Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary,Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.     

An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides.

We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.     

Synthesis of 9-fluorenemethyl boranophosphonodiphosphate via an H-phosphonate approach

9-Fluorenemethyl boranophosphonate 6 and its boranophosphonodiphosphate 7 were synthesized via an H-phosphonate approach. The method is efficient for the synthesis of acyclic compounds 6 & 7, and can be explored for the synthesis of nucleoside 5'-deoxy boranophosphonodiphosphate.     

Non‐Parametric Selected Ranked Set Sampling

A nonparametric selected ranked set sampling is suggested. The estimator of population mean based on the new approach is compared with that using the simple random sampling (SRS), the ranked set sampling (RSS) and the median ranked set sampling (MRSS) methods. The estimator of population mean using the new approach is found to be more efficient than its counter‐parts for almost all the cases considered.     

Effects of nerve growth factor on nerve regeneration through a vein graft across a gap.

The limited availability of donor sites for nerve grafts and their inherent associated morbidity continue to stimulate research toward finding suitable alternatives. In the following study, the effect of direct administration of nerve growth factor (NGF) into a nerve conduit across a gap was tested in a rat sciatic nerve model. A 1-cm segment of the right sciatic nerve in Sprague-Dawley rats was resected, and the gap was then bridged using one of three methods: group I (NGF-treated group, n = 12), a vein graft filled with NGF (100 ng in 0.3-ml phosphate buffered saline); group II (control group, n = 12), a vein graft filled with phosphate buffered saline only; group III (standard nerve graft, n = 11), a resected segment of the sciatic nerve. All animals were evaluated at 3 and 5 weeks by behavioral testing and at 5 weeks by electrophysiologic testing. At 3 weeks, sensory testing showed that the latency to a noxious stimulus in group I animals (8.0 +/- 5.4 sec, mean +/- SD) was significantly lower than that of group II animals (13.2 +/- 6.5 sec), indicating that sensory recovery was superior in the animals receiving NGF. The mean latency of animals in group III was 12.9 +/- 6.5 sec, but the difference between the latencies of group I and group III did not reach statistical significance. At 5 weeks, there was no difference in sensory testing between groups. Motor function in groups I and III as measured by walk pattern analysis was superior to that of group II at 5 weeks (toe spread ratios 0.66 +/- 0.09, 0.48 +/- 0.07, and 0.69 +/- 0.09 for groups I, II, and III, respectively). Mean motor conduction velocities across the 1-cm gap were 8.6 +/- 4.7 m/sec, 2.5 +/- 0.7 m/sec, and 6.9 +/- 2.9 m/sec in groups I, II, and III respectively. The difference between groups I and III was not statistically significant, but the motor conduction velocity of group II was significantly slower than that of either group I or III (p < 0.002). The positive effects of NGF on regeneration of nerves across a gap seen in this study suggest that it may be useful for treating peripheral nerve injuries in combination with autogenous vein grafts.     

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.     

Isolation and characterization of a highly effective bacterium Bacillus cereus b-525k for hexavalent chromium detoxification

The chromate resistant Gram-positive Bacillus cereus strain b-525k was isolated from tannery effluents, demonstrating optimal propagation at 37 °C and pH 8. The minimum inhibitory concentration (MIC) test showed that B. cereus b-525k can tolerate up to 32 mM Cr⁶⁺, and also exhibit the ability to resist other toxic metal ions including Pb²⁺ (23 mM), As³⁺ (21 mM), Zn²⁺ (17 mM), Cd²⁺ (5 mM), Cu²⁺ (2 mM), and Ni²⁺ (3 mM) with the resistance order as Cr ⁶⁺ > Pb²⁺ > As³⁺ >Zn²⁺ >Cd²⁺ >Ni²⁺ >Cu²⁺. B. cereus b-525k showed maximum biosorption efficiency (q) of 51 mM Cr⁶⁺/g after 6 days. Chromate stress elicited pronounced production of antioxidant enzymes such as catalase (CAT) 191%, glutathione transferase (GST) 192%, superoxide dismutase (SOD) 161%, peroxidase (POX) 199%, and ascorbate peroxidase (APOX) (154%). Within B. cereus b-525k, the influence of Cr⁶⁺ stress (2 mM) did stimulate rise in levels of GSH (907%) and non-protein thiols (541%) was measured as compared to the control (without any Cr⁶⁺ stress) which markedly nullifies Cr⁶⁺ generated oxidative stress. The pilot scale experiments utilizing original tannery effluent showed that B. cereus b-525k could remove 99% Cr⁶⁺ in 6 days, thus, it could be a potential candidate to reclaim the chromate contaminated sites.     

Species richness,abundance, distributional pattern and trait composition of butterfly assemblage change along an altitudinal gradient in the Gulmarg region of Jammu & Kashmir,India

Despite enormous diversity, abundance, and role in ecosystem processes, little is known about how butterflies differ across altitudinal gradients. For this, butterfly communities were investigated along an altitudinal gradient of 2700–3200 m a.s.l, along the Gulmarg region of Jammu & Kashmir, India. We aimed to determine how the altitudinal gradient and environmental factors affect the butterfly diversity and abundance. Our findings indicate that species richness and diversity are mainly affected by the synergism between climate and vegetation. Alpha diversity indices showed that butterfly communities were more diverse at lower elevations and declined significantly with increase in elevation. Overall, butterfly abundance and diversity is stronger at lower elevations and gradually keep dropping towards higher elevations because floristic diversity decreased on which butterflies rely for survival and propagation. A total of 2023 individuals of butterflies were recorded belonging to 40 species, represented by 27 genera and 05 families. Six survey sites (S I- S VI) were assessed for butterfly diversity from 2018 to 2020 in the Gulmarg region of Jammu & Kashmir. Across the survey, Nymphalidae was the most dominant family represented by 16 genera and 23 species, while Papilionidae and Hesperiidae were least dominant represented by 01 genera and 01 species each. Among the six collection sites selected, Site I was most dominant, represented by 16 genera and 21 species, while Site VI was least dominant, represented by 04 genera and 04 species.