Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR.

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.

Study of Efficacy of Injection Remdesivir in Patients of COVID-19.

INTRODUCTION: The coronavirus disease of 2019 (COVID-19) is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). World Health Organization (WHO) declared it a pandemic on 11th March 2020. Injectable remdesivir (RDV), a repurposed antiviral, was first accorded approval by the United States of America (USA) Food and Drug Administration (FDA) on 1st May 2020, for emergency use to treat suspected or laboratory-confirmed COVID-19 patients. Interim analysis of the Solidarity trial revealed no benefits in patients treated with RDV in any group of patients with COVID-19. Here, we have attempted to place our data on the efficacy of RDV in patients of COVID-19 with moderate to severe categories. MATERIALS AND METHODS: A retrospective review and data analysis of 100 COVID-19 patients with reverse transcriptase polymerase chain reaction (RT-PCR)/rapid antigen test positive was performed. Among them, 50 received RDV in addition to the standard treatment protocol (STP), while the remaining 50 received only the STP. STP is an injectable steroid and heparin, along with other supportive management. Prevalent government guidelines were followed as per usual for the classification of the patients and treatment protocol. Every day of hospitalization, the status of respiratory support was checked, and every 3rd-day inflammatory markers [C-reactive protein (CRP) and D-dimer] were measured until the patient was discharged or died. Statistical analysis of the data was done using online software. RESULTS: Age and comorbidity distribution in both groups ensures adequate matching between the two groups. A statistically significant difference in hospitalization days was obtained in RDV-treated patients (15 vs 19 days, p-value -0.003). Statistically significant differences were not found in mortality (6 vs 10 deaths, p-value -0.27) and reduction in oxygen (O2)/ventilatory support requirements (p-value -0.75) in the RDV group as compared to other groups. The difference in the value of CRP (p-value 0.001) and D-dimer (p-value 0.049) on day 5 is statistically significant in the RDV group as compared to the other groups. DISCUSSION: The finding of a reduction in days of hospitalization was similar to the Adaptive COVID-19 Treatment Trial (ACTT) 1 study conducted by Beigel et al. The mortality data were also comparable to those from WHO's Solidarity trial. No similarity was found in data on the reduction in ordinal scale from higher to lower scale for O2/ventilatory support on day 10 from 0. Similarity regarding the reduction in values of inflammatory markers on day 5 was found in studies conducted by Kannan et al. and Stoeckle et al. CONCLUSION: We found mortality benefit and reduction in O2 requirements/ventilatory support in RDV plus STP-administered cases as compared to STP only, but statistically, this difference is not significant, which suggests that mortality benefit in the RDV group in our study is merely by chance. Here, we can definitely conclude that days of hospital stay and inflammatory markers are reduced in the RDV plus STP-administered group, and the difference between the two groups is statistically significant, which suggests that early use of RDV could shorten the time to clinical improvement.

NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4.

Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix-helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.

Novel carbazole-stilbene hybrids as multifunctional anti-Alzheimer agents.

Molecules capable of engaging with multiple targets associated with pathological condition of Alzheimer's disease have proved to be potential anti-Alzheimer's agents. In our goal to develop multitarget-directed ligands for the treatment of Alzheimer's disease, a novel series of carbazole-based stilbene derivatives were designed by the fusion of carbazole ring with stilbene scaffold. The designed compounds were synthesized and evaluated for their anti-AD activities including cholinesterase inhibition, Aß aggregation inhibition, antioxidant and metal chelation properties. Amongst them, (E)-1-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(pyrrolidin-1-yl)ethyl)thiourea (50) appeared to be the best candidate with good inhibitory activities against AChE (IC50 value of 2.64 µM) and BuChE (IC50 value of 1.29 µM), and significant inhibition of self-mediated Aß1-42 aggregation (51.29% at 25 µM concentration). The metal chelation study showed that compound (50) possessed specific copper ion chelating property. Additionally, compound (50) exhibited moderate antioxidant activity. To understand the binding mode of 50, molecular docking studies were performed, and the results indicated strong non-covalent interactions of 50 with the enzymes in the active sites of AChE, BuChE as well as of the Aß1-42 peptide. Additionally, it showed promising in silico ADMET properties. Putting together, these findings evidently showed compound (50) as a potential multitarget-directed ligand in the course of developing novel anti-AD drugs.

Pheromone Perception: Mechanism of the Reversible Coil-Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1.

Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.

Production and biophysical characterization of a mini-membrane protein, Ost4V23D: A functionally important mutant of yeast oligosaccharyltransferase subunit Ost4p.

N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc. Complete loss of N-glycosylation is lethal in all organisms. In Saccharomyces cerevisiae, OST consists of nine non-identical protein subunits. Ost4p is the smallest subunit containing 36 residues. It bridges catalytic subunit Stt3p to Ost3p/Ost6p subunit. Mutation of Valine (V) at position 23 in Ost4p to Aspartate (D) causes defects in the N-glycosylation process. To understand the structure, function and role of Ost4p in N-glycosylation, characterization of Ost4p and its functionally important mutant/s are critical. We report the mutagenesis, heterologous overexpression, purification, reconstitution in DPC micelles and biophysical characterization of Ost4V23D and compare its secondary structure and conformation to that of Ost4p. CD and NMR data suggest that mutation of Val23 to Asp impacts the secondary structure and conformation of Ost4p.

Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

Association of Mean Platelet Volume With Vascular Complications in the Patients With Type 2 Diabetes Mellitus.

Background Diabetes mellitus (DM) is a global health concern that is predicted to involve over 10% of the adult population by the next decade. Vascular complications are the major source of mortality and morbidity in diabetics. Mean platelet volume (MPV) which indicates platelet activity may play a crucial role in the vascular effects of DM and, hence, can be used as a prognostic marker. We have attempted to study the association of MPV with the glycemic status, duration of diabetes, and presence of vascular complications in diabetics. Methods A cross-sectional study of 300 patients with type 2 DM aged ≥18 years admitted to the inpatient department of medicine was carried out in a tertiary care hospital. After subgrouping patients according to their glycemic status and MPV, the association between microvascular and macrovascular complications was studied. Results The majority of patients were >60 years of age and an increasing prevalence of vascular complications was noted with increasing age. Forty-six percent and 45% of the patients had microvascular and macrovascular complications, respectively. Ischemic heart disease (IHD) and retinopathy were the most common macrovascular and microvascular complications, respectively. Vascular complications in DM showed a significant association with MPV, fasting blood sugar (FBS), post-prandial blood sugar (PP2BS), glycated hemoglobin (HbA1c), and the duration of diabetes. Conclusion A high MPV was linked to poor glycemic control, a longer duration of diabetes, and an increased prevalence of vascular complications. Hence, MPV could be used as a cost-effective marker to predict vascular complications in patients with type 2 DM.

Carbazole-based semicarbazones and hydrazones as multifunctional anti-Alzheimer agents.

With the aim to combat a multi-faceted neurodegenerative Alzheimer's disease (AD), a series of carbazole-based semicarbazide and hydrazide derivatives were designed, synthesized and assessed for their cholinesterase (ChE) inhibitory, antioxidant and biometal chelating activity. Among them, (E)-2-((9-ethyl-9H-carbazol-3-yl)methylene)-N-(pyridin-2-yl)hydrazinecarbothioamide (62) and (E)-2-((9-ethyl-9H-carbazol-3-yl)methylene)-N-(5-chloropyridin-2-yl)hydrazinecarbothioamide (63) emerged as the premier candidates with good ChE inhibitory activities (IC50 values of 1.37 µM and 1.18 µM for hAChE, IC50 values of 2.69 µM and 3.31 µM for EqBuChE, respectively). All the test compounds displayed excellent antioxidant activity (reduction percentage of DPPH values for compounds (62) and (63) were 85.67% and 84.49%, respectively at 100 µM concentration). Compounds (62) and (63) conferred specific copper ion chelating property in metal chelation study. Molecular docking studies of compounds (62) and (63) indicate strong interactions within the active sites of both the ChE enzymes. Besides that, these compounds also exhibited significant in silico drug-like pharmacokinetic properties. Thus, taken together, they can serve as a starting point in the designing of multifunctional ligands in pursuit of potential anti-AD agents that might further prevent the progression of ADs.Communicated by Ramaswamy H. Sarma.

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

Structural and Functional Characterization of European Corn Borer, Ostrinia nubilalis, Pheromone Binding Protein 3.

Ostrinia nubilalis, a lepidopteran moth, also known as the European corn borer, has a major impact on the production of economically important crops in the United States and Europe. The female moth invites the male moth for mating through the release of pheromones, a volatile chemical signal. Pheromone binding proteins (PBPs) present in the male moth antennae are believed to pick up the pheromones, transport them across the aqueous sensillum lymph, and deliver them to the olfactory receptor neurons. Here we report for the first time the cloning, expression, refolding, purification, and structural characterization of Ostrinia nubilalis PBP3 (OnubPBP3). The recombinant protein showed nanomolar affinity to each isomer of the Ostrinia pheromones, E- and Z-11-tetradecenyl acetate. In a pH titration study by nuclear magnetic resonance, the protein exhibited an acid-induced unfolding at pH below 5.5. The molecular dynamics simulation study demonstrated ligand-induced conformational changes in the protein with both E- and Z-isomers of the Ostrinia pheromone. The simulation studies showed that while protein flexibility decreases upon binding to E-pheromone, it increases when bound to Z-pheromone. This finding suggests that the OnubPBP3 complex with E-pheromone is more stable than with Z-pheromone.

1H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D.

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high-mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as Congenital Disorders of Glycosylation. Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C, and 15N resonance assignments for Ost4 and Ost4V23D in dodecylphosphocholine micelles.

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase.

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell-cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

1H, 13C, and 15N resonance assignment and secondary structure of the pheromone-binding protein2 from the agricultural pest Ostrinia furnacalis (OfurPBP2).

Ostrinia furnacalis, a lepidopteran moth, is an invasive pest found in Asia, Australia, Africa, and parts of the United States. The O. furnacalis pheromone-binding protein2 (OfurPBP2), present in the male moth antenna, plays a role in the detection of female-secreted pheromone in a process that leads to mating. To understand the structural mechanism of pheromone binding and release in this pest, we have initiated characterization of OfurPBP2 by solution NMR. Here, we report the backbone resonance assignments and the secondary structural elements of OfurPBP2 at pH 6.5 using uniformly 13C, 15N-labeled protein with various triple resonance NMR experiments. The assignments are 97% completed for backbone and 88% completed for side-chain resonances. The secondary structure of OfurPBP2, based on backbone chemical shifts, consists of eight α-helices, including a well-structured C-terminal helix.

Further Studies on Triazinoindoles as Potential Novel Multitarget-Directed Anti-Alzheimer's Agents.

The inadequate clinical efficacy of the present anti-Alzheimer's disease (AD) drugs and their low impact on the progression of Alzheimer's disease in patients have revised the research focus from single targets to multitarget-directed ligands. A novel series of substituted triazinoindole derivatives were obtained by introducing various substituents on the indole ring for the development of multitarget-directed ligands as anti-AD agents. The experimental data indicated that some of these compounds exhibited significant anti-AD properties. Among them, 8-(piperidin-1-yl)-N-(6-(pyrrolidin-1-yl)hexyl)-5H-[1,2,4]triazino[5,6-b]indol-3-amine (60), the most potent cholinesterase inhibitor (AChE, IC50 value of 0.32 µM; BuChE, IC50 value of 0.21 µM), was also found to possess significant self-mediated Aß1-42 aggregation inhibitory activity (54% at 25 µM concentration). Additionally, compound 60 showed strong antioxidant activity. In the PAMPA assay, compound 60 exhibited blood-brain barrier penetrating ability. An acute toxicity study in rats demonstrated no sign of toxicity at doses up to 2000 mg/kg. Furthermore, compound 60 significantly restored the cognitive deficits in the scopolamine-induced mice model and Aß1-42-induced rat model. In the in silico ADMET prediction studies, the compound satisfied all the parameters of CNS acting drugs. These results highlighted the potential of compound 60 to be a promising multitarget-directed ligand for the development of potential anti-AD drugs.

Peritoneal dialysis for the prevention of fluid overload in infants after cardiac surgery - A systematic review and meta-analysis.

The objective is to compare peritoneal dialysis with standard care therapy for the prevention of fluid overload in infants after cardiac surgery. We searched published literature through the major database up to December 2017. Randomized controlled trials (RCTs), quasi-randomized trials, and observational studies were included in the study. The primary outcome measures were as follows: all-cause mortality and duration of hospitalization. Of the 392-citation retrieved, full text of 7 was finally assessed for eligibility. Of these, a total of five studies (RCTs = 3, and observational studies = 2) were included. There was no significant difference between the prophylactic PD and the standard therapy group for any of the primary outcomes. The present systematic review shows that prophylactic PD is not beneficial compared to standard care in infants postcardiac surgery for congenital heart disease. The GRADE evidence generated was of "very low quality."

Structure and Function Studies of Asian Corn Borer Ostrinia furnacalis Pheromone Binding Protein2.

Lepidopteran male moths have an extraordinarily sensitive olfactory system that is capable of detecting and responding to minute amounts of female-secreted pheromones over great distances. Pheromone-binding proteins (PBPs) in male antennae ferry the hydrophobic ligand across the aqueous lymph to the olfactory receptor neuron triggering the response. PBPs bind ligands at physiological pH of the lymph and release them at acidic pH near the receptor while undergoing a conformational change. In Anthereae polyphemus PBP1, ligand binding to the hydrophobic pocket and its release is regulated by two biological gates: His70 and His95 at one end of the pocket and C-terminus tail at the other end. Interestingly, in Asian corn borer Ostrinia furnacalis PBP2 (OfurPBP2), critical residues for ligand binding and release are substituted in both biological gates. The impact of these substitutions on the ligand binding and release mechanism in OfurPBP2 is not known. We report here overexpression of soluble OfurPBP2 and structural characterization at high and low pH by circular dichroism (CD) and NMR. Ligand binding and ab initio model development were carried out with fluorescence and small-angle X-ray scattering (SAXS) respectively. OfurPBP2 in solution at pH 6.5 is homogeneous, well-folded and has a compact globular shape.

Comparison of caudal and intravenous dexamethasone as adjuvants for caudal epidural block: A double blinded randomised controlled trial.

BACKGROUND AND AIMS: Dexamethasone has a powerful anti-inflammatory action with significant analgesic benefits. The aim of this study was to compare the efficacy of dexamethasone administered through intravenous (IV) and caudal route on post-operative analgesia in paediatric inguinal herniotomy patients. METHODS: One hundred and five paediatric patients undergoing inguinal herniotomy were included and divided into three groups. Each patient received a single caudal dose of ropivacaine 0.15%, 1.5 mL/kg combined with either corresponding volume of normal saline (Group 1) or caudal dexamethasone 0.1 mg/kg (Group 2) or IV dexamethasone 0.5 mg/kg (Group 3). Baseline, intra- and post-operative haemodynamic parameters, pain scores, time to rescue analgesia, total analgesic consumption and adverse effects were evaluated for 24 h after surgery. Unpaired Student's t-test and analysis of variance were applied for quantitative data and Chi-square test for qualitative data. Time to first analgesic administration was analysed by Kaplan-Meier survival analysis and log-rank test. RESULTS: Duration of analgesia was significantly longer (P < 0.001), and total consumption of analgesics was significantly lower (P < 0.001) in Group II and III compared to Group I. The incidence of nausea and vomiting was higher in Group I (31.4%) compared to Group II and III (8.6%). CONCLUSIONS: Addition of dexamethasone both caudally or intravenously as an adjuvant to caudal 0.15% ropivacaine significantly reduced the intensity of post-operative pain and prolonged the duration of post-operative analgesia with the significant advantage of caudal over IV route.

Malaria mortality in Africa and Asia: evidence from INDEPTH health and demographic surveillance system sites.

BACKGROUND: Malaria continues to be a major cause of infectious disease mortality in tropical regions. However, deaths from malaria are most often not individually documented, and as a result overall understanding of malaria epidemiology is inadequate. INDEPTH Network members maintain population surveillance in Health and Demographic Surveillance System sites across Africa and Asia, in which individual deaths are followed up with verbal autopsies. OBJECTIVE: To present patterns of malaria mortality determined by verbal autopsy from INDEPTH sites across Africa and Asia, comparing these findings with other relevant information on malaria in the same regions. DESIGN: From a database covering 111,910 deaths over 12,204,043 person-years in 22 sites, in which verbal autopsy data were handled according to the WHO 2012 standard and processed using the InterVA-4 model, over 6,000 deaths were attributed to malaria. The overall period covered was 1992-2012, but two-thirds of the observations related to 2006-2012. These deaths were analysed by site, time period, age group and sex to investigate epidemiological differences in malaria mortality. RESULTS: Rates of malaria mortality varied by 1:10,000 across the sites, with generally low rates in Asia (one site recording no malaria deaths over 0.5 million person-years) and some of the highest rates in West Africa (Nouna, Burkina Faso: 2.47 per 1,000 person-years). Childhood malaria mortality rates were strongly correlated with Malaria Atlas Project estimates of Plasmodium falciparum parasite rates for the same locations. Adult malaria mortality rates, while lower than corresponding childhood rates, were strongly correlated with childhood rates at the site level. CONCLUSIONS: The wide variations observed in malaria mortality, which were nevertheless consistent with various other estimates, suggest that population-based registration of deaths using verbal autopsy is a useful approach to understanding the details of malaria epidemiology.