Control of copula duration and sperm storage by female Queensland fruit flies

Copula duration and sperm storage patterns can directly or indirectly affect fitness of male and female insects. Although both sexes have an interest in the outcome, research has tended to focus on males. To investigate female influences, we compared copula duration and sperm storage of Queensland fruit fly females that were intact, or had been incapacitated through decapitation or abdomen isolation. We found that copulations were far longer when females had been incapacitated, indicating that constraints imposed on copula duration by intact females had been relaxed. Repeatability of copula duration for males was very low regardless of female treatment, and this is also consistent with strong female influence. Number of sperm in the spermathecae was not influenced by female treatment, suggesting that female abdominal ganglia control the transport of sperm to these long-term storage organs. However, more sperm were found in the ventral receptacles of incapacitated females compared to intact females. Overall, results implicate cephalic ganglia in regulation of copula duration and short-term sperm storage in the ventral receptacle and abdominal ganglia in regulation of long-term sperm storage in the spermathecae.     

Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells

Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at 'safe harbor' sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that ～10% of integrations of a lentivirally encoded β-globin transgene in β-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level β-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy.     

miRNA regulatory circuits in ES cells differentiation: a chemical kinetics modeling approach

MicroRNAs (miRNAs) play an important role in gene regulation for Embryonic Stem cells (ES cells), where they either down-regulate target mRNA genes by degradation or repress protein expression of these mRNA genes by inhibiting translation. Well known tables TargetScan and miRanda may predict quite long lists of potential miRNAs inhibitors for each mRNA gene, and one of our goals was to strongly narrow down the list of mRNA targets potentially repressed by a known large list of 400 miRNAs. Our paper focuses on algorithmic analysis of ES cells microarray data to reliably detect repressive interactions between miRNAs and mRNAs. We model, by chemical kinetics equations, the interaction architectures implementing the two basic silencing processes of miRNAs, namely "direct degradation" or "translation inhibition" of targeted mRNAs. For each pair (M,G) of potentially interacting miRMA gene M and mRNA gene G, we parameterize our associated kinetic equations by optimizing their fit with microarray data. When this fit is high enough, we validate the pair (M,G) as a highly probable repressive interaction. This approach leads to the computation of a highly selective and drastically reduced list of repressive pairs (M,G) involved in ES cells differentiation.     

(−)-Epigallocatechin-3-gallate (EGCG) attenuates peripheral nerve degeneration in rat sciatic nerve crush injury

Recently, we have shown that green tea (GT) consumption improves both reflexes and sensation in unilateral chronic constriction injury to the sciatic nerve. Considering the substantial neuroprotective properties of GT polyphenols, we sought to investigate whether (−)-epigallocatechin-3-gallate (EGCG) could protect the sciatic nerve and improve functional impairments induced by a crushing injury. We also examined whether neuronal cell apoptosis induced by the crushing injury is affected by EGCG treatment.     

Population genetic structure of the Plasmodium vivax circumsporozoite protein (Pvcsp) in Sri Lanka

Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710 bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~ 2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant.     

Angiotensin AT2 Receptor Contributes towards Gender Bias in Weight Gain

Obesity is a major disease condition, in turn leading to pathological changes collectively recognized as metabolic syndrome. Recently angiotensin receptor AT₂R has been associated negatively with body weight (BW) gain in male mice. However, the gender differences in AT₂R and BW changes have not been studied. To understand the gender based role of AT₂R involving BW changes, we fed male and female wild type (WT) and AT₂R knock out (AT₂KO) mice with C57BL6 background with high fat diet (HFD) for 16 weeks. The male AT₂KO had higher HFD calorie intake (WT: 1280±80; AT₂KO:1680±80 kcal) but gained less BW compared with the WT (WT: 13; AT₂KO: 6 g). Contrary to the male animals, the female AT₂KO mice with equivalent caloric intake (WT: 1424±48; AT₂KO:1456±80 kcal) gained significantly more BW than the WT mice (WT: 9 g; AT₂KO: 15 g). The male AT₂KO on HFD displayed lower plasma insulin level, less impaired glucose tolerance (GT), and higher plasma T3 compared with WT males on HFD; whereas the female AT₂KO mice on HFD showed elevated levels of plasma insulin, more impaired GT, lower plasma T3 and higher free fatty acid and hepatic triglycerides compared with WT females on HFD. Interestingly, compared with WT, AT₂KO female mice had significantly lower estrogen, which was further reduced by HFD. These results suggest that AT₂R in female mice via potentially regulating estrogen may have protective role against BW gain and impaired glucose tolerance and lipid metabolism.     

Biotransformation of Meloxicam by <Emphasis Type="Italic">Cunninghamella blakesleeana</Emphasis>: Significance of Carbon and Nitrogen Source

Influence of carbon and nitrogen source, on biotransformation of meloxicam was studied by employing Cunninghamella blakesleeana NCIM 687 with an aim to achieve maximum transformation of meloxicam and in search of new metabolites. The transformation was confirmed by HPLC and based on LC–MS–MS data and previous reports the metabolites were predicted as 5-hydroxymethyl meloxicam, 5-carboxy meloxicam and a novel metabolite. The quantification of metabolites was performed using HPLC peak areas. The results obtained indicate that glucose as carbon source, ammonium nitrate as nitrogen source, were found to be optimum for maximum transformation of meloxicam. The study suggests the significance of these factors in biotransformation of meloxicam using microbial cultures. The fermentation was scaled up to 1 l level.