A novel interaction between FRMD7 and CASK: evidence for a causal role in idiopathic infantile nystagmus.

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder of eye movement that can be caused by mutations in the FRMD7 gene that encodes a FERM domain protein. FRMD7 is expressed in the brain and knock-down studies suggest it plays a role in neurite extension through modulation of the actin cytoskeleton, yet little is known about its precise molecular function and the effects of IIN mutations. Here, we studied four IIN-associated missense mutants and found them to have diverse effects on FRMD7 expression and cytoplasmic localization. The C271Y mutant accumulates in the nucleus, possibly due to disruption of a nuclear export sequence located downstream of the FERM-adjacent domain. While overexpression of wild-type FRMD7 promotes neurite outgrowth, mutants reduce this effect to differing degrees and the nuclear localizing C271Y mutant acts in a dominant-negative manner to inhibit neurite formation. To gain insight into FRMD7 molecular function, we used an IP-MS approach and identified the multi-domain plasma membrane scaffolding protein, CASK, as a FRMD7 interactor. Importantly, CASK promotes FRMD7 co-localization at the plasma membrane, where it enhances CASK-induced neurite length, whereas IIN-associated FRMD7 mutations impair all of these features. Mutations in CASK cause X-linked mental retardation. Patients with C-terminal CASK mutations also present with nystagmus and, strikingly, we show that these mutations specifically disrupt interaction with FRMD7. Together, our data strongly support a model whereby CASK recruits FRMD7 to the plasma membrane to promote neurite outgrowth during development of the oculomotor neural network and that defects in this interaction result in nystagmus.

Unravelling the Tripartite Interactions Among Hepatitis E Virus RNA, miR-140 and hnRNP K.

In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140.

Variable neutralizing antibody responses to 10 SARS-CoV-2 variants in natural infection with wild- type (B.1) virus, Kappa (B.1.617.1), and Delta (B.1.617.2) variants and COVISHIELD vaccine immunization in India: utility of the MSD platform.

For the efficacy of COVID-19 vaccines, emergence of variants accumulating immune-escape mutations remains a major concern. We analyzed the anti-variant (n = 10) neutralization activity of sera from COVID-19 patients infected with Wuhan (B.1), Kappa, and Delta variants and COVISHIELD vaccine recipients with (prepositives) or without (prenegatives) prior antibody positivity using V- PLEX ACE2 Neutralization Kit from MSD. MSD and PRNT50 correlated well (r = 0.76-0.83, p < 0.0001). Despite the least antibody positivity in Kappa patients, anti-variant neutralizing antibody (Nab) levels in the responders were comparable with Delta patients. Vaccinees sampled at 1 month (PD2-1) and 6 months (PD2-6) post-second dose showed the highest seropositivity and Nab levels against the Wuhan strain. At PD2-1, the responder rate was variant-dependent and 100% respectively in prenegatives and prepositives. Nab levels against B.1.135.1, B.1.620, B.1.1.7+E484K (both groups), AY.2 (prenegatives), and B.1.618 (prepositives) were lower than that of Wuhan. At PD2-6, positivity decreased to 15.6%-68.8% in the prenegatives; 3.5%-10.7% of prepositives turned negative for the same four variants. As against the decline in Nab levels in 9/10 variants (prenegatives), a further reduction was seen against the same four variants in the prepositives. These variants possess immune-evasion-associated mutations in the RBD/S region. In conclusion, our data show that the Nab response of patients to multiple variants depends on the infecting variant. We confirm superiority of hybrid immunity in neutralizing multiple variants. Depending on the infecting variant pre- or postvaccination, immune response to different vaccines in different populations will vary and impact protection against emerging variants. The MSD platform provides an excellent alternative to live virus/pseudovirus neutralization tests.

Uncovering the Roles of miR-214 in Hepatitis E Virus Replication.

Viral pathogenesis is a complex event and its regulation involve dynamic interactions with various host factors, of which microRNAs are the key players. In the current study, we have identified the functional importance of an interplay between hepatitis E virus (HEV) and miR-214. Computational analysis indicated that miR-214 binding site is significantly conserved among HEV and related RNA viruses. Intact miR-214 binding site is imperative for HEV replication. miR-214 is an essential host factor for HEV replication. Herein, we demonstrate that miR-214 interacts directly with HEV RNA to enhance HEV replication and HEV genome translation. Augmented translation results in increased levels of HEV ORF2, which is a factor responsible for upregulation of miR-214. HEV usurps host cellular machinery for improving viral fitness and elevates miR-214 expression for amplifying the expression of proviral host factor intracellular active thrombin. This is because miR-214 represses the expression of the negative regulator of thrombin, i.e., protein C. Another viral factor, HEV ORF3, also contributes to the enhancement of intracellular active thrombin. Furthermore, miR-214 directly targets antiviral host factor 2'-5'-oligoadenylate synthetase. Conclusively, we identified a novel mechanism of positive regulation of HEV replication. miR-214 interacts directly with HEV genome and fine-tunes host factors expression. This results in outweighing the proviral factors on the proviral-antiviral axis probably for generating virus supportive environment.

Relevance of sonochemistry or ultrasound (US) as a proficient means for the synthesis of fused heterocycles.

Medicinal chemistry has been benefited by combinatorial chemistry and high throughput parallel synthesis. The use of sonochemistry under controlled conditions has been proved beneficial for medicinal chemistry and drug discovery process since it dramatically reduces reaction times, from days or hours to minutes. In addition, sonochemistry synthesis provides higher yields, lower cost, easy workups and greater purity as compared to lower yields, tedious workups, longer reaction times, lesser purity and termination of many by-products in the conventional thermal methods.