A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

The development of indicators to measure the quality of care in geriatric rehabilitation.

Quality of care is an essential aspect of geriatric rehabilitation. Usually, there are national standards for the quality of care or indicators to measure the quality of care. However, this is not the case for geriatric rehabilitation. Therefore, the aim of this study was to develop structure, process, and outcome indicators to measure the quality of geriatric rehabilitation. To develop quality indicators for geriatric rehabilitation, a literature search was performed to identify indicators for all types of rehabilitation that can also be suitable for geriatric rehabilitation. Thereafter, in the qualitative phase, different stakeholders were inte. Indicators from the literature and indicators developed based on the interviews were merged and processed in a questionnaire. Through this questionnaire, elderly care physicians and managers of geriatric rehabilitation facilities were asked to rate the indicators on relevance and feasibility. Indicators that were considered relevant and feasible by the respondents were included in the final quality indicator set for geriatric rehabilitation. Thirty-six indicators suitable for geriatric rehabilitation were identified from the literature. Additionally, 55 quality indicators were developed based on the interviews. Merging the indicators and omitting duplicates resulted in 69 quality indicators. Analysis of the data from the questionnaires resulted in a final set of 27 quality indicators for geriatric rehabilitation that consists of 17 structure, 8 process, and 2 outcome indicators. This study contributes to the quality of geriatric rehabilitation by providing a first set of quality indicators ready to use in practice. Follow-up research is recommended and may include an assessment of the applicability, reliability, and validity of the developed indicator set.

Structural Insights into Molecular Recognition by Human Chemokine CCL19.

The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7-70) at 2.50 Å resolution, revealing molecular details crucial for protein-protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30's and 40's loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5-11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40's loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine's function and inhibitor development.

[Clinical characteristics and prognosis in papillary thyroid carcinoma patients with recurrent laryngeal nerve invasion].

Objective: To investigate the clinical characteristics and risk factors of postoperative recurrence in papillary thyroid carcinoma (PTC) patients with recurrent laryngeal nerve (RLN) invasion. Methods: The data of PTC patients with recurrent laryngeal nerve invasion treated in Beijing Tongren Hospital, Capital Medical University from January 2006 to December 2019 were retrospectively analyzed. The acoustic parameters were compared between different subgroups. Kaplan-Meier method was used to calculate the overall survival (OS) and the recurrence-free rate (RFS), and univariate and multivariate Cox regression analyses were performed to determine the risk factors for postoperative recurrence. Results: A total of 150 PTC patients were enrolled in the final analysis, including 102 females and 48 males, with an average age of (53.5±13.7) years, and 62 patients (41.3%) aged over 55 years. There were 88 cases with stage Ⅰ, and 62 cases with stage Ⅲ. Fifty-five patients presented with preoperative vocal cord paralysis. There were 75 cases appearing adhesion between tumor or lymph node and recurrent laryngeal nerve while 75 cases presented with direct invasion. The comparisons of acoustic parameters showed that patients with RLN invasion had higher jitter compared with patients without RLN invasion [2.3% (1.4%, 3.2%) vs 1.8% (0.8%, 2.6%), P<0.001]. Moreover, patients with preoperative vocal cord paralysis (VCP) had higher jitter[3.1% (2.2%, 4.6%) vs 2.0% (1.1%, 2.8%), P<0.001] and shimmer [7.1% (4.9%, 9.9%) vs 5.5% (4.2%, 7.3%), P<0.001] and shorter maximum phonation time (MPT) [8.0 (6.0, 10.0) s vs 12.0 (10.0, 15.3) s, P<0.001] compared with patients without preoperative VCP. However, there was no statistical difference in acoustic parameters between cases with RLN adhesion and RLN invasion (all P>0.05). Postoperative follow-up time ranged between 12-196 months, with an average of (65.0±35.9) months. Sixteen patients (10.7%) had recurrence or metastasis, and 8 cases (5.3%) died of recurrence or metastasis. The 5-year OS rate was 95.1%, and the 10-year OS rate was 92.8%. The 5-year RFS rate was 88.9%, and the 10-year RFS rate was 86.2%. Univariate Cox analysis showed that age of onset ≥ 55 years, preoperative recurrent laryngeal nerve palsy, laryngeal, trachea or esophageal invasion were the risk factors for postoperative recurrence of PTC with RLN invasion (all P<0.05). Multivariate Cox analysis showed that age of onset ≥ 55 years (OR=1.060, 95%CI: 1.011-1.110, P=0.015) was an independent risk factor. Conclusions: Age of onset ≥ 55 years is an independent risk factor for postoperative recurrence in PTC patients with RLN invasion. Preoperative acoustic parameters may provide reference for evaluation of RLN function.

[The immune-modulation therapy of autoimmune liver diseases].

Autoimmune liver diseases are a panel of chronic inflammatory diseases caused by abnormal immune attack against hepatocytes or bile duct epithelial cell, including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), IgG4-related sclerosing cholangitis (IgG4-SC), and overlap syndrome. Currently main drug treatments of autoimmune liver diseases contain corticosteroids, immunosuppressant and other immune-modulation therapy. Using anti-CD20 to deplete B cells has achieved biochemical improvement in patients with IgG4-SC or PBC. There are several clinical trials focus on immune cell transfusion and novel target drug therapy finished or being conducted.

Metallocenyl 7-ACA Conjugates: Antibacterial Activity Studies and Atomic-Resolution X-ray Crystal Structure with CTX-M ß-Lactamase.

The conjugation of organometallic groups to current ß-lactam antibiotics is a field of increasing study due to the ability of certain organometallic groups to enhance the antibiotic potency of these drugs. Herein, we report the antibacterial properties of two metallocenyl (ferrocenyl and ruthenocenyl) 7-aminocephalosporanic acid (7-ACA) antibiotic conjugates. Continuing a trend we found in our previous studies, the ruthenocenyl conjugate showed greater antibacterial activity than its ferrocenyl counterpart. Compared with the previously published 7-aminodesacetoxycephalosporanic acid (7-ADCA) conjugates, the 3-acetyloxymethyl group significantly improved the compounds' activity. Furthermore, the Rc-7-ACA compound was more active against clinical Staphylococcus aureus isolates than the ampicillin reference. Noticeably, neither of the two new compounds showed an undesirable toxic effect in HeLa and L929 cells at the concentrations at which they displayed strong antibacterial effects. The antibacterial activity of the two metallocenyl 7-ACA derivatives was further confirmed by scanning electron microscopy (SEM). SEM micrographs showed that bacteria treated with metallocenyl 7-ACA derivatives feature cell wall damage and morphology changes. Using a CTX-M-14 ß-lactamase competition assay based on nitrocefin hydrolysis, we showed that the Rc-7-ACA bound more favorably to CTX-M-14 than its ferrocenyl counterpart, again confirming the superiority of the ruthenocenyl moiety over the ferrocenyl one in interacting with proteins. We also report a 1.47 Å resolution crystal structure of Rc-7-ACA in complex with the CTX-M-14 E166A mutant, an enzyme sharing a similar active site configuration with penicillin-binding proteins, the molecular target of ß-lactam antibiotics. These results strengthen the case for the antibacterial utility of the Rc and Fc groups.

Integrated analysis of different mRNA and miRNA profiles in human hypopharyngeal squamous cell carcinoma sensitive and resistant to chemotherapy.

The purpose of this study was to identify potential miRNAs and mRNAs involved in chemotherapy insensitivity in hypopharyngeal squamous cell carcinoma (HSCC) and to explore the underlying mechanisms involved to provide diagnostic markers and therapeutic targets for HSCC. We used microarrays to identify differences in both the mRNA and miRNA expression profiles between a group (twelve patients) sensitive to chemotherapy and a resistant group (nine patients). We then employed bioinformatics tools to examine the functions and pathways involved. The genes and miRNAs most related to chemotherapy sensitivity in HSCC were screened. Finally, a miRNA-mRNA-phenotype network was constructed with an integrated analysis based on the identified miRNAs and mRNAs. Nine differentially expressed miRNAs and one hundred differentially expressed mRNAs were identified, and the functions of these genes and miRNAs were predicted. Bioinformatics analysis revealed a regulatory network consisting of eight genes and two miRNAs that influenced HSCC chemosensitivity. According to our analysis, CCL4L1 may be a potential molecular marker for HSCC chemotherapy, and excess CCL4L1 leads to the upregulation of PRAME and the downregulation of miR-375, thus decreasing HSPB8 expression and promoting chemotherapy sensitivity. Our work provides reliable data for further studies investigating the mechanism of HSCC chemotherapy sensitivity.

[Research and development of new traditional Chinese medicine drugs for certain syndromes based on "theoretical innovation"].

Compound prescription of traditional Chinese medicine(TCM), the main form in prevention and treatment of diseases in TCM, has unique advantages in medical practice. New TCM drugs for certain syndromes serve as an important carrier of inheritance and innovation in TCM. The research and development of new TCM drugs for certain syndromes should be based on the theory of TCM, guided by clinical value, and based on the principle of quality, safety and effectiveness. Through the innovative understanding of disease development rules, clinicians carry out academic theory innovation to guide clinical practice, aiming to effectively promote academic innovation and the development of new TCM drugs for certain syndromes. In this paper, we expounded some understanding of the innovation system of TCM, and analyzed the research value of new TCM drugs for certain syndromes. Based on theoretical innovation, the overall research model was preliminarily put forward. Subsequently, a concrete discussion from three aspects, including pharmaco-logy and toxicology, pharmacy and clinic research, was made on the specific process and existing problems of new drug research and development of TCM. Our research group attempts to establish a new drug innovation ecosystem for TCM syndrome, with the purpose of providing reference for other researchers.

[Clinical observation of isolated immunoglobulin G4-related sclerosing cholangitis and immunoglobulin G4 sclerosing cholangitis combined autoimmune pancreatitis].

Objective: To compare and analyze patient's general condition, laboratory testing and therapeutic responses of isolated immunoglobulin G4- related sclerosing cholangitis (IgG4-SC) and immunoglobulin G4 sclerosing cholangitis combined autoimmune pancreatitis (IgG4-SC/AIP). Methods: A retrospective study was conducted on IgG4-SC patients who attended outpatient and inpatients department of our hospital from April 2014 to March 2018 and their demographic characteristics, laboratory testing, and therapeutic responses were statistically analyzed. Normal distribution of continuous variables was compared with t-test, non-normal distribution of continuous variables was compared using the Mann-Whitney U test, and the categorical variables were compared with χ (2) test. Results: 29 IgG4-SC patients were included, including 19-isolated IgG4-SC and 10 IgG4-SC combined AIP (IgG4-SC/AIP). The average age of onset in the isolated IgG4-SC group was (46.06±19.03) years which was lower than IgG4-SC/AIP group (62.60±15.11), t = -2.360, P < 0.05. The median IgG4 in IgG4-SC/AIP patients is higher than that in isolated IgG4-SC, respectively 10.87 (3.73 ~ 20.13) and 3.14 (2.37 ~ 4.78)g/L(U = 159.000, P < 0.05). IgG4/IgG ratio is higher in IgG4-SC/AIP, than that in isolated IgG4-SC, respectively 0.62(0.23 ~ 0.86) and 0.16(0.10 ~ 0.21), U = 130.000, P < 0.05. Liver cirrhosis was more common in isolated IgG4-SC group (47%) than the IgG4-SC/AIP group (0), χ (2) = 9.637, P < 0.05. The median biochemical response time of isolated IgG4-SC group was 3.00 (2.00 to 4.00) months, which was longer than 1.00 (1.00 to 1.25) months of IgG4-SC/AIP group, U = 30.000, P < 0.05. The biochemical recurrence rate of isolated IgG4-SC group was 32%, which was lower than that of IgG4-SC/AIP (χ (2) = 6.461, P < 0.05). Conclusion: Serum IgG4 level and IgG4/IgG ratio were higher in patients with IgG4-SC/AIP group, and therapeutic responses in isolated IgG4-SC patients were worse than that of IgG4-SC/AIP patients. The efficacy of glucocorticoid monotherapy and immunosuppressive agents combined with glucocorticoid therapy demonstrated no considerable difference in IgG4-SC patients.

Emerging technologies to measure neighborhood conditions in public health: implications for interventions and next steps.

Adverse neighborhood conditions play an important role beyond individual characteristics. There is increasing interest in identifying specific characteristics of the social and built environments adversely affecting health outcomes. Most research has assessed aspects of such exposures via self-reported instruments or census data. Potential threats in the local environment may be subject to short-term changes that can only be measured with more nimble technology. The advent of new technologies may offer new opportunities to obtain geospatial data about neighborhoods that may circumvent the limitations of traditional data sources. This overview describes the utility, validity and reliability of selected emerging technologies to measure neighborhood conditions for public health applications. It also describes next steps for future research and opportunities for interventions. The paper presents an overview of the literature on measurement of the built and social environment in public health (Google Street View, webcams, crowdsourcing, remote sensing, social media, unmanned aerial vehicles, and lifespace) and location-based interventions. Emerging technologies such as Google Street View, social media, drones, webcams, and crowdsourcing may serve as effective and inexpensive tools to measure the ever-changing environment. Georeferenced social media responses may help identify where to target intervention activities, but also to passively evaluate their effectiveness. Future studies should measure exposure across key time points during the life-course as part of the exposome paradigm and integrate various types of data sources to measure environmental contexts. By harnessing these technologies, public health research can not only monitor populations and the environment, but intervene using novel strategies to improve the public health.

Hospital and geographic variability in two colorectal cancer surgery outcomes: complications and mortality after complications.

BACKGROUND: The purpose of this study was to describe hospital and geographic variation in 30-day risk of surgical complications and death among colorectal cancer (CRC) patients and the extent to which patient-, hospital-, and census-tract-level characteristics increased risk of these outcomes. METHODS: We included patients at least 66 years old with first primary stage I-III CRC from the 2000-2005 National Cancer Institute's Surveillance, Epidemiology, and End Results data linked with 1999-2005 Medicare claims. A multilevel, cross-classified logistic model was used to account for nesting of patients within hospitals and within residential census tracts. Outcomes were risk of complications and death after a complication within 30 days of surgery. RESULTS: Data were analyzed for 35,946 patients undergoing surgery at 1,222 hospitals and residing in 12,187 census tracts; 27.2 % of patients developed complications, and of these 13.4 % died. Risk-adjusted variability in complications across hospitals and census tracts was similar. Variability in mortality was larger than variability in complications, across hospitals and across census tracts. Specific characteristics increased risk of complications (e.g., census-tract-poverty rate, emergency surgery, and being African-American). No hospital characteristics increased complication risk. Specific characteristics increased risk of death (e.g. census-tract-poverty rate, being diagnosed with colon (versus rectal) cancer, and emergency surgery), while hospitals with at least 500 beds showed reduced death risk. CONCLUSIONS: Large, unexplained variations exist in mortality after surgical complications in CRC across hospitals and geographic areas. The potential exists for quality improvement efforts targeted at the hospital and/or census-tract levels to prevent complications and augment hospitals' ability to reduce mortality risk.

Overexpression of protein kinase B/AKT induces phosphorylation of p70S6K and 4E-BP1 in goat fetal fibroblasts.

Protein kinases regulate many processes, including cell growth, metabolism, molecular interactions, and cell proliferation. Protein kinase B (PKB)/AKT (v-AKT mouse thymoma viral oncogene homolog) is an upstream component of mammalian target of rapamycin (mTOR) signaling and mediates pathophysiological processes in several signaling pathways. This study aimed to construct and overexpress a eukaryotic goat AKT expression vector in goat fetal fibroblasts and examine the effects of AKT on the phosphorylation of p70S6K and 4E-BP1. AKT was subcloned into the expression vector pIRES2-DsRed2 to generate pIRES2-DsRed2-AKT, which was transfected into goat fetal fibroblasts with LipofectamineTM 2000. AKT was measured by reverse transcription-polymerase chain reaction in the transgenic cells, and the expression of AKT and phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) were analyzed by Western blot. Cell clones that stably emitted red fluorescence were obtained after transfection for 48 h, and the exogenous gene was verified. Exogenous AKT was transcribed, and AKT was overexpressed, inducing the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) in goat fetal fibroblasts. Thus, the overexpression of AKT activates mTOR signaling in goat cells.

Drug Discovery in the Field of ß-Lactams: An Academic Perspective.

ß-Lactams are the most widely prescribed class of antibiotics that inhibit penicillin-binding proteins (PBPs), particularly transpeptidases that function in peptidoglycan synthesis. A major mechanism of antibiotic resistance is the production of ß-lactamase enzymes, which are capable of hydrolyzing ß-lactam antibiotics. There have been many efforts to counter increasing bacterial resistance against ß-lactams. These studies have mainly focused on three areas: discovering novel inhibitors against ß-lactamases, developing new ß-lactams less susceptible to existing resistance mechanisms, and identifying non-ß-lactam inhibitors against cell wall transpeptidases. Drug discovery in the ß-lactam field has afforded a range of research opportunities for academia. In this review, we summarize the recent new findings on both ß-lactamases and cell wall transpeptidases because these two groups of enzymes are evolutionarily and functionally connected. Many efforts to develop new ß-lactams have aimed to inhibit both transpeptidases and ß-lactamases, while several promising novel ß-lactamase inhibitors have shown the potential to be further developed into transpeptidase inhibitors. In addition, the drug discovery progress against each group of enzymes is presented in three aspects: understanding the targets, screening methodology, and new inhibitor chemotypes. This is to offer insights into not only the advancement in this field but also the challenges, opportunities, and resources for future research. In particular, cyclic boronate compounds are now capable of inhibiting all classes of ß-lactamases, while the diazabicyclooctane (DBO) series of small molecules has led to not only new ß-lactamase inhibitors but potentially a new class of antibiotics by directly targeting PBPs. With the cautiously optimistic successes of a number of new ß-lactamase inhibitor chemotypes and many questions remaining to be answered about the structure and function of cell wall transpeptidases, non-ß-lactam transpeptidase inhibitors may usher in the next exciting phase of drug discovery in this field.

Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance.

SARS-CoV-2 main protease, M pro , is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. M pro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a M pro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length M pro protein as substrates, we demonstrate that the binding of M pro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the M pro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of M pro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying M pro and substrate interactions, and offers important insights into M pro function, resistance development, and inhibitor design.

Are neighborhood conditions associated with HIV management?

OBJECTIVES: HIV infection has become a manageable chronic disease as a result of treatment advances. Secondary prevention efforts have proved inadequate to reduce the estimated incidence of new HIV infections. Epidemiological data suggest that geographical clustering of new HIV infections is a common phenomenon, particularly in urban areas among populations of low socioeconomic status. This study aimed to assess the relationship between neighbourhood conditions and HIV management and engagement in high-risk behaviours. METHODS: During routine out-patient HIV clinic visits, 762 individuals from the St Louis metropolitan area completed behavioural assessments in 2008. Biomedical markers were abstracted from their medical records. Multi-level analyses were conducted based on individuals' census tracts. RESULTS: The majority of the sample were male and African American. In the adjusted models, individuals residing in neighbourhoods with higher poverty rates were more likely to have lower CD4 cell counts and be current smokers. In neighbourhoods with higher rates of unemployment, individuals were less likely to have a current antiretroviral prescription. In more racially segregated neighbourhoods, individuals reported more depressive symptoms. CONCLUSIONS: Despite the advances in HIV disease management, neighbourhood characteristics contribute to disparities in HIV care. Interventions that address neighbourhood conditions as barriers to HIV management may provide improved health outcomes.

Inbreeding and genetic diversity in three imported Swine breeds in china using pedigree data.

The accumulation of inbreeding and the loss of genetic diversity is a potential problem in the modern swine breeds in China. Therefore, the purpose of this study was to analyze the pedigrees of Chinese Duroc (CD), Landrace (CL) and Yorkshire (CY) swine to estimate the past and current rates of inbreeding, and to identify the main causes of genetic diversity loss. Pedigree files from CD, CL and CY containing, 4529, 16,776 and 22,600 records, respectively, were analyzed. Pedigree completeness indexes of the three breeds, accounting for one generation back, were 83.72, 93.93 and 93.59%, respectively. The estimated average annual inbreeding rates for CD, CL and CY in recent three years were 0.21, 0.19 and 0.13%, respectively. The estimated average percentage of genetic diversity loss within each breed in recent three years was about 8.92, 2.19, and 3.36%, respectively. The average relative proportion of genetic diversity loss due to unequal contributions of founders in CD, CL and CY was 69.09, 57.95 and 60.57%, and due to random genetic drift was 30.91, 42.05 and 39.43%, respectively. The estimated current effective population size for CD, CL and CY was 76, 117 and 202, respectively. Therefore, CD has been found to have lost considerable genetic diversity, demanding priority for optimizing the selection and mating to control future coancestry and inbreeding. Unequal contribution of founders was a major cause of genetic diversity loss in Chinese swine breeds and random genetic drift also showed substantial impact on the loss of diversity.

A Qualitative Study Exploring Rehabilitant and Informal Caregiver Perspectives of a Challenging Rehabilitation Environment for Geriatric Rehabilitation.

There is a trend toward formalization of the rehabilitation process for older rehabilitants in a Challenging Rehabilitation Environment (CRE). This concept involves the comprehensive organization of care, support, and environment in rehabilitation wards. So far, literature about the principles of CRE is scarce. This study aims to explore the opinions of rehabilitants and informal caregivers regarding CRE, through a qualitative study between 2019 and 2020. Three telephone interviews were conducted with informal caregivers, and also 3 focus groups with 15 rehabilitants and 3 informal caregivers, all with recent experience in rehabilitation. Nine themes emerged regarding the rehabilitation process: (1) rehabilitant (attention for resilience, motivation, cognitive and emotional aspects), (2) rehabilitant centered (goal setting, physical and cognitive functioning and coping), (3) informal caregivers (involving and attention for resilience and relation), (4) communication (aligning the rehabilitation process), (5) exercise (increasing intensity by using task-oriented exercise, patient-regulated exercise, and group training), (6) peer support (learning experiences and recognition), (7) daily schedule (influence on the planning and activities outside therapy), (8) nutrition (energy for rehabilitation), and (9) eHealth (makes rehabilitation more challenging and fun). Regarding organizational processes, 4 themes were identified: (1) environmental aspects (single bedrooms, shared room for activities and therapy options on the ward), (2) staff aspects (small team with an emphatic supportive and motivating attitude), (3) organizational aspects (organized in an efficient way), and (4) return home (the discharge process should be well prepared for instance with home visits). Organizing excellent rehabilitation care requires a thorough understanding of the concept of CRE, as it is a complex and comprehensive concept that concerns the whole rehabilitation process. Its effectiveness and efficiency should be researched in prospective studies.

A Qualitative Study Exploring Professional Perspectives of a Challenging Rehabilitation Environment for Geriatric Rehabilitation.

There is a trend towards the formalization of the rehabilitation process for older rehabilitants in a Challenging Rehabilitation Environment (CRE). This concept involves the comprehensive organization of care, support, and environment on rehabilitation wards. So far, literature on the principles of the CRE is scarce. This study aims to explore the perspectives regarding the CRE of healthcare professionals through a qualitative study. Therefore, between 2018 and 2020, six international and 69 Dutch professionals were interviewed in focus groups, and 180 professionals attended workshops on two Dutch congresses. Data were thematically analyzed using ATLAS.ti. Seven themes emerged regarding the rehabilitation processes: (1) rehabilitant (attention for cognitive functioning and resilience); (2) goals (setting personal goals); (3) exercise (increasing exercise intensity); (4) daily schedule (following the daily rhythm); (5) involving the client system (involving informal caregivers); (6) nutrition (influences rehabilitation capability); and (7) technology (makes rehabilitation more safe and challenging). Regarding organizational aspects, four main themes were identified: (1) environmental aspects (encourages exercises); (2) staff aspects (interdisciplinary team); (3) organizational aspects (implementing CRE requires a shared vision); and (4) factors outside the ward (a well-prepared discharge process). To offer effective rehabilitation, all elements of the CRE should be applied. To improve the CRE, specific interventions need to be developed and implemented. Consequently, the effectiveness and efficiency of the CRE need to be measured with validated tools.

Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir.

The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring Mpro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km < 10-fold change) while being resistant to nirmatrelvir (Ki > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that Mpro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in Mpro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir.

The SARS-CoV-2 main protease (M pro ) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M pro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M pro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k cat /K m <10-fold change) and resistance to nirmatrelvir (K i >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M pro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance. One Sentence Summary: Paxlovid resistant SARS-CoV-2 viruses with mutations in the main protease have been identified from clinical isolates.