Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.     

Synthesis and characterization of pH-sensitive hydrogel composed of carboxymethyl chitosan for colon targeted delivery of ornidazole

In the present study, carboxymethyl chitosan was prepared from chitosan, crosslinked with glutaraldehyde and evaluated in vitro as a potential carrier for colon targeted drug delivery of ornidazole. Ornidazole was incorporated at the time of crosslinking of carboxymethyl chitosan. The chitosan was evaluated for its degree of deacetylation (DD) and average molecular weight; which were found to be 84.6% and 3.5×10(4) Da, respectively. The degree of substitution on prepared carboxymethyl chitosan was found to be 0.68. All hydrogel formulations showed more than 85% and 74% yield and drug loading, respectively. The swelling behaviour of prepared hydrogels checked in different pH values, 1.2, 6.8 and 7.4, indicated pH responsive swelling characteristic with very less swelling at pH 1.2 and quick swelling at pH 6.8 followed by linear swelling at pH 7.4 with slight increase. In vitro release profile was carried out at the same conditions as in swelling and drug release was found to be dependant on swelling of hydrogels and showed biphasic release pattern with non-fickian diffusion kinetics at higher pH. The carboxymethylation of chitosan, entrapment of drug and its interaction in prepared hydrogels were checked by FTIR, (1)H NMR, DSC and p-XRD studies, which confirmed formation of carboxymethyl chitosan from chitosan and absence of any significant chemical change in ornidazole after being entrapped in crosslinked hydrogel formulations. The surface morphology of formulation S6 checked before and after dissolution, revealed open channel like pores formation after dissolution.     

Expression and immunoaffinity purification of recombinant dengue virus 2 NS1 protein as a cleavable SUMOstar fusion

Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S^∗-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S^∗-tag increasing protein solubility and secretion compared with a non-S^∗-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S^∗-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S^∗-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions.     

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the β-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.     

Synthesis, structural properties and insulin-enhancing potential of bis(quercetinato)oxovanadium(IV) conjugate

An oxovanadium complex of quercetin (2), exhibits highly potent insulin-enhancing activity in streptozotocin-induced diabetic mice. It also mimics mitogenic potential of insulin as evaluated by [H(3)]thymidine uptake assay making an effective, orally active insulin-enhancing agent for the treatment of both type 1 and type 2 diabetes without any noticeable toxic effects.     

Symbols, Meaning, and Origins of Mind

The mind maps symbols and the extra-symbolic relationships amongst them to specific meanings. When symbols of various levels are placed in a hierarchical ordering, one may look at such ordered classes as distinct worlds where one class represents objects and the other represents the objects’ corresponding meanings. However, such an explanation can only be partial because the number of potential levels in such an ordering is infinite and, therefore, it engenders problems of recursion and infinite regress. There are also logical problems in the form of paradoxes that emanate from the consideration of sets of sets. Given that most prior studies only consider symbols that are classical objects in associative relationships, we argue that there is a need to also consider objects with shifting boundaries and quantum objects. We believe that objects belonging to each of these three classes—that is classical objects, objects with shifting boundaries, and quantum objects—play a role in the workings of the mind.     

Ex Vivo Neurogenesis within Enteric Ganglia Occurs in a PTEN Dependent Manner

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU⁺ cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.     

Metalloantimalarials: synthesis, X-ray crystal structure of potent antimalarial copper (II) complex of arylazo-4-hydroxy-1,2-naphthoquinone

The crystal structure of copper (II) complex of 3-arylazo-4-hydroxy-1,2-naphthoquinone is reported. The in vitro antimalarial activity of analogous compounds against Plasmodium falciparum 3D7 strain reveals correlation with metal redox couple, suggesting component of parasitic electron transport chain as a possible target.     

Cortical 5-HT1A receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Inhibitors of Escherichia coli serine acetyltransferase block proliferation of Entamoeba histolytica trophozoites

The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells.