A Novel Agarase, Gaa16B, Isolated from the Marine Bacterium Gilvimarinus agarilyticus JEA5, and the Moisturizing Effect of Its Partial Hydrolysis Products

We recently identified a β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 ∘C and pH 6–7, respectively, and the protein was highly stable at 55 ∘C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.

Use of the Codon Table to Quantify the Evolutionary Role of Random Mutations

The various biases affecting RNA mutations during evolution is the subject of intense research, leaving the extent of the role of random mutations undefined. To remedy this lacuna, using the codon table, the number of codons representing each amino acid was correlated with the amino acid frequencies in different branches of the evolutionary tree. The correlations were seen to increase as evolution progressed. Furthermore, the number of RNA mutations that resulted in a given amino acid mutation were found to be correlated with several widely used amino acid similarity tables (used in sequence alignments). These correlations were seen to increase when the observed codon usage was factored in.

The First Feline Immunodeficiency Virus in Siberian Tigers (Panthera Tigris Altaica) from China

Research on feline immunodeficiency virus (FIV) from tigers is scant throughout the world. In this study, 320 captive Siberian tigers were tested for FIV by nested PCR, and three Siberian tigers were FIV-positive. This is the first time FIV has been detected in Siberian tigers in China. The phylogenetic analysis of three FIV genes, gag-p26, pol-RT, and pol-RNAse, revealed that the Siberian tiger FIV had the minimum genetic divergence, the closest genetic relationship and the highest amino acid similarity with subtype A FIV strains from domestic cats, suggesting that the Siberian tiger FIV may have been transmitted by stray cats.

Novel Siphoviridae Bacteriophages Infecting Bacteroides uniformis Contain Diversity Generating Retroelement

Intestinal phages are abundant and important components of gut microbiota, yet the isolated and characterized representatives that infect abundant gut bacteria are sparse. Here we describe the isolation of human intestinal phages infecting Bacteroidesuniformis. Bacteroides is one of the most common bacterial groups in the global human gut microbiota; however, to date not many Bacteroides specific phages are known. Phages isolated in this study belong to a novel viral genus, Bacuni, within the Siphoviridae family. Their genomes encode diversity-generating retroelements (DGR), which were shown in other bacteriophages to promote phage adaptation to rapidly changing environmental conditions and to broaden their host range. Three isolated phages showed 99.83% genome identity but one of them infected a distinct B. uniformis strain. The tropism of Bacuni phages appeared to be dependent on the interplay of DGR mediated sequence variations of gene encoding putative phage fimbrial tip proteins and mutations in host genes coding for outer-membrane proteins. We found prophages with up to 85% amino acid similarity over two-thirds of the Bacuni phage genome in the B. acidifaciens and Prevotella sp. genomes. Despite the abundance of Bacteroides within the human microbiome, we found Bacuni phages only in a limited subset of published gut metagenomes.

Adenoviral-Vectored Mayaro and Chikungunya Virus Vaccine Candidates Afford Partial Cross-Protection From Lethal Challenge in A129 Mouse Model

Mayaro (MAYV) and chikungunya viruses (CHIKV) are vector-borne arthritogenic alphaviruses that cause acute febrile illnesses. CHIKV is widespread and has recently caused large urban outbreaks, whereas the distribution of MAYV is restricted to tropical areas in South America with small and sporadic outbreaks. Because MAYV and CHIKV are closely related and have high amino acid similarity, we investigated whether vaccination against one could provide cross-protection against the other. We vaccinated A129 mice (IFNAR −/−) with vaccines based on chimpanzee adenoviral vectors encoding the structural proteins of either MAYV or CHIKV. ChAdOx1 May is a novel vaccine against MAYV, whereas ChAdOx1 Chik is a vaccine against CHIKV already undergoing early phase I clinical trials. We demonstrate that ChAdOx1 May was able to afford full protection against MAYV challenge in mice, with most samples yielding neutralizing PRNT80 antibody titers of 1:258. ChAdOx1 May also provided partial cross-protection against CHIKV, with protection being assessed using the following parameters: survival, weight loss, foot swelling and viremia. Reciprocally, ChAdOx1 Chik vaccination reduced MAYV viral load, as well as morbidity and lethality caused by this virus, but did not protect against foot swelling. The cross-protection observed is likely to be, at least in part, secondary to cross-neutralizing antibodies induced by both vaccines. In summary, our findings suggest that ChAdOx1 Chik and ChAdOx1 May vaccines are not only efficacious against CHIKV and MAYV, respectively, but also afford partial heterologous cross-protection.

A Novel Neoagarotriose-Producing Agarase from the Marine Bacterium Gilvimarinus Agarilyticus JEA5

Background The degradation of agar by bacterial agarases has many commercial and academic applications. We recently identified a novel neoagarotriose-producing β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. This is the first report to describe neoagarotriose production from β-agarase.Results The Gaa16B agarase, which belongs to the glycoside hydrolase 16 (GH16) family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. The coding region of Gaa16B is 1800 bp long, encoding 600 amino acids, and exhibits features typical of agarases belonging to the GH16 family. A recombinant Gaa16B lacking the carbohydrate binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified as a maltose-binding protein (MBP) fusion protein. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 °C and pH 6–7, respectively, and the protein was highly stable at 55 °C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/ml and 953 U/mg, respectively. Thin layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose, neoagarotriose, and neoagarobiose, and the production of neoagarotriose by rGaa16Bc was successfully validated by high-resolution electrospray ionization mass spectrometry.Conclusion The biochemical properties of Gaa16B and the results of the hydrolytic pattern analysis suggest that Gaa16B could be useful to produce functional neoagaro-oligosaccharides for industrial applications.

The Product Specificities of Maize Terpene Synthases TPS4 and TPS10 Are Determined Both by Active Site Amino Acids and Residues Adjacent to the Active Site

Terpene synthases make up a large family of enzymes that convert prenyl diphosphates into an enormous variety of terpene skeletons. Due to their electrophilic reaction mechanism—which involves the formation of carbocations followed by hydride shifts and skeletal rearrangements—terpene synthases often produce complex mixtures of products. In the present study, we investigate amino acids that determine the product specificities of the maize terpene synthases TPS4 and TPS10. The enzymes showed 57% amino acid similarity and produced different mixtures of sesquiterpenes. Sequence comparisons and structure modeling revealed that out of the 43 amino acids forming the active site cavity, 17 differed between TPS4 and TPS10. While combined mutation of these 17 residues in TPS4 resulted in an enzyme with a product specificity similar to TPS10, the additional mutation of two amino acids next to the active site led to a nearly complete conversion of TPS4 into TPS10. These data demonstrate that the different product specificities of TPS4 and TPS10 are determined not only by amino acids forming the active site cavity, but also by neighboring residues that influence the conformation of active site amino acids.

Nucleotide Analogues as Inhibitors of SARS-CoV Polymerase

SummarySARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency.1 Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved heptatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2.2 Here, using model polymerase extension experiments, we demonstrate that the activated triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the activated triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected two other anti-viral agents, Alovudine and AZT (an FDA approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two HIV reverse transcriptase inhibitors, 3’-fluoro-3’-deoxythymidine triphosphate and 3’-azido-3’-deoxythymidine triphosphate (the active triphosphate forms of Alovudine and AZT), to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.

Structural modeling of 2019-novel coronavirus (nCoV) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to SARS-CoV and related SARS-like coronaviruses

The 2019 novel coronavirus (2019-nCoV) is currently causing a widespread outbreak centered on Hubei province, China and is a major public health concern. Taxonomically 2019-nCoV is closely related to SARS-CoV and SARS-related bat coronaviruses, and it appears to share a common receptor with SARS-CoV (ACE-2). Here, we perform structural modeling of the 2019-nCoV spike glycoprotein. Our data provide support for the similar receptor utilization between 2019-nCoV and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains, which we predict to be proteolytically-sensitive. We suggest this loop confers fusion activation and entry properties more in line with MERS-CoV and other coronaviruses, and that the presence of this structural loop in 2019-nCoV may affect virus stability and transmission.

Molecular Docking Study of Akar Kuning (Arcangelisia flava) Secondary Metabolites as Src Inhibitor

Proto-oncogene tyrosine-protein kinase Src is also known as simply Src is a tyrosine kinase protein which is one of the targets in various cancer therapies such as leukemia. Meanwhile, akar kuning (Arcangelisia flava) has gained significant attention as a medicinal plant that has a cytotoxic effect on various types of cancer cells. This study aims to determine the potential of secondary metabolites of akar kuning as Src inhibitors. Molecular docking was carried out using Autodock Vina 1.1.2 with 2HCK receptors, that quercetin and dasatinib were used as reference ligands. The docking results showed that the highest affinity was shown by berberine with a ΔG value of -9.0 kcal/mol, exceeded quercetin and dasatinib. However, the highest amino acid similarity to quercetin and dasatinib was produced by jatrorrhizine, with 93.33% and 73.91%, respectively. Interestingly, berberine is the ligand with the third-highest similarity after jatrorrhizine and palmatine, while jatrorrhizine has the second-highest affinity after berberine. The results concluded that the combination of berberine and jatrorrhizine is predicted to be optimally used as an Src inhibitor in cancer therapy.