Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

Targeted Whole Exome Sequencing in Children With Early-Onset Epilepsy: Parent Experiences.

This study investigated the experiences of 25 caregivers of children with early-onset, treatment-resistant epilepsy who pursued whole exome sequencing to determine the impact of the test results on their child's treatment. Caregivers who consented to be recontacted were recruited from a previous study investigating the diagnostic yield of whole exome sequencing. A semistructured interview addressed questions based on one of 2 study phases. The first phase discussed the decision-making process for genetic testing (15 interviews), which revealed 4 major themes: (1) prognosis, (2) engagement, (3) concerns, and (4) autonomy. The second phase discussed the impact of genetic testing on treatment (10 interviews), which revealed 3 major themes: (1) testing features, (2) emotional impact, and (3) treatment outcomes. Overall, parents pursued genetic testing to obtain a clear prognosis, inform treatment decisions, engage with other families, and exercise autonomy. Caregivers felt that early testing is warranted to inform their child's diagnostic odyssey.

Regional distribution and maturation of tau pathology among phenotypic variants of Alzheimer's disease.

Alzheimer's disease neuropathologic change (ADNC) is clinically heterogenous and can present with a classic multidomain amnestic syndrome or focal non-amnestic syndromes. Here, we investigated the distribution and burden of phosphorylated and C-terminally cleaved tau pathologies across hippocampal subfields and cortical regions among phenotypic variants of Alzheimer's disease (AD). In this study, autopsy-confirmed patients with ADNC, were classified into amnestic (aAD, N = 40) and non-amnestic (naAD, N = 39) groups based on clinical criteria. We performed digital assessment of tissue sections immunostained for phosphorylated-tau (AT8 detects pretangles and mature tangles), D421-truncated tau (TauC3, a marker for mature tangles and ghost tangles), and E391-truncated tau (MN423, a marker that primarily detects ghost tangles), in hippocampal subfields and three cortical regions. Linear mixed-effect models were used to test regional and group differences while adjusting for demographics. Both groups showed AT8-reactivity across hippocampal subfields that mirrored traditional Braak staging with higher burden of phosphorylated-tau in subregions implicated as affected early in Braak staging. The burden of phosphorylated-tau and TauC3-immunoreactive tau in the hippocampus was largely similar between the aAD and naAD groups. In contrast, the naAD group had lower relative distribution of MN423-reactive tangles in CA1 (ß = - 0.2, SE = 0.09, p = 0.001) and CA2 (ß = - 0.25, SE = 0.09, p = 0.005) compared to the aAD. While the two groups had similar levels of phosphorylated-tau pathology in cortical regions, there was higher burden of TauC3 reactivity in sup/mid temporal cortex (ß = 0.16, SE = 0.07, p = 0.02) and MN423 reactivity in all cortical regions (ß = 0.4-0.43, SE = 0.09, p < 0.001) in the naAD compared to aAD. In conclusion, AD clinical variants may have a signature distribution of overall phosphorylated-tau pathology within the hippocampus reflecting traditional Braak staging; however, non-amnestic AD has greater relative mature tangle pathology in the neocortex compared to patients with clinical amnestic AD, where the hippocampus had greatest relative burden of C-terminally cleaved tau reactivity. Thus, varying neuronal susceptibility to tau-mediated neurodegeneration may influence the clinical expression of ADNC.

Mechanical Stiffness Controls Dendritic Cell Metabolism and Function.

Stiffness in the tissue microenvironment changes in most diseases and immunological conditions, but its direct influence on the immune system is poorly understood. Here, we show that static tension impacts immune cell function, maturation, and metabolism. Bone-marrow-derived and/or splenic dendritic cells (DCs) grown in vitro at physiological resting stiffness have reduced proliferation, activation, and cytokine production compared with cells grown under higher stiffness, mimicking fibro-inflammatory disease. Consistently, DCs grown under higher stiffness show increased activation and flux of major glucose metabolic pathways. In DC models of autoimmune diabetes and tumor immunotherapy, tension primes DCs to elicit an adaptive immune response. Mechanistic workup identifies the Hippo-signaling molecule, TAZ, as well as Ca2+-related ion channels, including potentially PIEZO1, as important effectors impacting DC metabolism and function under tension. Tension also directs the phenotypes of monocyte-derived DCs in humans. Thus, mechanical stiffness is a critical environmental cue of DCs and innate immunity.