A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, the Omicron variant (Pango lineage B.1.1.529), was identified in South Africa in late September 2021. This variant has ...multiple spike protein deletions and mutations, with 15 amino acid substitutions detected in the receptor-binding domain (RBD). These RBD substitutions are hypothesized to increase infectivity and reduce antibody affinity, which is supported by recent data showing that the Omicron variant spreads faster than the Delta variant (Pango lineage B.1.617.2). Thus, this increase in infectivity should lead to Omicron being the dominant variant and developing screening tests that discriminate between Omicron and Delta variants is urgently needed. In this study, we successfully developed a novel screening assay using high-resolution melting analysis, in which two genotypes at G446/L452 and S477/T478 RBD were determined (G446S/L452 and S477N/T478K for Omicron; G446/L452R and S477/T478K for Delta). Using synthetic DNA fragments, we confirmed both melting point and melting peak shape of the RBD Omicron variant was distinguishable from those of wild-type and the Delta variant. Although this study was conducted without clinical samples, these results suggest that our high-resolution melting (HRM)-based genotyping method can readily identify the Omicron and Delta variants. This simple method should contribute to the rapid identification of SARS-CoV-2 variants and thus prevent potential widespread infection and inflow of the Omicron variant.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus causing coronavirus disease 2019 (COVID-19), has been expanding globally since late 2019. SARS-CoV-2, an RNA virus, ...has a genome sequence that can easily undergo mutation. Several mutated SARS-CoV-2 strains, including those with higher infectivity than others, have been reported. To reduce SARS-CoV-2 transmission, it is crucial to trace its infection sources. Here, we developed a simple, easy-to-use genotyping method to identify SARS-CoV-2 variants using a high-resolution melting (HRM) analysis.
We investigated five mutation sites, A23403G, G25563T, G26144T, T28144C, and G28882A, which are known strain determinants according to GISAID clades (L, S, V, G, GH, and GR).
We first employed synthetic DNA fragments containing the five characteristic sites for HRM analysis. All sequences clearly differentiated wild-type from mutant viruses. We then confirmed that RNA fragments were suitable for HRM analysis following reverse transcription. Human saliva did not negatively affect the HRM analysis, which supports the absence of a matrix effect.
Our results indicate that this HRM-based genotyping method can identify SARS-CoV-2 variants. This novel assay platform potentially paves the way for accurate and rapid identification of SARS-CoV-2 infection sources.
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.
Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic ...diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC50 values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.
Purpose
To evaluate the association between the sagittal alignment of the pelvis and residual knee flexion contracture after total knee arthroplasty (TKA). This is important as a flexion contraction ...can be associated with the risk of poor outcomes and patient satisfaction after TKA.
Methods
This was a retrospective, case–control, study of 200 osteoarthritic knees, contributed by 200 patients, over a mean follow-up of 2.4 years. The following factors were compared between patients ‘with’ (46 knees) and ‘without’ (154 knees) a residual flexion contracture ≥ 10° after TKA: age, sex, pelvic incidence (PI), anterior femoral bowing, femoral component flexion angle (FFA), and patient-reported outcomes. Logistic regression and receiver operating characteristic curve analyses were used to identify predictive factors.
Results
The following factors were predictive of a residual flexion contracture ≥ 10°: a pelvic incidence ≥ 55° (odds ratio, 1.29; 95% confidence interval, 1.05−1.59;
P
= 0.031) and the FFA (odds ratio, 1.08; 95% confidence interval, 1.03−1.14;
P
= 0.044). A pelvic incidence cutoff of 55° yielded a significant between-group difference, with a sensitivity of 78.4% and specificity of 89.9% to differentiate a residual knee flexion contracture ≥ 10° (
P
= 0.001), patient satisfaction (
P
= 0.029), EuroQol 5-Dimension score (
P
= 0.028), anterior femoral curvature (
P
= 0.031), and Knee Injury and Osteoarthritis Outcome Score-Joint Replacement score (
P
= 0.046).
Conclusion
A pelvic incidence > 55° is associated with a residual knee flexion contracture ≥ 10° after TKA. The significance of the pelvic incidence measurement as a possible predictor of TKA outcome was highlighted, including its impact on patient satisfaction.
Level of evidence
III.
Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic ...potential were determined. A pellet containing either 2-chloro-17β-estradiol (2-ClE2) or 4-chloro-17β-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17β-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.
•4-FE2, like E2, induced mammary tumors in ACI rats whereas 2-FE2 did not.•Both 4-FE2 and 2-FE2 showed high uterotrophic potency.•Estrogenic potential may not be the sole factor driving mammary ...tumorigenesis.•The carcinogenic effect may occur through the metabolic activation of 2−OHE2.
Fluorination preventing metabolic hydroxylation of 17β-estradiol (E2) was applied to investigate the mechanisms underlying estrogen-induced carcinogenesis. Either 2-fluoro-17β-estradiol (2-FE2) or 4-fluoro-17β-estradiol (4-FE2) was administered subcutaneously for 52 weeks to August Copenhagen Irish (ACI) rats, the preferred animal model for human breast cancer. 4-FE2 induced frequent mammary tumors whereas 2-FE2 did not. The cumulative incidence of mammary tumors in rats treated with 4-FE2 was comparable to that observed with E2. The carcinogenic results were supported by histological examination of mammary glands of fluorinated estrogen-treated ACI rats. To evaluate the estrogenic potential of the fluorinated estrogens, 2-FE2 or 4-FE2 was administrated subcutaneously to ovariectomized rats. Both 4-FE2 and 2-FE2 showed high uterotrophic potency. Our results indicate that estrogenic potential may not be the sole factor driving mammary tumorigenesis. Since fluorination inhibits metabolic hydroxylation of E2 at the substituted position, the carcinogenic effect may occur through the metabolic activation of 2-hydroxylated E2, in combination with the compound’s estrogenic potency.
Purpose
The purpose of this study was to compare the hindfoot alignment and symptoms in patients with pre-existing moderate and severe knee deformities after total knee arthroplasty (TKA).
Methods
...Eighty knees of 75 patients who underwent TKA for varus osteoarthritis were enrolled retrospectively and evaluated the following pre-operatively and at 2 years post-operatively: the American Orthopaedic Foot and Ankle Society ankle–hindfoot scale (pain and function scores), calcaneal pitch, and naviculocuboid overlap as an indicator of hindfoot alignment. The knees were divided into two groups according to the pre-operative hip–knee–ankle angle defined as the angle between the mechanical axis of the femur and the tibia: group M with genu varus of ≤6°, and group S with varus >6°.
Results
The pain (
p
= 0.03) and function (
p
= 0.02) scores improved in group M; however, in group S, these measures did not change. The differences between the groups were not significant concerning the pre-operative calcaneal pitch and naviculocuboid overlap. The post-operative pitch (
p
= 0.03) and the overlap (
p
= 0.04) in group M was significantly greater and less than those in group S, respectively. Although the pitch (
p
< 0.01) and the overlap (
p
= 0.03) increased in group M, these did not change in group S. Post-operative hindfoot pain and valgus remained in patients in group S.
Conclusions
For pre-existing moderate knee deformities, a relationship was observed between post-operative knee alignment and compensatory hindfoot alignment, whereas patients with severe deformities experienced persistent post-operative hindfoot pain and valgus alignment. It was concluded that evaluations and managements of residual symptoms after TKA including the hindfoot are important. These findings are clinically relevant that perioperative evaluation of the hindfoot should be required in knee surgery. To help improve the outcomes of TKA, clinicians may consider perioperative intervention in the insole and/or physical therapy of the foot and ankle.
Level of evidence
Therapeutic study, Level III.
•Total knee arthroplasty (TKA) can lead to an ‘artificial’ joint perception.•An artificial perception is associated with lower patient satisfaction.•A gap difference ≥1.0 mm (distraction force, 1.5 ...BMI) improved outcomes.•Soft tissue balance during TKA is predictive of patient-reported outcomes.
We aimed to evaluate the association between the flexion–extension gap difference and patient-reported outcome measures after total knee arthroplasty (TKA).
This was a retrospective case–control study of 60 cruciate-retaining single-radius TKAs. Soft tissue balancing was measured using an offset seesaw tensioner and centre-type digital knee balancer under joint distraction forces of 20–50 lbf and 1.5 times body mass index (1.5 BMI). At the last follow up of 2.0–6.5 (mean, 4.5) years postoperatively, patients were asked if they perceived their knee joint as ‘natural’ (26 knees) or ‘artificial’ (34 knees). Age, sex, and the flexion–extension gap were compared between the two groups. A receiver operating characteristic curve was used to determine cut-off values of variables predictive of a natural joint perception.
Natural joint perception was associated with a greater flexion–extension gap difference under a distraction force of 1.5 BMI (P = 0.016), higher knee function (Knee Injury and Osteoarthritis Outcome Score for Joint Replacement, P = 0.019), and quality of life (EuroQol 5-Dimension, P = 0.029). A gap difference of 0.95 mm under 1.5 BMI distraction predicted a natural joint perception post-TKA (sensitivity, 97.1%; specificity, 88.5%). Using a gap threshold of 1.0 mm under a 1.5 BMI distraction force yielded significant between-group differences in postoperative flexion (P = 0.040), satisfaction (P = 0.043), knee joint function (P < 0.001), quality of life (P = 0.032), and posterior femoral condylar offset (P = 0.037)
A flexion–extension gap difference ≥1.0 mm under a distraction force of 1.5 BMI predicted superior outcomes, including patient satisfaction, after cruciate-retaining TKA. It was suggested that posterior femoral condylar offset could influence this finding.
Bitter taste receptors (TAS2Rs) are expressed by oral cavity cells in mammals and classically function as sensors for bitter compounds. There are 25 functional isoforms of human TAS2Rs, with ...individual bitter ligands. Each human TAS2R isoform is distributed in several tissues, such as the airway epithelia and gastrointestinal tract, and plays an important role in physiological functions. However, quantification of each isoform is difficult because of highly homologous sequences between some TAS2R isoforms. Therefore, differentiating the isoforms by their expression levels is suitable for clarifying the tissue-specific effects of bitter compounds. In this study, we developed a real-time quantitative PCR (qPCR) method to determine the expression of each TAS2R isoform. Using plasmid standards harboring each isoform, we confirmed that the current assay can quantify the gene expression of each isoform, with negligible interference from other isoforms. In addition, our methods can successfully discriminate between the mRNA expression of each isoform in human cell lines and tissues. Therefore, this qPCR method can successfully quantify the mRNA level of each TAS2R isoform. This method will contribute to a better understanding of the molecular mechanisms underlying the TAS2R ligand-activated signal transduction.
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