Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has been reported worldwide and novel SARS-CoV-2 variants continue to emerge. A novel SARS-CoV-2 strain, the Delta variant (B.1.617.2), is spreading worldwide. The Delta variant has reportedly high infectivity and immune evasion potency. In June 2021, the World Health Organization categorized it as a variant of concern (VOC). Therefore, it is vital to develop tests that can exclusively identify the Delta variant. Here, we developed a rapid screening assay to detect characteristic mutations observed in the Delta variant using high-resolution melting (HRM) analysis. In this assay, we determined L452R and T478K, among which T478K is an identifier of the Delta variant since L452R is seen in other strains (Kappa and Epsilon variants). Additionally, nested PCR-based HRM analysis, which involved RT-PCR (1st PCR) and HRM analysis (2nd PCR), was developed to improve the specificity and sensitivity. Our method discriminated between the L452R mutant and wild-type L452. In addition, HRM analysis distinguished the T478K mutant from the wild-type T478. Seven clinical samples containing the Delta variant were successfully identified as L452R/T478K mutants. These results indicate that this HRM-based genotyping method can identify the Delta variant. This simple method should contribute to rapid identification of the Delta variant and the prevention of infection spread.

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1

ABSTRACT. Claudin-16 is one of the tight junction protein claudins and has been shown to contribute to reabsorption of divalent cations in the human kidney. In cattle, total deficiency of claudin-16 causes severe renal tubular dysplasia without aberrant metabolic changes of divalent cations, suggesting that bovine claudin-16 has some roles in renal tubule formation and paracellular transport that are somewhat different from those expected from the pathology of human disease. As the first step to clarify these roles, we examined the expression and distribution of claudin-16 and several other major claudin subtypes, claudins 1-4 and 10, in bovine renal tubular segments by immunofluorescence microscopy. Claudin-16 was exclusively distributed to the tight junction in the tubular segment positive for TammHorsfall glycoprotein, the thick ascending limb (TAL) of Henle's loop, and was found colocalized with claudins 3, 4, and 10. This study also demonstrates that bovine kidneys possess segment-specific expression patterns for claudins 2-4 and 10 that are different from those reported for mice. Particularly, distribution of claudin-4 in the TAL and distal convoluted tubules was characteristic of bovine nephrons as were differences in the expression patterns of claudins 2 and 3. These findings demonstrate that the total lack of claudin-16 in the TAL segment is the sole cause of renal tubular dysplasia in cattle and suggest that the tight junctions in distinct tubular segments including the TAL have barrier functions in paracellular permeability that are different among animal species. KEY WORDS: cattle, claudin-16, paracellular pathway, renal tubule, tight junction.J. Vet. Med. Sci. 68 (5): [453][454][455][456][457][458][459][460][461][462][463] 2006 Tight junctions (TJs) are located at the apicalmost region of lateral membranes of epithelial cells, and play a central role in sealing the intercellular space in epithelial cellular sheets. TJs create the primary barrier to the diffusion of solutes and water through the paracellular pathway and maintain cell polarity as a boundary between the apical and basolateral plasma membrane domains [7,26,29,34]. On freeze-fracture electron microscopy, TJs are visualized as a continuous anastomosing network of intramembranous particle strands, i.e., TJ strands, and complementary grooves [28]. The major components of TJ strands are the integral membrane proteins occludin and claudins, all of which have four transmembrane spans and two intervening extracellular loops [19]. Claudins consist of a family of more than 20 homologous subtypes, and they show tissue-specific and segment-specific distribution patterns in epithelia [19,29] such as those of the gastrointestinal tract [23] and renal tubules [15].Among these claudin proteins, claudin-16, formerly paracellin-1, has been shown to be responsible for inherited disease. Various mutations of the CLDN16 gene were reported to be the cause for familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) in the human [27]. Simon...

Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1- and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH2) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.

Preparation of epitaxial PLZT thin films on sapphire has been investigated, and excellent ferroelectric properties such as piezoelectricity and electrooptic effect with high transparency were obtained in thin films. Moreover, a preparation process was developed involving the multitarget sputtering method, and strict control of film composition and epitaxial growth with the buffer layer of graded composition were performed. Using these PLZT thin films, some optical applications, including an acoustooptic deflector and an electrooptic guided-light switch, are shown.