Identification of novel small-molecule inhibitors of SARS-CoV-2 by chemical genetics.

There are only eight approved small molecule antiviral drugs for treating COVID-19. Among them, four are nucleotide analogues (remdesivir, JT001, molnupiravir, and azvudine), while the other four are protease inhibitors (nirmatrelvir, ensitrelvir, leritrelvir, and simnotrelvir-ritonavir). Antiviral resistance, unfavourable drug‒drug interaction, and toxicity have been reported in previous studies. Thus there is a dearth of new treatment options for SARS-CoV-2. In this work, a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity. One compound, designated 172, demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern. Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease (3CLpro) by binding to an allosteric site and reduces 3CLpro dimerization. A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro. In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice. Overall, this study identified an alternative druggable site on the SARS-CoV-2 3CLpro, proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

Ultrapotent class I neutralizing antibodies post Omicron breakthrough infection overcome broad SARS-CoV-2 escape variants.

BACKGROUND: The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. METHODS: Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model). FINDINGS: By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. INTERPRETATION: Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

The significance of recurrent de novo amino acid substitutions that emerged during chronic SARS-CoV-2 infection: an observational study.

BACKGROUND: De novo amino acid substitutions (DNS) frequently emerge among immunocompromised patients with chronic SARS-CoV-2 infection. While previous studies have reported these DNS, their significance has not been systematically studied. METHODS: We performed a review of DNS that emerged during chronic SARS-CoV-2 infection. We searched PubMed until June 2023 using the keywords "(SARS-CoV-2 or COVID-19) and (mutation or sequencing) and ((prolonged infection) or (chronic infection) or (long term))". We included patients with chronic SARS-CoV-2 infection who had SARS-CoV-2 sequencing performed for at least 3 time points over at least 60 days. We also included 4 additional SARS-CoV-2 patients with chronic infection of our hospital not reported previously. We determined recurrent DNS that has appeared in multiple patients and determined the significance of these mutations among epidemiologically-significant variants. FINDINGS: A total of 34 cases were analyzed, including 30 that were published previously and 4 from our hospital. Twenty two DNS appeared in ≥3 patients, with 14 (64%) belonging to lineage-defining mutations (LDMs) of epidemiologically-significant variants and 10 (45%) emerging among chronically-infected patients before the appearance of the corresponding variant. Notably, nsp9-T35I substitution (Orf1a T4175I) emerged in all three patients with BA.2.2 infection in 2022 before the appearance of Variants of Interest that carry nsp9-T35I as LDM (EG.5 and BA.2.86/JN.1). Structural analysis suggests that nsp9-T35I substitution may affect nsp9-nsp12 interaction, which could be critical for the function of the replication and transcription complex. INTERPRETATION: DNS that emerges recurrently in different chronically-infected patients may be used as a marker for potential epidemiologically-significant variants. FUNDING: Theme-Based Research Scheme [T11/709/21-N] of the Research Grants Council (See acknowledgements for full list).

Work and efficiency fluctuations in a quantum Otto cycle with idle levels.

We study the performance of a quantum Otto heat engine with two spins coupled by a Heisenberg interaction, taking into account not only the mean values of work and efficiency but also their fluctuations. We first show that, for this system, the output work and its fluctuations are directly related to the magnetization and magnetic susceptibility of the system at equilibrium with either heat bath. We analyze the regions where the work extraction can be done with low relative fluctuation for a given range of temperatures, while still achieving an efficiency higher than that of a single spin system heat engine. In particular, we find that, due to the presence of "idle" levels, an increase in the interspin coupling can either increase or decrease fluctuations, depending on the other parameters. In all cases, however, we find that the relative fluctuations in work or efficiency remain large, implying that this microscopic engine is not very reliable as a source of work.

Characterizing fitness and immune escape of SARS-CoV-2 EG.5 sublineage using elderly serum and nasal organoid.

SARS-CoV-2 Omicron variant has evolved into sublineages. Here, we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However, there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population.

[METATARSUS ADDUCTUS - A COMMON DEFORMITY - WHAT TO DO AND WHEN?]

INTRODUCTION: The most common foot deformity in newborns is the forefoot adduction deformity (FAD), where the hindfoot foot is in a normal position. The diagnosis for this problem is mainly based on a physical examination. The use of imaging methods has been described, but no advantage was shown with their utilization in determining the diagnosis and guiding treatment. Several classification systems have been proposed to characterize the degree of severity. The classifications are based on the degree of deviation and the flexibility of the foot. Early diagnosis and early treatment, if necessary, are extremely important to improve the chances of treatment success. Treatment depends on the severity of the deformity. For mild deformities the treatment is conservative - follow-up or stretching of the foot. The usual treatment for severe deformities is serial casting. Several orthoses have recently been proposed to address the problem and these demonstrated similar results, higher comfort and satisfaction, lower cost and a similar side effect profile. Surgical treatments to correct the deformity are reserved for cases where conservative treatment failed and for older children. This review aims to summarize the current knowledge on the subject, describe the ways to diagnose and classify the deformity, and present the variety of ways to treat the problem including the use of innovative braces. In addition, we will offer a protocol for the treatment of the deformity that is accepted in our institution. The protocol will assist primary care physicians to both diagnose and treat appropriate deformities, and know when a specialist referral is necessary.

Review of Current Concepts in Metatarsus Adductus.

Metatarsus adductus (MA), the most common congenital foot deformity, involves adduction of the forefoot at the tarsometatarsal joint, with normal hindfoot alignment. Early diagnosis is important because treatment is more successful if initiated before age 9 months. Treatment of MA depends on deformity severity, in which mild to moderate deformity can be treated conservatively. Current standard of care for severe or rigid deformity involves referral by primary care physicians to specialists for management by casting and splinting. Recently, several orthoses have demonstrated equal effectiveness to casting and may allow for primary care physicians to treat MA without the need for referral. In this review article, we provide an overview of MA and discuss diagnosis and treatment. We also discuss novel devices and suggest how they may affect the future management of severe and rigid MA. [Pediatr Ann. 2024;53(4):e152-e156.].

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman's ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

NfL reliability across laboratories, stage-dependent diagnostic performance and matrix comparability in genetic FTD: a large GENFI study.

BACKGROUND: Blood neurofilament light chain (NfL) is increasingly considered as a key trial biomarker in genetic frontotemporal dementia (gFTD). We aimed to facilitate the use of NfL in gFTD multicentre trials by testing its (1) reliability across labs; (2) reliability to stratify gFTD disease stages; (3) comparability between blood matrices and (4) stability across recruiting sites. METHODS: Comparative analysis of blood NfL levels in a large gFTD cohort (GENFI) for (1)-(4), with n=344 samples (n=148 presymptomatic, n=11 converter, n=46 symptomatic subjects, with mutations in C9orf72, GRN or MAPT; and n=139 within-family controls), each measured in three different international labs by Simoa HD-1 analyzer. RESULTS: NfL revealed an excellent consistency (intraclass correlation coefficient (ICC) 0.964) and high reliability across the three labs (maximal bias (pg/mL) in Bland-Altman analysis: 1.12±1.20). High concordance of NfL across laboratories was moreover reflected by high areas under the curve for discriminating conversion stage against the (non-converting) presymptomatic stage across all three labs. Serum and plasma NfL were largely comparable (ICC 0.967). The robustness of NfL across 13 recruiting sites was demonstrated by a linear mixed effect model. CONCLUSIONS: Our results underline the suitability of blood NfL in gFTD multicentre trials, including cross-lab reliable stratification of the highly trial-relevant conversion stage, matrix comparability and cross-site robustness.

Humoral and cellular immunity against different SARS-CoV-2 variants in patients with chronic kidney disease.

Chronic kidney disease (CKD) patients are at higher risk of severe COVID-19. Humoral and cellular immunity from prior infection or vaccination are important for protection, but the neutralizing antibody (nAb) response against SARS-CoV-2 variants is impaired. We investigated the variant-specific nAb and T cell immunity among CKD patients. Adult CKD patients were recruited between August and October 2022. nAb against the SARS-CoV-2 (ancestral strains and four Omicron sublineages) and T cell response were measured using the live virus neutralization assay and interferon-gamma release assay (IGRA). The correlation between nAb/T-cell response and subsequent infection after recruitment were also determined. Among the 88 recruited patients, 95.5% had prior infection or had completed the primary vaccine series. However, only 77.3% had detectable nAb against at least one SARS-CoV-2 strains, 59.1% tested positive in IGRA, and 52.3% had detectable nAb and tested positive in the IGRA. The nAb geometic mean titers (GMTs) against XBB.1, BA.5 and BA.2.3.20 were significantly lower than those against BA.2 and ancestral strain. Prior SARS-CoV-2 infection was associated with elevated nAb and T cell response. More kidney transplant recipients (KTRs) showed absent nAb and T cell response (36.8% vs. 10.1%), despite a higher prevalence of vaccine booster in this population (94.7% vs. 50.7%). Lower levels of nAb titer and T cell response were significantly associated with subsequent infection. A considerable proportion of CKD patients, especially KTRs, showed absence of humoral and cellular protective immunity against SARS-CoV-2. Strategies to improve immunogenicity in this population are urgently needed.

Nephrology in Veterinary Medicine.

Veterinary nephrology is a specialized field of veterinary medicine providing a high level of care for animals with all types of kidney disease. Veterinarians complete extensive training to become board-certified in veterinary nephrology-urology. Companion animal nephrology is the most advanced field; however, all species are afflicted by a variety of renal disorders. Most naturally occurring animal kidney diseases have similar disorders found in people; where veterinary research is lacking, clinical management is often modified from standard of care in people. Veterinarians have become adept at scaling down procedures to safely perform them on dogs and cats weighing only a few kilograms. Advanced diagnostics (renal biopsy, cystoscopy, fluoroscopic studies, etc. ) and therapeutics (renal replacement therapy, interventional endourology, etc. ) are commonly performed within the practice of veterinary nephrology-urology. Collaboration between veterinary and human nephrologists may advance both disciplines and improve care for people and animals alike.

The T1 Ratio of Marrow (TROM) as a Novel Tool to Identify Metastatic from Nonmalignant Marrow Lesions of the Spine: A Pilot Study.

Objective The purpose of this study was to analyze quantitative values of normal and abnormal marrow on T1-weighted images of spine, to propose a ratio for T1 values of abnormal to normal vertebrae, and to assess whether this ratio could be helpful in predicting presence of neoplastic lesions in the spine. Materials and Methods One-hundred randomly selected magnetic resonance imagings of lumbar spine without infection, fracture, and tumor were selected to form normal cohort. A second cohort of 100 metastasis of lumbar spine was identified. Ratio of T1 value of vertebral body to the T1 value of the inferior vertebral body was performed for normal cohort from D11 to L5. Ratio of T1 value of metastasis to adjacent normal vertebral body was done for metastatic cohort. Data was analyzed using standard t -test and kappa was performed for intra- and inter-observer reliability. Results A decline in T1 value of abnormal to normal marrow was seen in patients with metastasis that was statistically significant. We call this the T1 ratio of marrow (TROM). The sensitivity and accuracy with the cutoff value of TROM at 0.7 (92% sensitivity, 97.1% accuracy) are better than at 0.6 (75% sensitivity, 96.2% accuracy) or 0.5 (47% sensitivity, 93.2% accuracy). A subset analysis of the other T1 hypointense benign lesions including atypical hemangiomas and focal marrow hyperplasia, however, revealed overlapping TROM values with the metastatic cohort. Conclusion Using the TROM on T1-weighted images could not confidently differentiate malignant from benign T1 hypointense lesions of the spine.

Transparency films: intraoperative templating to prevent limb deformity.

Operative management of fractures and malunions can be challenging when restoring native anatomy is not straightforward. Comminuted fractures and managing deformity correction in the setting of osteolysis, callus, and even complete fracture healing must include careful planning. Preoperative planning has been popularized and taught as an integral part of a surgeon's skill set, with critical evaluation and assessment of the implemented plan being the final step in the process. We present a robust, reproducible, and cost-effective technique for intraoperative fracture fixation assessment with case examples, used routinely at our institution.

Measurement Analysis of Ice Ball Size during CT-Guided Cryoablation Procedures for Better Prediction of Final Ice Ball Size and Avoidance of Complications.

Introduction Cryoablation is the destruction of living tissue by the application of extreme freezing temperature. There has been an increase in the use of cryoablation in the management of musculoskeletal lesions, in particular fibromatosis. Aim This study aimed to measure the average and relative increase in size of the cryoablation ice ball after the first (10 minutes) and second freeze cycles (20 minutes) to accurately predict the size of the ice ball between first and second freezes to help prevent any unwanted damage of the nearby skin and neurovascular structures. This is especially important when ablating in relatively small body parts such as in the appendicular skeleton. Material and Methods Eight patients treated with cryoablation over a 12-month period for fibromatosis were, included in the study. The size and volume of the ice ball were measured during the first and second cycle of cryoablation. Results The average patient age of the cohort was 35.6 years old (min 28 and max 43). There was female predominance in the study (3:2, F:M). There was a significant increase (26%) in the linear dimensions and almost doubling in the volume of the ice ball between freeze cycles ( p -value = 0.0037 for dimensions and p -value = 0.0002 for volumes). Conclusion This pilot study is a preliminary attempt to predict the eventual size of the ice ball during cryoablation procedures when treating cases of fibromatosis. This should help in planning cryoablation to ensure decrease morbidity by preventing injury to adjacent critical structures (neurovascular bundle and skin).

Global prevalence of SARS-CoV-2 3CL protease mutations associated with nirmatrelvir or ensitrelvir resistance.

BACKGROUND: Nirmatrelvir-ritonavir (Paxlovid) and ensitrelvir are 3-chymotrypsin-like cysteine protease (3CLpro) inhibitors which have been approved for the treatment of COVID-19 in 2021 and 2022, respectively. Previous studies have identified 3CLpro mutations that are associated with reduced susceptibility to these antivirals. The aim of the current study was to estimate the global prevalence of 3CLpro inhibitor-resistant SARS-CoV-2 strains. METHODS: We compiled a list of 3CLpro mutations which have been associated with nirmatrelvir or ensitrelvir resistance based on either viral replication or 3CLpro activity assays, and determined their prevalence among 13.4 million sequences deposited in GISAID as of December 14, 2022, about 1 year after the approval of nirmatrelvir-ritonavir. We analyzed the prevalence for different time periods, SARS-CoV-2 lineages and geographical locations. FINDINGS: Overall, 0.5% (67,095/13,446,588) of the sequences contained at least one mutation that was shown to affect the inhibitory activity of nirmatrelvir or ensitrelvir on viral replication or 3CLpro activity. We did not observe any increasing trend of resistance after the widespread clinical use of nirmatrelvir-ritonavir. G15S (2070 per million) and T21I (1386 per million) were the most prevalent mutations, and these mutations were dominant in some SARS-CoV-2 lineages. E166V and S144E, previously shown to affect the inhibitory activity of nirmatrelvir on viral replication or protease activity by > 100-folds, were found in <1 per million sequences. INTERPRETATION: Our data suggest that 3CLpro inhibitor resistance is currently rare. However, continuous global genotypic and phenotypic surveillance would be crucial in the early detection of resistant mutants. FUNDING: Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, the Emergency Key Program of Guangzhou Laboratory (See acknowledgements for full list).