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The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

Although Caesarean section has greatly improved obstetric care throughout the world, there is a great aversion to caesarean section in developing countries. This study was carried out to examine the rate at which pregnant women with Pre-eclampsia decide to choose caesarean section over natural birth.A descriptive study was conducted among a sample of 112 pregnant women with Pre-eclampsia, aged 18-40 years, whose gestation period has exceeded 20 weeks and are registered in the hospitals. The sample selection was purposive.Data collection was done through self-administered structured questionnaire. Data analysis was presented using simple percentages.94% of respondents had no prior information about Pre-eclampsia and its associated complications. Pre-eclampsia has a prevalence rate of 12.3% among respondents in Ado-Ekiti as at the time this study was conducted. 38% of respondent favored caesarean section because they believed it is safer, less painful with little or no complications. If caesarean section is given as an only medical option for safe childbirth to respondents who preferred natural birth, 46.4% would opt for religious interventions while 10.7% would opt for traditional healthcare.Respondents who preferred natural birth would not choose caesarean section because of fear, cultural belief, religious beliefs and the cost. Level of education, religion and employment status were found to influence respondents' decision to choose. Hence the need to encourage research in awareness creation, patient education and best ways to assist patients decide on mode of delivery and plan on how to finance treatment.

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue ‘flaps’. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

Three aquaporin water channel proteins, AQP3, AQP4 and AQP15 were localized to cells within the kidney of the spiny dogfish, Squalus acanthias, using an immunohistochemical approach. Dogfish kidney has two zones, the bundle zone (including five nephron segment bundles) and the sinus zone (with two major loops). In order to discriminate between the two loops, the cilia occurring in the first proximal/intermediate loop were labeled with two antibodies including an anti-acetylated tubulin antibody. The second late distal tubule loop (LDT) was identified, as the nephron in that region has no luminal cilia. Strong staining of the rabbit anti-dogfish AQP3, AQP4 (AQP4/2) or AQP15 polyclonal antibodies localized to LDT tubules. These antibodies were further co-stained with a mouse anti-Na+,K+-ATPase a5 monoclonal antibody, as Na+,K+-ATPase has previously been suggested to localize to the early distal tubule (EDT) and LDT and a mouse anti-NKCC T4 antibody, as NKCC2 was previously suggested to be located in the EDT and the second half of the LDT. In the LDT, strong AQP4/2 and AQP15 antibody staining localized together with the strong Na+,K+-ATPase antibody staining, whereas strong AQP3 antibody staining was largely separate but with an overlapping distribution. Very low levels of AQP4/2 antibody basal membrane staining was also detected in the first proximal /intermediate loop of the sinus zone. There was no mouse anti-NKCC T4 antibody staining apparent in the LDT. In the convoluted part of the bundle zone, the AQP4/2 and Na+,K+-ATPase but not the AQP3 or AQP15 antibodies stained tubule segments, with both AQP4/2 and Na+,K+-ATPase staining the EDT, and with low-level AQP4/2 staining of two other tubules of the bundle, which were most likely to be the proximal 1a (PIa) and intermediate II (IS II) tubules. The AQP4/2 antibody also stained the EDT in the straight bundle zone. The mouse anti-NKCC T4 antibody stained the apical region of EDT tubules in the convoluted bundle zone, suggesting that the antibody was binding to the NKCC2 cotransporter. The AQP15 antibody appeared to bind to the peritubular sheath surrounding bundles in the bundle zone. Due to the AQP4/2 antibody staining in the EDT that immediately proceeds and continues into the LDT, this suggested that the strong AQP4/2, AQP15 and Na+,K+-ATPase antibody staining was located at the beginning of the LDT and therefore the strong AQP3 was located at the end of the LDT. The staining of all three AQP antibodies was blocked by the peptide-antigen used to make each one, suggesting that all the staining is specific to each antibody.

INTRODUCTION Hypoxemia is a life-threatening condition and is commonly seen in children with severe pneumonia. A government-led, NGO-supported, multifaceted oxygen improvement program was implemented to increase access to oxygen therapy in 29 hospitals in Kaduna, Kano, and Niger states. The program installed pulse oximeters and oxygen concentrators, trained health care workers, and biomedical engineers (BMEs), and provided regular feedback to health care staff through quality improvement teams. OBJECTIVE The aim of this study is to evaluate whether the program increased screening for hypoxemia with pulse oximetry and prescription of oxygen for patients with hypoxemia. METHODOLOGY The study is an uncontrolled before-after interventional study implemented at the hospital level. Medical charts of patients under 5 admitted for pneumonia between January 2017 and August 2018 were reviewed and information on patient care was extracted using a standardized form. The preintervention period of this study was defined as 1 January to 31 October 2017 and the postintervention period as 1 February to 31 August 2018. The primary outcomes of the study were whether blood-oxygen saturation measurements (SpO2 ) were documented and whether children with hypoxemia were prescribed oxygen. RESULTS A total of 3418 patient charts were reviewed (1601 during the preintervention period and 1817 during the postintervention period). There was a significant increase in the proportion of patients with SpO2 measurements after the interventions were conducted (adjusted odds ratio [aOR] 5.0; 4.3-5.7, P

Plasmodium falciparum heat shock protein 90 (PfHsp90) has been investigated as a potential target of antimalarial drug action using naturally occurring compounds. In this study, we performed in silico screening of 236 phytochemicals of Azadirachta indica, a plant known to possess antimalarial activity, and identified fourteen (14) potential non-carcinogenic, non-mutagenic, non-teratogenic and non-genotoxic phytochemicals. These phytochemicals were docked into the ATP-binding site of PfHsp90 using Autodock vina, and docked poses were rescored using PLANTS ChemPlp, X-Score version 1.2 and NNScore version 2.0. Consensus analysis of the scores using rank-by-rank and rank-by-number and receptor-ligand interaction assessment using LigPlot, led to the identification of margolone, margolonone, nimbinone, nimbione, nimosone and sugiol as best ranked potential interacting partners of PfHsp90. Molecular dynamic simulations of PfHsp90-ligand complexes for the six phytochemicals were performed using NAMD 2.9. The RMSD analysis of simulations trajectories, the ligand interaction analysis of receptor-ligand complex, and the free energy of binding with MMPBSA.py script and Bennett's acceptance ratio method (BAR) confirmed that these six phytochemicals may have potential to functionally interact with PfHsp90. However, though sharing several similar interacting residues with standard control binders yet the higher number of hydrogen bonds, higher level of sustained stability during molecular dynamics simulations and better free energy of binding suggest that margolonone, nimbinone and nimbione may have higher functional interaction potential with PfHsp90. Therefore, these phytochemicals may serve as potential leads in antimalarial drug design and development.

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3′ and 5′ ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3′-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5′-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3′-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5′-terminal 5-nt barcode extensions for a set of 20 barcoded 3′ adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

MicroRNAs play important roles in animal development. Numerous conditional knockout (cKO) studies of Dicer have been performed to interrogate the functions of microRNA during mammalian development. However, because Dicer was recently implicated in the biogenesis of endogenous siRNAs in mammals, it raises the question whether the Dicer cKO defects can be attributable to the loss of microRNAs. Previously, we and others conditionally targeted Dicer and identified its critical roles in embryonic skin morphogenesis. Here, we focus explicitly on microRNAs by taking a parallel strategy with Dgcr8 , encoding an essential component of the microprocessor complex that is exclusively required for microRNA biogenesis. With this comparative analysis, we show definitively that the Dicer - and Dgcr8 -null skin defects are both striking and indistinguishable. By deep sequencing analysis of microRNA depletion in both Dicer - and Dgcr8 -null skin, we demonstrate that most abundantly expressed skin microRNAs are dependent on both Dicer and DGCR8. Our results underscore a specific importance of microRNAs in controlling mammalian skin development.

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.

As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.

Distinct classes of small RNAs, 20 to 32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.