Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Biosynthesis of fluorescent gold nanoparticles using an edible freshwater red alga,Lemanea fluviatilis (L.) C.Ag. and antioxidant activity of biomatrix loaded nanoparticles

Biosynthesis of gold nanoparticles has been accomplished via reduction of an aqueous chloroauric acid solution with the dried biomass of an edible freshwater epilithic red alga, Lemanea fluviatilis (L.) C.Ag., as both reductant and stabilizer. The synthesized nanoparticles were characterized by UV–visible, powder X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), and dynamic light scattering (DLS) studies. The UV–visible spectrum of the synthesized gold nanoparticles showed the surface plasmon resonance (SPR) at around 530 nm. The powder XRD pattern furnished evidence for the formation of face-centered cubic structure of gold having average crystallite size 5.9 nm. The TEM images showed the nanoparticles to be polydispersed, nearly spherical in shape and have sizes in the range 5–15 nm. The photoluminescence spectrum of the gold nanoparticles excited at 300 nm showed blue emission at around 440 nm. Gold nanoparticles loaded within the biomatrix studied using a modified 2,2-diphenyl-1-picrylhydrazyl (DPPH) method exhibited pronounced antioxidant activity.     

Cloning, expression, and purification of Brucella suis outer membrane proteins

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.     

Alternative bisphosphonate targets and mechanisms of action

As the number of bisphosphonates continues to increase, they have found widespread use in an increasing number of clinical conditions. Ongoing examination of their targets and mechanisms of action has revealed that this surprisingly diverse class of drugs has effects beyond those first described for osteoclasts. These additional targets include osteoblasts, osteocytes, angiogenesis, and gammadelta T lymphocytes of the human immune system. The immune system effects are specifically targeted to gammadelta T cells and are reminiscent of the effects seen after ingestion of tea beverage. BP effects on such alternate targets may explain not only their antiresorptive effect, but also their effect on bone quality, tumors, and microbes.     

Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.     

Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Targeted disruption of the mouse Asna1 gene results in embryonic lethality

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.     

Bilateral Common Carotid Artery Ligation Transiently Changes Brain Lipid Metabolism in Rats

Brain lipid metabolism was studied in rats following permanent bilateral common carotid artery ligation (BCCL), a model for chronic cerebral hypoperfusion. Unesterified (free) fatty acids (uFA) and acyl-CoA concentrations were measured 6 h, 24 h, and 7 days after BCCL or sham surgery, in high energy-microwaved brain. In BCCL compared to sham rats, cytosolic phospholipase A(2) (cPLA(2)) immunoreactivity in piriform cortex, and concentrations of total uFA and arachidonoyl-CoA, an intermediate for arachidonic acid reincorporation into phospholipids, were increased only at 6 h. At 24 h, immunoreactivity for secretory phospholipase A(2) (sPLA(2)), which may regulate blood flow, was increased near cortical and hippocampal blood vessels. BCCL did not affect levels of brain IB(4)+ microglia, glial fibrillary acidic protein (GFAP)+ astrocytes, cyclooxygenase-2 (COX-2) immunoreactivity at any time, but increased cPLA(2) immunoreactivity in one region at 6 h. Thus, BCCL affected brain lipid metabolism transiently, likely because of compensatory sPLA(2)-mediated vasodilation, without producing evidence of neuroinflammation.