An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

Size- and Age-Dependent Neurotoxicity of Engineered Metal Nanoparticles in Rats

Earlier we showed that chronic administration of engineered nanoparticles (NPs) from metals, e.g., Cu, Ag, or Al (50–60 nm, 50 mg/kg, i.p. daily for 1 week) alter blood–brain barrier (BBB) disruption and induce brain pathology in adult rats (age 18 to 22 weeks). However, effects of size-dependent neurotoxicity of NPs in vivo are still largely unknown. In present investigation, we examined the effects of different size ranges of the above-engineered NPs on brain pathology in rats. Furthermore, the fact that age is also an important factor in brain pathology was also investigated in our rat model. Our results showed that small-sized NPs induced the most pronounced BBB breakdown (EBA +480 to 680 %; radioiodine +850 to 1025 %), brain edema formation (+4 to 6 %) and neuronal injuries (+30 to 40 %), glial fibrillary acidic protein upregulation (+40 to 56 % increase), and myelin vesiculation (+30 to 35 % damage) in young animals as compared to controls. Interestingly, the oldest animals (30 to 35 weeks of age) also showed massive brain pathology as compared to young adults (18 to 20 weeks old). The Ag and Cu exhibited greater brain damage compared with Al NPs in all age groups regardless of their size. This suggests that apart from the size, the composition of NPs is also important in neurotoxicity. The very young and elderly age groups exhibited greater neurotoxicity to NPs suggests that children and elderly are more vulnerable to NPs-induced brain damage. The NPs-induced brain damage correlated well with the upregulation of neuronal nitric oxide synthase activity in the brain indicating that NPs-induced neurotoxicity may be mediated via increased production of nitric oxide, not reported earlier.     

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.     

Phylogenetic relationships in the cricket tribe Xenogryllini (Orthoptera,Gryllidae, Eneopterinae) and description of the Indian genus Indigryllus gen. nov.

The subfamily Eneopterinae is known greatly for its diversified acoustic modalities and disjunct distribution. Within Eneopterinae, tribe Lebinthini is the most studied group, due to its highest species diversity (ca. 150 species in 12 genera), endemic distribution on the islands of Southeast Asia and of the South West Pacific, males’ ability to produce high‐frequency calling songs, and evolution of females’ vibrational response. To investigate the distribution pattern and diversification of acoustic and behavioral attributes in a larger frame, clear understanding of phylogenetic relationships within other tribes of Eneopterinae is vital. In this study, we focus on the tribe Xenogryllini, sister group of Lebinthini. Xenogryllini, as opposed to Lebinthini, is known by fewer species (11 species in two genera), distributed widely in continental Asia and Africa, and for producing low‐frequency calling songs. We describe a new genus Indigryllus with a new species of the tribe Xenogryllini, discovered from the southwest of India. We used eight molecular genetic markers to reconstruct the phylogenetic relationships. The resultant phylogenetic tree is used to compare and discuss distribution patterns and acoustic modalities between Lebinthini and Xenogryllini.     

Role of UDP-N-Acetylglucosamine2-Epimerase/N-Acetylmannosamine Kinase (GNE) in β1-Integrin-Mediated Cell Adhesion

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L). The subcellular distribution of recombinant GNE and its mutant showed differential localization in the cell. The effect of mutation on GNE function was investigated by studying hyposialylation of cell membrane receptor, β1-integrin. Hyposialylated β1-integrin localized to internal vesicles that was restored upon supplementation with sialic acid. Fibronectin stimulation caused migration of hyposialylated β1-integrin to the cell membrane and co-localization with focal adhesion kinase (FAK) leading to increased focal adhesion formation. This further activated FAK and Src, downstream signaling molecules and led to increased cell adhesion. This is the first report to show that mutation in GNE affects β1-integrin-mediated cell adhesion process in GNE mutant cells.     

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

Background

Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level.

Methods

MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction.

Results

Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2 × 10⁴ M^- 1 for DNA and 1.06 × 10³ M^− 1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin.

Conclusions

The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin.

General significance

The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.     

Neuroadaptations in the Striatal Proteome of the Rat Following Prolonged Excessive Sucrose Intake

Obesity is a contemporary health problem of rapidly increasing prevalence. One possible cause of obesity is loss of control over consumption of highly palatable foodstuffs, perhaps mirroring the processes involved in drug addiction. Accordingly, the striatum may be a key neural substrate involved in both food and drug craving. We hypothesised here that prolonged exposure to 10 % sucrose solution might cause neuroadaptations in the striatum that are analogous to those previously reported following prolonged exposure to alcohol or recreational drugs. Male Wistar rats were given constant access to 10 % sucrose solution (in addition to normal lab chow and tap water) for 8 months and were compared with control rats receiving no sucrose access. Rats in the sucrose group typically drank more than 100 ml of sucrose solution per day and showed 13 % greater body weight than controls at the end of the 8 months. Striatal dopamine (DA) concentrations were decreased in the sucrose group rats relative to controls. Differential expression of 18 proteins was identified in the striatum of the sucrose group rats relative to controls. Down regulated proteins included pyridoxal phosphate phosphatase, involved in DA synthesis, and glutathione transferase, involved in free radical scavenging. Up regulated proteins included prolactin (which is under negative regulation by DA) and adipose differentiation-related protein, involved in fat synthesis. We hypothesise that DA-related neuroadaptations in the striatum caused by prolonged sucrose intake may partly drive compulsive intake and seeking of high palatability foodstuffs, in a similar way to that observed with drug and alcohol addictions.