A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

Drug delivery into the cochlear apex: Improved control to sequentially affect finely spaced regions along the entire length of the cochlear spiral.

BACKGROUND: Administering pharmaceuticals to the scala tympani of the inner ear is a common approach to study cochlear physiology and mechanics. We present here a novel method for in vivo drug delivery in a controlled manner to sealed ears. NEW METHOD: Injections of ototoxic solutions were applied from a pipette sealed into a fenestra in the cochlear apex, progressively driving solutions along the length of scala tympani toward the cochlear aqueduct at the base. Drugs can be delivered rapidly or slowly. In this report we focus on slow delivery in which the injection rate is automatically adjusted to account for varying cross sectional area of the scala tympani, therefore driving a solution front at uniform rate. RESULTS: Objective measurements originating from finely spaced, low- to high-characteristic cochlear frequency places were sequentially affected. Comparison with existing methods(s): Controlled administration of pharmaceuticals into the cochlear apex overcomes a number of serious limitations of previously established methods such as cochlear perfusions with an injection pipette in the cochlear base: The drug concentration achieved is more precisely controlled, drug concentrations remain in scala tympani and are not rapidly washed out by cerebrospinal fluid flow, and the entire length of the cochlear spiral can be treated quickly or slowly with time. CONCLUSIONS: Controlled administration of solutions into the cochlear apex can be a powerful approach to sequentially effect objective measurements originating from finely spaced cochlear regions and allows, for the first time, the spatial origin of CAPs to be objectively defined.

Rothmund-Thomson syndrome in siblings: evidence for acquired in vivo mosaicism.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by skin abnormalities that appear in infancy, skeletal abnormalities, juvenile cataracts and other manifestations of premature aging, and a predisposition to malignancy. The diagnosis is made on clinical grounds as no consistent laboratory test has been identified. Chromosome studies have been reported for only three patients with RTS and in two of these three, trisomy 8 mosaicism was found. We performed a variety of cytogenetic and molecular genetic studies on two siblings with RTS and on their phenotypically normal parents. Two chromosomally abnormal clones involving either trisomy 8 or i(8q) were found in both patients with RTS. These clones were present in vivo, as they were seen in interphase buccal smears and lymphocytes from unstimulated preparations using both conventional cytogenetic studies and fluorescence in situ hybridization (FISH) with a centromere probe for chromosome 8. These results suggest that RTS is associated with in vivo clonal chromosomal rearrangements causing an acquired somatic mosaicism.

Macular suprastructure, stereociliary bonding and kinociliary/stereociliary coupling in rat utricular macula.

This report considers rat utricular macular suprastructure after chemical treatment with the sodium salt of N,N-Naphthaloylhydroxylamine (NHA), used alone or in combination with tannic acid (TA). NHA and TA preserve calcium and complex carbohydrate-protein molecules, respectively. Macromolecules of supramacular substance appear comparable morphologically to material of the external lamina of glycocalyx. Similar material crosslinks stereocilia with approximately equal to 58 nm periodicity and couples parts of kinocilia with specific stereocilia. Particles which occur within kinocilia at certain attachment sites connect to dynein arms of the kinocilia. Interstereociliary connections are aligned with internal linkers to actin. Thus, a basis for dynamic communication between kinocilia and stereociliary actin has been shown. The mechanism would appear to be calcium dependent. The findings support the concept that kinocilia are motile and lead to the stereociliary tuft in vestibular hair cells.

The suprastructure of the saccular macula.

Inclusion of tannic acid in the primary fixative fortuitously preserved the macular suprastructure, from the macular surface to the otoconia, in rat. Results demonstrate that fluid-filled channels around the stereociliary tufts and a slit-like space immediately above the macula are artefacts of tissue preparation. Filaments fill the area between, and show linkage to, the macular surface and the more peripheral, closely knit layer identified here as the true otoconial membrane. The material also passes through the otoconial membrane at various sites to surround the otoconia and extends well beyond the maculas in all directions. Although it may prove later to be endolymph, it is referred to here as supramacular substance. Kinocilia and the tallest stereocilia as well as the otoconia are tethered to the otoconial membrane. Findings were confirmed in decalcified material. Results are discussed in terms of functional and biochemical implications, and by analogy to man-made acceleration sensing devices.

Innervation patterns in rat saccular macula. A structural basis for complex sensory processing.

Serial sections through the anterior part of rat saccular macula were reconstructed as montages. Findings are that type II hair cells are integrated into the neural circuitry of type I cells, chiefly by synapses with neighboring calyces and their collaterals; and that complex interactions between afferent- and efferent-type nerve elements take place. Three basic types of nerve/calyx pattern are present: U-type nerves lose their myelin before they enter the macula and have complex calyces with several collaterals; M-type nerves are myelinated up to the calyx, which lacks collaterals; and M/U-type nerves have short, unmyelinated segments proximal to their calyces, which have few collaterals. Both afferent- and efferent-type collaterals spring from calyces, chiefly from those of U-type nerves. Type II cells are presynaptic both to electron-lucent and to vesiculated terminals; some synapses are reciprocal. Electron-lucent boutons sometimes are presynaptic to calyces and to type II hair cells; and morphologically afferent-to-afferent kinds of synapses occur in the neuroepithelium. The anatomical findings indicate that complex information processing must occur in mammalian gravity receptors.

Otoconia as test masses in biological accelerometers: what can we learn about their formation from evolutionary studies and from work in microgravity?

This paper reviews previous findings and introduces new material about otolith end organs that help us to understand their functioning and development. In particular, we consider the end organs as biological accelerometers. The otoconia are dealt with as test masses whose substructure and evolutionary trend toward calcite may prove significant in understanding formation requirements. Space-flight helps illuminate the influence of gravity, while right-left asymmetry is suggested by study of certain rat strains.

Removal of an N-terminal peptide from mitochondrial aspartate aminotransferase abolishes its interactions with mitochondria in vitro.

Treatment of mitochondrial aspartate aminotransferase from rat liver with trypsin leads to specific cleavage of the bonds between residues 26 and 27, and residues 31 and 32. The proteolysed enzyme has only a small residual catalytic activity, but retains a conformation similar to that of the native form as judged by accessibility and reactivity of cysteine residues. Proteolysis abolishes the ability of the enzyme either to bind to mitochondria or to be imported into the organelles. This suggests that the N-terminal segment of the native enzyme is essential for both of these functions, at least in the model system used to study the import process.

Permanent deficits in lordosis behavior in female rats with lesions of the ventromedial nucleus of the hypothalamus.

Bilateral lesions of the ventromedial nucleus of the hypothalamus (VMN) significantly attentuated lordosis behavior in the female rat. The degree of this deficit was significantly correlated with the amount of damage to the VMN. There was no improvement in lordosis performance even after an extended recovery period.

Antarctic environment and nail growth.

Nail growth of all fingers was measured on 13 men wintering in Antarctica. A mean rate of 109-5 micrometer/day was found with the longer digits having faster growth. No significant difference was demonstrated between the warmer and colder months of the study period. Monthly variations suggested that subjects had an individual rate irrespective of environmental influences. Comparison with previous studies both in polar and temperature conditions showed growth rates in Antarctica have increased progressively; present values being comparable with observations in temperate climates.