Development of a hybrid alphavirus-SARS-CoV-2 pseudovirion for rapid quantification of neutralization antibodies and antiviral drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S)-pseudotyped viruses are commonly used for quantifying antiviral drugs and neutralizing antibodies. Here, we describe the development of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirion, which is a non-replicating SARS-CoV-2 virus-like particle composed of viral structural proteins (S, M, N, and E) and an RNA genome derived from a fast-expressing alphaviral vector. We validated Ha-CoV-2 for rapid quantification of neutralization antibodies, antiviral drugs, and viral variants. In addition, as a proof of concept, we used Ha-CoV-2 to quantify the neutralizing antibodies from an infected and vaccinated individual and found that the one-dose vaccination with Moderna mRNA-1273 greatly increased the anti-serum titer by approximately 6-fold. The post-vaccination serum can neutralize all nine variants tested. These results demonstrate that Ha-CoV-2 can be used as a robust platform for the rapid quantification of neutralizing antibodies against SARS-CoV-2 and its emerging variants.

Brief report: S6 ribosomal protein phosphorylation in autistic frontal cortex and cerebellum: a tissue array analysis.

Few autistic brain samples are available for study, limiting investigations into molecular and histopathological abnormalities associated with this common disease. To facilitate distribution of samples, we have constructed a tissue array containing cerebral and cerebellar cores from 5 autistic children, 1 girl with Rett syndrome, and 5 age-matched controls. To demonstrate the utility of this resource, we examined phosphorylation of the S6 ribosomal protein, a signaling event regulated by the genes mutated in tuberous sclerosis and Cowden disease. We hypothesized that the molecular pathways altered in these inherited conditions associated with autism might be dysregulated in sporadic autistic cases as well. However, no consistent alterations in S6 phosphorylation were detected in autistic tissues compared to controls in the brain regions analyzed.

beta2-adrenergic receptor activation and genetic polymorphisms in autism: data from dizygotic twins.

Gestational and genetic factors can contribute to autism during infancy and early childhood through their effects on fetal brain development. Previous twin studies have shown strong genetic components for the development of autism, a disorder that can have multiple causes. We investigated the effects of prenatal overstimulation of the beta2-adrenergic receptor in dizygotic twins who were exposed to terbutaline, a selective beta2-adrenergic receptor agonist used to treat premature labor, as a gestational factor. As a possible genetic mechanism, we studied two beta2-adrenergic receptor polymorphisms in twins from whom DNA was available: glycine substitution at codon 16 (16G) and glutamic acid substitution at codon 27 (27E), which show diminished desensitization in vivo compared with the wild-type receptor. Continuous terbutaline exposure for 2 weeks or longer was associated with increased concordance for autism spectrum disorders in dizygotic twins (relative risk = 2.0), with a further increase in the risk for male twins with no other affected siblings (relative risk = 4.4). A significant association was found between the presence of 16G and 27E polymorphisms in autistic patients compared with population controls (P = .006). Prenatal overstimulation of the beta2-adrenergic receptor by terbutaline or by increased signaling of genetic polymorphisms of the beta2-adrenergic receptor that have diminished desensitization can affect cellular responses and developmental programs in the fetal brain, leading to autism.

Diagnostic method validation: High resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene.

OBJECTIVES: According to OECD guidelines methods implemented in a diagnostic laboratory should be properly validated prior their implementation. For this purpose we selected genotyping by High Resolution Melting (HRM) of small amplicons using common variants in MTHFR as a model. DESIGN AND METHODS: We selected previously typed samples on which selected analytical validation-related parameters relevant to DNA diagnostics - specificity, sensitivity, precision, robustness and ability to perform reliable calls were evaluated. RESULTS: Correct genotype was assigned in 375/381 (98.4%) for c.677 C>T (rs1801133: C>T; p.A222 V) and in 102/104 (98.1%) for c.1298 A>C (rs1801131: A>C; p.E429A) of all cases. Low analytical failure rate and very high specificity/sensitivity were achieved. Similarly, precision and robustness were consistent. CONCLUSIONS: We have successfully validated HRM of small amplicons using common MTHFR variants as a model. We proved that this technique is highly reliable for routine diagnostics and our diagnostic validation strategy can serve as a model for other applications.