Biophysical properties of the isolated spike protein binding helix of human ACE2

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel β-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1^mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.     

Defining the Catalytic Activity of Nanoceria in the P23H-1 Rat,a Photoreceptor Degeneration Model

Purpose

Inorganic catalytic nanoceria or cerium oxide nanoparticles (CeNPs) are bona fide antioxidants that possess regenerative radical scavenging activities in vitro. Previously, we demonstrated that CeNPs had neuroprotective and anti-angiogenic properties in rodent retinal degeneration and neovascularization models. However, the cellular mechanisms and duration of the catalytic activity of CeNPs in preventing photoreceptor cell loss are still unknown. In this study, we sought to answer these questions using the P23H-1 rat, an autosomal dominant retinitis pigmentosa (adRP) model.

Methods

A single dose of either saline or CeNPs was delivered intravitreally into the eyes of P23H-1 rats at 2–3 weeks of age. Retinal functions were examined at 3 to 7 weeks post injection. We quantified retinal proteins by Western blot analyses and counted the number of apoptotic (TUNEL+) profiles in the outer nuclear layer (ONL) of retinal sections. We measured free 8-isoprostanes to quantify lipid peroxidation in retinal tissues.

Results

We observed increased rod and cone cell functions up to three weeks post injection. Apoptotic cells were reduced by 46%, 56%, 21%, and 24% at 3, 7, 14, 21 days, respectively, after CeNPs injection compared to saline. Additionally, reduction of lipid peroxidation in the retinas of CeNPs-treated vs saline-treated animals was detected 14 days post injection.

Conclusions

We validated that CeNPs were effective in delaying loss of photoreceptor cell function in an adRP rat model. This represents the fourth rodent retinal disease model that shows delay in disease progression after a single application of CeNPs. We further demonstrated that CeNPs slowed the rate of photoreceptor cell death. We deduced that the catalytic activity of CeNPs in vivo in this rat model to be undiminished for at least 7 days and then declined over the next 14 days after CeNPs administration.     

Effect of TIM-3 Blockade on the Immunophenotype and Cytokine Profile of Murine Uterine NK Cells

NK cells are the most abundant lymphocyte population in the feto-maternal interface during gestation. The uterine NK cells (uNK) are transient, have a unique immunophenotype and produce a number of cytokines. These cytokines play an important role in establishment and maintenance of vascular remodeling and tolerance associated with successful pregnancy. The uNK cells also express TIM-3 during gestation and blockade of TIM-3 expression results in fetal loss in mice. In this study we determined the effect of TIM-3 blockade on uNK cells. Specifically we observed surface receptor phenotype and cytokine production by uNK cells following TIM-3 blockade. Our results show that TIM-3 plays a role in regulating the uNK cells and contributes to the maintenance of tolerance at the feto-maternal interface.     

Impact of bifunctional chelators on biological properties of <Superscript>111</Superscript>In-labeled cyclic peptide RGD dimers

The present study describes the synthesis and biological evaluation of ¹¹¹In(DOTA-3P-RGD₂) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), ¹¹¹In(DTPA-3P-RGD₂) (DTPA = diethylenetriaminepentaacetic acid) and ¹¹¹In(DTPA-Bn-3P-RGD₂) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α_vβ₃ expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the ¹¹¹In-labeled 3P-RGD₂. IC₅₀ values of DOTA-3P-RGD₂, DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂ were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting ¹¹¹InCl₃ with the RGD peptide conjugates in NH₄OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD₂, successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15–20 min. For DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂, the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for ¹¹¹In(DOTA-3P-RGD₂) and ~200 mCi/mg (or ~400 mCi/μmol) for ¹¹¹In(DTPA-3P-RGD₂). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both ¹¹¹In(DTPA-3P-RGD₂) and ¹¹¹In(DTPA-Bn-3P-RGD₂) showed a much faster tumor washout and poorer T/B ratios than ¹¹¹In(DOTA-3P-RGD₂). The tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) is integrin α_vβ₃- and RGD-specific. ¹¹¹In(DOTA-3P-RGD₂) is metabolically stable while only ~25% of ¹¹¹In(DTPA-Bn-3P-RGD₂) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that ¹¹¹In(DTPA-3P-RGD₂) can be an effective integrin α_vβ₃-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.     

Effects of entrapment on nucleic acid content,cell morphology,cell surface property,and stress of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.     

Influence of plant growth hormones on the growth of Mucor rouxii and chitosan production

Supplementation of molasses-salt medium with plant growth hormones, viz., indoleacetic acid, indolebutyric acid, kinetin and gibberellic acid, increased chitosan production by Mucor rouxii as well as its growth at different optimum concentrations. The increase in yield of chitosan was found to range from 34% to 69% and mycelial growth from 12% to 17.4%. Gibberellic acid was the most potent in this respect. Sixty-nine percent more chitosan over the control could be obtained from 1l of the medium supplemented with 3mg gibberellic acid. Degree of acetylation of chitosan ( approximately 13%) was not changed due to addition of hormone in the medium but weight average molecular weight of chitosan increased by more than 50%. Thus, the plant growth hormones add a value to chitosan by increasing its molecular weight.     

NMR insights into folding and self-association of Plasmodium falciparum P2

The eukaryotic 60S-ribosomal stalk is composed of acidic ribosomal proteins (P1 and P2) and neutral protein P0, which are thought to be associated as a pentameric structure, [2P1, 2P2, P0]. Plasmodium falciparum P2 (PfP2) appears to play additional non-ribosomal functions associated with its tendency for homo-oligomerization. Recombinant bacterially expressed PfP2 protein also undergoes self-association, as shown by SDS-PAGE analysis and light scattering studies. Secondary structure prediction algorithms predict the native PfP2 protein to be largely helical and this is corroborated by circular dichroism investigation. The (1)H-(15)N HSQC spectrum of native P2 showed only 43 cross peaks compared to the expected 138. The observed peaks were found to belong to the C-terminal region, suggesting that this segment is flexible and solvent exposed. In 9 M urea denaturing conditions the chain exhibited mostly non-native β structural propensity. (15)N Relaxation data for the denatured state indicated substantial variation in ms-μs time scale motion along the chain. Average area buried upon folding (AABUF) calculations on the monomer enabled identification of hydrophobic patches along the sequence. Interestingly, the segments of slower motion in the denatured state coincided with these hydrophobic patches, suggesting that in the denatured state the monomeric chain undergoes transient hydrophobic collapse. The implications of these results for the folding mechanism and self-association of PfP2 are discussed.     

Sulfated polysaccharides from Laminaria angustata: Structural features and in vitro antiviral activities

Sulfated polysaccharides potently inhibit the infectivity of herpes simplex virus (HSV) in cultured cells. In this study, we have analyzed sulfated xylogalactofucan and alginic acid containing fractions generated from Laminaria angustata, a marine alga. The xylogalactofucan that has apparent molecular mass of 56 ± 5 kDa and unusually low sulfate content contains, inter alia, 1,3-, 1,4- and 1,2-linked fucopyranosyl residues. The algin (molecular mass: 32 ± 5 kDa) contains gulo- (55.5%) and mannuronic (44.5%) acid residues. Introduction of sulfate groups enhanced the macromolecules capability to inhibit the infection of cells by HSV-1. The 50% inhibitory concentration (IC₅₀) values of these macromolecules against HSV-1 were in the range of 0.2-25 μg ml⁻¹ and they lacked cytotoxicity at concentrations up to 1000 μg ml⁻¹. The sulfate content appeared to be an important hallmark of anti-HSV-1 activity. Our results suggest the feasibility of inhibiting HSV attachment to cells by direct interaction of polysaccharides with viral particles.     

Characterization and crop production efficiency of diazotrophic bacterial isolates from coastal saline soils

Use of eco-friendly area specific salt tolerant bioinoculants is better alternatives to chemical fertilizer for sustainable agriculture in coastal saline soils. We isolated diverse groups of diazotrophic bacteria from coastal saline soils of different forest and agricultural lands in the Sundarbans, West Bengal, India, to study their effect on crop productivity in saline soils. Phenotypic, biochemical and molecular identifications of the isolates were performed. The isolates produced indole acetic acid, phosphatase, and solubilized insoluble phosphates. Sequence analysis of 16S rDNA identified the SUND_BDU1 strain as Agrobacterium and the strains SUND_LM2, Can4 and Can6 belonging to the genus Bacillus. The ARA activity, dinitrogen fixation and presence of nifH genes indicated they were diazotrophs. Field trials with these strains as bioinoculants were carried out during 2007-2009, with rice during August-December followed by Lady's finger during April-June. Microplots, amended with FYM inoculated with four bioinoculants individually were compared against sole FYM (5 t ha(-1)) and a sole chemical fertilizer (60:30:30 kg ha(-1) NPK) treated plot. The strain Can6 was by far the best performer in respect of yield attributes and productivity of studied crops.