Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

NTRC and thioredoxins m1/m2 underpin the light acclimation of plants on proteome and metabolome levels.

During photosynthesis, plants must manage strong fluctuations in light availability on different time scales, leading to long-term acclimation and short-term responses. However, little is known about the regulation and coordination of these processes and the modulators involved. In this study, we used proteomics, metabolomics, and reverse genetics to investigate how different light environmental factors, such as intensity or variability, affect long-term and short-term acclimation responses of Arabidopsis (Arabidopsis thaliana) and the importance of the chloroplast redox network in their regulation. In the wild type, high light, but not fluctuating light, led to large quantitative changes in the proteome and metabolome, accompanied by increased photosynthetic dynamics and plant growth. This finding supports light intensity as a stronger driver for acclimation than variability. Deficiencies in NADPH-thioredoxin reductase C (NTRC) or thioredoxins m1/m2, but not thioredoxin f1, almost completely suppressed the re-engineering of the proteome and metabolome, with both the induction of proteins involved in stress and redox responses and the repression of those involved in cytosolic and plastid protein synthesis and translation being strongly attenuated. Moreover, the correlations of protein or metabolite levels with light intensity were severely disturbed, suggesting a general defect in the light-dependent acclimation response, resulting in impaired photosynthetic dynamics. These results indicate a previously unknown role of NTRC and thioredoxins m1/m2 in modulating light acclimation at proteome and metabolome levels to control dynamic light responses. NTRC, but not thioredoxins m1/m2 or f1, also improves short-term photosynthetic responses by balancing the Calvin-Benson cycle in fluctuating light.

Determining the Optimal Intramedullary Screw Canal Fill Ratio in Length Unstable Metacarpal Fractures: A Biomechanical Investigation.

PURPOSE: Intramedullary (IM) screw fixation is gaining popularity in the treatment of metacarpal fractures. Despite its rapid adoption, there is a paucity of evidence regarding parameters to optimize effectiveness. This study aimed to quantify the relationship between stability, IM screw size, and canal fill using a cadaveric model. METHODS: Thirty cadaveric metacarpals (14 index, 13 middle, and three ring fingers; mean age: 58.3 years, range: 48-70) were selected to allow for canal fill ratios of 0.7-1.1 for screws sized 3.0, 3.5, and 4.5 mm. Metacarpals underwent a 45° volar-dorsal osteotomy at the midpoint before fixation with an IM screw. Specimens were subjected to 100 cycles of loading at 10 N, 20 N, and 30 N before load-to-failure testing. Correlation coefficients for angular displacement on the final cycle at each load, peak load to failure, and average stiffness were assessed. RESULTS: Correlation coefficients for the angular displacement on the 100th cycle were as follows: 10 N, R = 0.62, 20 N, R = 0.57, and 30N, R = 0.58. Correlation values for peak load to failure as a function of canal fit were as follows: 3.0 mm, R = 0.5, 3.5 mm, R = 0.17, and 4.5 mm, R = 0.44. The canal fill ratio that intersected the line-of-best fit at an angular deformity of 10° was 0.74. Average peak forces for 3.0-, 3.5-, and 4.5-mm screws were 79.5, 136.5, and 179.6 N, respectively. Average stiffness for each caliber was 14.8, 33.4, and 52.3 N/mm. CONCLUSIONS: Increasing screw diameter and IM fill resulted in more stable fixation, but marginal gains were seen in ratios >0.9. A minimum fill ratio of 0.74 was sufficient to withstand forces of early active motion with angular deformity <10°. CLINICAL RELEVANCE: An understanding of the relationship of IM fill ratio of metacarpal screws to fracture stability may provide a framework for clinicians to optimally size these implants.

The impact of social deprivation on healthcare utilization patterns following rotator cuff repair.

BACKGROUND: Disparities in social determinants of health have been linked to worse patient reported outcomes, higher pain, and increased risk of revision surgery following rotator cuff repair. Identification of perioperative predictors of increased healthcare utilization is of particular interest to surgeons to improve outcomes and mitigate the total cost of care. The effect of social deprivation on healthcare utilization has not been fully characterized. METHODS: This is a retrospective review of a single institution's experience with primary rotator cuff repair between 2012 and 2020. Demographic variables (age, race, gender, American Society of Anesthesiologists (ASA) score) and healthcare utilization (hospital readmission, emergency department visits, follow-up visits, telephone calls) were recorded within 90 days of surgery. The Area Deprivation Index (ADI) was recorded, and patients were separated into terciles according to their relative level of social deprivation. Outcomes were then stratified based on ADI tercile and compared. RESULTS: A total of 1695 patients were included. The upper, middle, and lower terciles of ADI consisted of 410, 767, and 518 patients, respectively. The most deprived tercile had greater emergency department visitation and office visitation within 90 days of surgery relative to the least and intermediate deprived terciles. Higher levels of social deprivation were independent risk factors for increased emergency department (ED) visitation and follow-up visitation. There was no difference in 90-day readmission rates or telephone calls made between the least, intermediate, and most deprived patients. CONCLUSIONS: Patients with higher levels of deprivation demonstrated greater postoperative hospital utilization. We hope to use these results to identify risk factors for increased hospital use, guide clinical decision making, increase transparency, and manage patient outcomes following rotator cuff repair surgery.

The impact of social deprivation on rotator cuff repair outcomes.

BACKGROUND: Rotator cuff tears are a common orthopedic injury, and the role of social determinants of health (SDoH) in surgical outcomes remains underexplored. The goal of this study was to investigate the correlation between social deprivation, measured by the Area Deprivation Index (ADI), and outcomes following arthroscopic rotator cuff repair. METHODS: We conducted a retrospective chart review on patients undergoing primary arthroscopic rotator cuff repair at a level 1 academic center between 2006 and 2019. Patient demographics (age, gender, race), comorbidities, ADIs, range of motion, visual analog pain scores, and patient-reported outcomes (Simple Shoulder Test [SST], American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form [ASES], and Quick Disabilities of the Arm, Shoulder, and Hand questionnaire [QuickDASH]) were collected. Patients were stratified into terciles based on their relative level of deprivation. Statistical analysis was performed using analysis of variance, t tests, χ2 tests, and univariate or multivariate logistic regression. RESULTS: A total of 322 patients were included in this study. The most deprived group had a higher prevalence of diabetes compared to the least and intermediately deprived group (P < .001). Massive tear occurrence was greater in the least deprived group (P = .003) compared to the most deprived group. There was no difference in objective outcomes between groups. Patient-reported outcomes (SST, ASES, and QuickDASH scores) were worse in the most deprived group compared with the least and intermediate deprived groups. CONCLUSION: Social deprivation significantly affects patient-reported outcomes in rotator cuff repair surgery. Although clinician-reported outcomes were consistent, patients' perceptions varied based on social determinants. Integrating SDoH considerations in orthopedic care is a promising next step in securing equitable approaches. However, more research is needed to validate and expand these findings.

SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells.

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.

A dominant mutation in ß-AMYLASE1 disrupts nighttime control of starch degradation in Arabidopsis leaves.

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

In brief: It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. Abstract: An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

Variable detection of Omicron-BA.1 and -BA.2 by SARS-CoV-2 rapid antigen tests.

During 2022, the COVID-19 pandemic has been dominated by the variant of concern (VoC) Omicron (B.1.1.529) and its rapidly emerging subvariants, including Omicron-BA.1 and -BA.2. Rapid antigen tests (RATs) are part of national testing strategies to identify SARS-CoV-2 infections on site in a community setting or to support layman's diagnostics at home. We and others have recently demonstrated an impaired RAT detection of infections caused by Omicron-BA.1 compared to Delta. Here, we evaluated the performance of five SARS-CoV-2 RATs in a single-centre laboratory study examining a total of 140 SARS-CoV-2 PCR-positive respiratory swab samples, 70 Omicron-BA.1 and 70 Omicron-BA.2, as well as 52 SARS-CoV-2 PCR-negative swabs collected from March 8th until April 10th, 2022. One test did not meet minimal criteria for specificity. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 4.2 × 104 to 9.2 × 105 RNA copies subjected to the RAT for Omicron-BA.1 compared to 1.3 × 105 to 1.5 × 106 for Omicron-BA.2. Overall, intra-assay differences for the detection of Omicron-BA.1-containing and Omicron-BA.2-containing samples were non-significant, while a marked overall heterogeneity among the five RATs was observed. To score positive in these point-of-care tests, up to 22-fold (LoD50) or 68-fold (LoD95) higher viral loads were required for the worst performing compared to the best performing RAT. The rates of true-positive test results for these Omicron subvariant-containing samples in the highest viral load category (Ct values < 25) ranged between 44.7 and 91.1%, while they dropped to 8.7 to 22.7% for samples with intermediate Ct values (25-30). In light of recent reports on the emergence of two novel Omicron-BA.2 subvariants, Omicron-BA.2.75 and BJ.1, awareness must be increased for the overall reduced detection rate and marked differences in RAT performance for these Omicron subvariants.

Ten rapid antigen tests for SARS-CoV-2 widely differ in their ability to detect Omicron-BA.4 and -BA.5.

Since late 2021, the variant landscape of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been dominated by the variant of concern (VoC) Omicron and its sublineages. We and others have shown that the detection of Omicron-BA.1 and -BA.2-positive respiratory specimens by rapid antigen tests (RATs) is impaired compared to Delta VoC-containing samples. Here, in a single-center retrospective laboratory study, we evaluated the performance of ten most commonly used RATs for the detection of Omicron-BA.4 and -BA.5 infections. We used 171 respiratory swab specimens from SARS-CoV-2 RNA-positive patients, of which 71 were classified as BA.4 and 100 as BA.5. All swabs were collected between July and September 2022. 50 SARS-CoV-2 PCR-negative samples from healthy individuals, collected in October 2022, showed high specificity in 9 out of 10 RATs. When assessing analytical sensitivity using clinical specimens, the 50% limit of detection (LoD50) ranged from 7.6 × 104 to 3.3 × 106 RNA copies subjected to the RATs for BA.4 compared to 6.8 × 104 to 3.0 × 106 for BA.5. Overall, intra-assay differences for the detection of these two Omicron subvariants were not significant for both respiratory swabs and tissue culture-expanded virus isolates. In contrast, marked heterogeneity was observed among the ten RATs: to be positive in these point-of-care tests, up to 443-fold (BA.4) and up to 56-fold (BA.5) higher viral loads were required for the worst performing RAT compared to the best performing RAT. True-positive rates for Omicron-BA.4- or -BA.5-containing specimens in the highest viral load category (Ct values < 25) ranged from 94.3 to 34.3%, dropping to 25.6 to 0% for samples with intermediate Ct values (25-30). We conclude that the high heterogeneity in the performance of commonly used RATs remains a challenge for the general public to obtain reliable results in the evolving Omicron subvariant-driven pandemic.