Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.     

Phosphorylation of eIF2 alpha in <Emphasis Type="Italic">Sf9</Emphasis> cells: a stress,survival and suicidal signal

An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2α) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2α phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2α phosphorylation. Infection of Sf9 cells by wt and a mutant Δpk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2α phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Δpk2 baculovirus that lacks pk2, an inhibitor of eIF2α kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2α phosphorylation, thereby suggesting that eIF2α phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2α phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2α phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2α, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2α phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.     

K<Emphasis Type="Italic">i</Emphasis> DoQ: using docking based energy scores to develop ligand based model for predicting antibacterials

Background  

Identification of novel drug targets and their inhibitors is a major challenge in the field of drug designing and development. Diaminopimelic acid (DAP) pathway is a unique lysine biosynthetic pathway present in bacteria, however absent in mammals. This pathway is vital for bacteria due to its critical role in cell wall biosynthesis. One of the essential enzymes of this pathway is dihydrodipicolinate synthase (DHDPS), considered to be crucial for the bacterial survival. In view of its importance, the development and prediction of potent inhibitors against DHDPS may be valuable to design effective drugs against bacteria, in general.     

Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na⁺, K⁺-ATPase activity, α₁ and β₁ subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na⁺, K⁺-ATPase α₁ subunit abundance increased in IR24 and IR48 cortical tissue and β₁ subunit decreased in IR48. In IR24 medullary tissue, α₁ abundance increased and returned to control values in IR48 while β₁ abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in α₁ mRNA in cortex and medulla that normalised after 24 h of reperfusion. β₁ mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult α₁ and β₁ protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na⁺, K⁺-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.     

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.