Synthesis, incorporation efficiency, and stability of disulfide bridged functional groups at RNA 5'-ends

Modified guanosine monophosphates have been employed to introduce various functional groups onto RNA 5'-ends. Applications of modified RNA 5'-ends include the generation of functionalized RNA libraries for in vitro selection of catalytic RNAs, the attachment of photoaffinity-tags for mapping RNA-protein interactions or active sites in catalytic RNAs, or the nonradioactive labeling of RNA molecules with fluorescent groups. While in these and in similar applications a stable linkage is desired, in selection experiments for generating novel catalytic RNAs it is often advantageous that a functional group is introduced reversibly. Here we give a quantitative comparison of the different strategies that can be applied to reversibly attach functional groups via disulfide bonds to RNA 5'-ends. We report the preparation of functional groups with disulfide linkages, their incorporation efficiency into an RNA library, and their stability under various conditions.     

Plant proteomics in India and Nepal: current status and challenges ahead

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.     

Replication-compromised cells require the mitotic checkpoint to prevent tetraploidization

Replication stress often induces chromosome instability. In this study, we explore which factors in replication-compromised cells promote abnormal chromosome ploidy. We expressed mutant forms of either polymerase α (Polα) or polymerase δ (Polδ) in normal human fibroblasts to compromise DNA replication. Cells expressing the mutant Polα-protein failed to sustain mitotic arrest and, when propagated progressively, down-regulated Mad2 and BubR1 and accumulated 4N-DNA from the 2N-DNA cells. Significantly, a population of these cells became tetraploids. The Polα mutant expressing cells also exhibited elevated cellular senescence markers, suggesting as a mechanism to limit proliferation of the tetraploids. Expression of the Polδ mutant also caused cells to accumulate 4N-DNA. In contrast to the Polα mutant expressing cells, the Polδ mutant expressing cells expressed sufficient levels of Mad2, BubR1, and cyclin B1 to sustain mitotic arrest, and these cells had normal chromosome ploidy. Together, these results suggest that replication-compromised cells depend on the mitotic checkpoint to prevent mitotic slippage that could result in tetraploidization.     

Meta-analysis of Genetic Structure and Association of Prolactin Gene with Performance Traits in Dairy Cattle in India

The present meta-analysis was carried to provide the more reliable estimates of gene frequency and association of Rsa 1 generated candidate genotype of prolactin gene within exon-3 with performance traits in 1198 Indian dairy cows using data from 15 published studies. Six genetic models viz., codominant (AA vs. AB, AA vs. BB and AB vs. BB), dominant (AA+AB vs. BB), completely over dominant (AA+BB vs. AB) and recessive (AA vs. AB+BB) were used to obtain standardized mean difference (SMD) between genotypes. Meta-analysis showed that the gene frequency of A allele (156 bp) was 0.60 (95% confidence interval (CI) 0.54, 0.65). In association analysis, cows with AB genotype [SMD?=?0.65, 95% CI 0.00, 1.30] had significantly (P?<?0.05) higher lactation milk yield (LMY) as compared to BB genotype, whereas AA and AB genotypes had similar trend. Likewise, AA?+?AB also had larger effect [SMD?=?2.31, 95% CI 0.21, 4.10] on LMY as compared to BB. Cows with AB genotype had significantly lower age at first calving (AFC) with small effect [SMD (AA vs. AB)?=?1.38, 95% CI 0.06, 2.70] and medium effect [SMD (AB vs. BB)?=????3.83, 95% CI???6.41,???1.24] as compared to cows with AA and BB genotypes, respectively. This finding was confirmed under dominant and completely over dominant models. In case of fat%, AA genotype showed negative effect (SMD?=????0.51, 95% CI???0.84,???0.17) under recessive model. It was concluded that the propagation of allele A is promising to help dairy farmers to improve the genetic quality of their dairy cows.

     

Fatty acid composition of the postmortem prefrontal cortex of adolescent male and female suicide victims

Prior epidemiological, prospective intervention, and peripheral and central fatty acid composition studies suggest that omega-3 fatty acid deficiency may be associated with the pathoaetiology of depression and suicide. In the present study, we determined the fatty acid composition of the postmortem prefrontal cortex (PFC) of adolescent male and female suicide victims and age-matched controls. Fatty acid composition (wt% total fatty acids) and concentrations (μmol/g) were determined in the postmortem PFC (Brodmann area 10) of male and female adolescent (aged 13–20 years) suicide victims (n=20) and age-matched controls (n=20) by gas chromatography. None of the major polyunsaturated fatty acids including the principle brain omega-3 fatty acid, docosahexaenoic acid (DHA), monounsaturated fatty acids, or saturated fatty acids differed significantly between adolescent suicide victims and controls before or after segregation by gender. The arachidonic acid (AA, 20:4n-6): DHA ratio and adrenic acid (22:4n-6) composition were negatively correlated with age at death in controls but not in suicides, and males exhibited a greater AA:DHA ratio irrespective of cause-of-death. These results demonstrate that adolescent male and female suicide victims do not exhibit DHA deficits in the postmortem PFC relative to age-matched controls, and suggest that suicide victims do not exhibit the normal age-related decrease in adrenic acid composition and the AA:DHA ratio.     

An Investigation into the Strength of the Association and Agreement Levels between Subjective and Objective Sleep Duration in Adolescents

Study Objectives

The majority of adolescent sleep research has utilized self-reported sleep duration and some have based information on a solitary question. Whilst some have claimed to have validated sleep survey data with objective actigraphy measures in adolescents, the statistical approach applied only demonstrates the strength of the association between subjective and objective sleep duration data and does not reflect if these different methods actually agree.

Methods

Data were collected as part of the Midlands Adolescents Schools Sleep Education Study (MASSES). Adolescents (n=225) aged 11-13 years provided estimates for weekday, weekend and combined sleep duration based on self-reported survey data, a 7-day sleep diary, and wrist-worn actigraphy.

Results

We assessed the strength of the relationship as well as agreement levels between subjective and objectively determined sleep duration (weekday, weekend and combined). Subjective diary sleep duration was significantly correlated with actigraphy estimates for weekday and weekend sleep duration r=0.30, p≤0.001 and r=0.31, p≤0.001 respectively. Pitman’s test demonstrated no significant difference in the variance between weekend sleep duration (r=0.09, p=0.16) and combined sleep duration (r=0.12, p=0.08) indicating acceptable agreement between actigraphy and sleep diary sleep duration only. Self-reported sleep duration estimates (weekday, weekend and combined) did not agree with actigraphy determined sleep duration.

Conclusions

Sleep diaries are a cost-effective alternative to survey/questionnaire data. Self-reported measures of sleep duration in adolescents do not agree with actigraphy measures and should be avoided where possible. Previous adolescent sleep studies that have utilized self-reported survey data may not provide a complete representation of sleep on the outcome measure of interest.     

Comparative assessment of in situ bioremediation potential of cadmium resistant acidophilic Pseudomonas putida 62BN and alkalophilic Pseudomonas monteilli 97AN strains on soybean

A study was undertaken to examine the effects of the acidophilic strain 62BN (pH 5.5) and alkalophilic strain 97AN (pH 9.0) on remediation of cadmium and their subsequent effects on soybean (Glycine max var. PS-1347) in acidic and alkaline soils, respectively. The effect of cadmium on soybean plants was studied in acidic (pH 6.3 ± 0.2) and alkaline (8.5 ± 0.2) soil amended with 124 μM CdCl₂ concentration, respectively, and the cadmium toxicity was evident from stunted growth, poor rooting, and cadmium accumulation in each case. Furthermore, 16S ribosomal DNA (rDNA) sequencing identified 62BN as a Pseudomonas putida strain and 97AN as a Pseudomonas monteilli strain. In situ studies showed that on seed bacterization, both the P. putida 62BN strain and P. monteilli 97AN strain were able to enhance plant growth in terms of agronomical parameters, in the presence of cadmium in acidic and alkaline soils, respectively. Apart from this, strain 62BN and 97AN reduced cadmium concentration in plant and soil significantly (p < 0.05) in their respective soil types. Further comparative analysis revealed that P. putida 62BN was more effective than P. monteilli 97AN strain in remediation of cadmium. The bacterial strains offer promise as inoculants to improve the growth of plants in the presence of toxic Cd concentrations in the environment with their optimum pH.     

Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814)

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

Compromised base excision repair pathway in Mycobacterium tuberculosis imparts superior adaptability in the host

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host’s dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.