Chotanagpur Plateau, eastern India. This is the first fossil evidence of relict family Juglandaceae from the Indian Cenozoic. We determine its taxonomic position on the basis of detailed macromorphological comparison with similar extant and fossil specimens and discuss its palaeoclimatic significance in terms of the present-day distribution of modern analogous species. We assign this Pliocene winged fruit specimen to the morphogenus Palaeocarya sect. Monocosta Manchester and describe it as a new species, namely Palaeocarya indica Hazra, Hazra M & Khan sp. nov. Palaeocarya sect. Monocosta has rich fossil records from the Cenozoic sediments of Europe, North America, and eastern Asia (China, Korea), but the modern analog, Engelhardia, is presently native only to India and neighboring Southeast Asia. We discuss the possible causes of disappearance of Engelhardia from the present-day vegetation of Chotanagpur Plateau. Its disappearance may be related to the gradual intensification of monsoonal rainfall seasonality since the Pliocene. Here, we also review in detail the biogeographic history of Palaeocarya sect. Monocosta and suggest its possible migration routes.     

Amino acid substitution in alpha-helix 7 of Cry1Ac delta-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner.

Insecticidal proteins or delta-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight alpha-helices and is implicated in pore formation in insect midgut epithelial membranes. All the delta-endotoxins share a common hydrophobic motif of eight amino acids in alpha-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac delta-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Mn‐catalase (Alr0998) protects the photosynthetic,nitrogen‐fixing cyanobacterium Anabaena PCC7120 from oxidative stress

Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat⁺. The Alr0998 protein could be immunodetected in AnKat⁺ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat⁺ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat⁺ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H₂O₂ caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO₂ fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat⁺ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase.     

Synthesis and pharmacological evaluation of N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide as cyclooxygenase inhibitors

A series of novel N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide derivatives were synthesized via converting the readily available 4-hydroxy coumarin to the corresponding ethyl 2-(2-oxo-2H-chromen-4-yloxy)propanoate followed by hydrolysis and then reacting with different substituted amines. The molecular structures of two representative compounds, that is, 3 and 5l were confirmed by single crystal X-ray diffraction study. All the compounds synthesized were evaluated for their cyclooxygenase (COX) inhibiting properties in vitro. The compound 5i showed balanced selectivity towards COX-2 over COX-1 inhibition and good docking scores when docked into the COX-2 protein.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Chemically characterised Artemisia nilagirica (Clarke) Pamp. essential oil as a safe plant-based preservative and shelf-life enhancer of millets against fungal and aflatoxin contamination and lipid peroxidation

Abstract

The study recommends the Artemisia nilagirica (Clarke) Pamp. essential oil (ANEO) as plant-based shelf-life enhancer of millets against fungal, aflatoxin B₁ (AFB₁) contamination and lipid peroxidation with favourable safety profile. Chemical characterisation of ANEO through GC-MS, recorded 1,5-heptadiene-4-one,3,3,6-trimethyl (32.72%)as the main compound, followed by Artemisia alcohol (13.40%), alpha lonone (4.55%), benzene, methyl (1-methylethyl) (2.97%) and 1-cyclohexene-1-carboxaldehyde,4-(1-methylethyenyl) (2.23%). The mycoflora analysis of millet samples showed Aspergillus flavus strain[LHP(R)-5] as the most AFB₁ secreting strain. The ANEO inhibited growth and AFB₁ production by the toxigenic strain at 1.4 and 1.0?µL?mL^?1, respectively, and also possess broad fungitoxic spectrum. The decrement in membrane ergosterol content, enhanced leakage of cellular Ca²⁺, K⁺ and Mg²⁺ ions along with SEM and TEM study of ANEO-treated cell denotes plasma membrane as antifungal site of action. The ANEO also showed strong antioxidant activity as determined by DPPH^? and ABTS^?+ assays having IC₅₀ value 2.51 and 1.07?µL?mL^?1, respectively. More than 70.78% protection of Ragi samples from fungal contamination was observed during in situ trial. The ANEO showed favourable safety profile with high LD₅₀ value (7528.10?µL?kg^?1) for male mice and also exhibited non-phytotoxicity for Ragi seeds germination.     

Estimating biofilm reaction kinetics using hybrid mechanistic-neural network rate function model

This work describes an alternative method for estimation of reaction rate of a biofilm process without using a model equation. A first principles model of the biofilm process is integrated with artificial neural networks to derive a hybrid mechanistic-neural network rate function model (HMNNRFM), and this combined model structure is used to estimate the complex kinetics of the biofilm process as a consequence of the validation of its steady state solution. The performance of the proposed methodology is studied with the aid of the experimental data of an anaerobic fixed bed biofilm reactor. The statistical significance of the method is also analyzed by means of the coefficient of determination (R²) and model efficiency (ME). The results demonstrate the effectiveness of HMNNRFM for estimating the complex kinetics of the biofilm process involved in the treatment of industry wastewater.     

Ameliorative Effect of Fisetin on Cisplatin-Induced Nephrotoxicity in Rats via Modulation of NF-κB Activation and Antioxidant Defence

Nephrotoxicity is a dose-dependent side effect of cisplatin limiting its clinical usage in the field of cancer chemotherapy. Fisetin is a bioactive flavonoid with recognized antioxidant and anti-inflammatory properties. In the present study, we investigated the potential renoprotective effect and underlying mechanism of fisetin using rat model of cisplatin-induced nephrotoxicity. The elevation in serum biomarkers of renal damage (blood urea nitrogen and creatinine); degree of histopathological alterations and oxidative stress were significantly restored towards normal in fisetin treated, cisplatin challenged animals. Fisetin treatment also significantly attenuated the cisplatin-induced IκBα degradation and phosphorylation and blocked the NF-κB (p65) nuclear translocation, with subsequent elevation of pro-inflammatory cytokine, TNF-α, protein expression of iNOS and myeloperoxidase activities. Furthermore, fisetin markedly attenuated the translocation of cytochrome c protein from the mitochondria to the cytosol; decreased the expression of pro-apoptotic proteins including Bax, cleaved caspase-3, cleaved caspase-9 and p53; and prevented the decline of anti-apoptotic protein, Bcl-2. The cisplatin-induced mRNA expression of NOX2/gp91phox and NOX4/RENOX and the NADPH oxidase enzyme activity were also significantly lowered by fisetin treatment. Moreover, the evaluated mitochondrial respiratory enzyme activities and mitochondrial antioxidants were restored by fisetin treatment. Estimation of platinum concentration in kidney tissues revealed that fisetin treatment along with cisplatin did not alter the cisplatin uptake in kidney tissues. In conclusion, these findings suggest that fisetin may be used as a promising adjunct candidate for cisplatin use.