Spermatogenic cell-specific type 1 hexokinase (HK1S) is essential for capacitation-associated increase in tyrosine phosphorylation and male fertility in mice.

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.

A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers.

The sensitivity of LC-MS in quantifying target proteins in plasma/tissues is significantly hindered by coeluted matrix interferences. While antibody-based immuno-enrichment effectively reduces interferences, developing and optimizing antibodies are often time-consuming and costly. Here, by leveraging the orthogonal separation capability of Field Asymmetric Ion Mobility Spectrometry (FAIMS), we developed a FAIMS/differential-compensation-voltage (FAIMS/dCV) method for antibody-free, robust, and ultrasensitive quantification of target proteins directly from plasma/tissue digests. By comparing the intensity-CV profiles of the target vs coeluted endogenous interferences, the FAIMS/dCV approach identifies the optimal CV for quantification of each target protein, thus maximizing the signal-to-noise ratio (S/N). Compared to quantification without FAIMS, this technique dramatically reduces endogenous interferences, showing a median improvement of the S/N by 14.8-fold for the quantification of 17 representative protein drugs and biomarkers in plasma or tissues and a 5.2-fold median increase in S/N over conventional FAIMS approach, which uses the peak CV of each target. We also discovered that the established CV parameters remain consistent over months and are matrix-independent, affirming the robustness of the developed FAIMS/dCV method and the transferability of the method across matrices. The developed method was successfully demonstrated in three applications: the quantification of monoclonal antibodies with subng/mL LOQ in plasma, an investigation of the time courses of evolocumab and its target PCSK9 in a preclinical setting, and a clinical investigation of low abundance obesity-related biomarkers. This innovative and easy-to-use method has extensive potential in clinical and pharmaceutical research, particularly where sensitive and high-throughput quantification of protein drugs and biomarkers is required.

Two-dimensional Mo1-xB2 with ordered metal vacancies obtained for advanced thermoelectric applications based on first-principles calculations.

The study and development of high thermoelectric properties is crucial for the next generation of microelectronic and wearable electronics. Derived from the recent experimental realization of layers of transition metal molybdenum and boride, we report the theoretical realization of advanced thermoelectric properties in two-dimensional (2D) transition metal boride Mo1-xB2 (x = 0, 0.05, 0.10, 0.125, 0.15)-based defect sheets. The introduction of metal vacancies results in stronger d-p exchange interactions and hybridization between the Mo-d and B-p atoms. Meanwhile, the ordered metal vacancies enabled transition metal borides (n-type Mo0.9B2) to widen the d-bandwidth and raise the d-band center, leading to a relatively high carrier mobility of 3262 cm2 V-1 s-1 and conductivity twice that of a bug-free n-type MoB2 layer, which indicates that it presents good electronic and thermal transport properties. Furthermore, investigations of the thermoelectric performance exhibit a maximum ZT of up to 3.29, which is superior to those of currently reported 2D materials. Modulation by defect engineering suggests that 2D transition metal boride sheets with ordered metal vacancies have promising applications in microelectronics, wearable electronics and thermoelectric devices.

The 50% and 95% effective dose of remimazolam tosilate for anaesthesia induction in sleep disorders patients undergoing laparoscopic cholecystectomy: an up-and-down sequential allocation trial.

PURPOSE: Previous reports argue that preoperative sleep conditions of patients can influence the dosage of general anaesthesia drugs. Therefore, we aimed to investigate the dose-effect relationship of preoperative sleep disorders on the induction of general anaesthesia with remimazolam tosilate and calculate the Median effective (ED50) and 95% effective (ED95) dosages. METHODS: Included in our study were 56 patients who underwent laparoscopic cholecystectomy at our hospital. A separate group of 27 patients with sleep disorders (SD group) and 29 patients without sleep disorders (NSD group) using the Pittsburgh Sleep Quality Index (PSQI) were also included. According to the Dixon 'up-and-down' design, patients received remimazolam at preselected concentrations starting at 0.2 mg/kg. After the administration of remimazolam, loss of consciousness was observed. By observing whether consciousness disappeared within a minute, we adjusted the dose of remimazolam by 0.1 mg/kg (up and down) in the following patient. The Median effective dose (ED50), 95% effective dose (ED95), and 95% confidence interval (CI) of remimazolam for effective sedation were calculated. RESULTS: The ED50 of remimazolam was 0.226 mg/kg (95%CI 0.221-0.232 mg/kg) in the SD group and 0.191 mg/kg (95%CI, 0.183-0.199 mg/kg) in the NSD group. The ED95 of remimazolam was 0.237 mg/kg (95%CI 0.231-0.262 mg/kg) in the SD group and 0.209 mg/kg (95%CI 0.200-0.254 mg/kg) in the NSD group. CONCLUSIONS: In the SD group, the ED50 and ED95 of remimazolam during anaesthesia induction were 0.226 and 0.237 mg/kg, respectively. The induction dose of remimazolam in the SD group was significantly higher than that in the NSD group.

Bio-Inspired Trace Hydroxyl-Rich Electrolyte Additives for High-Rate and Stable Zn-Ion Batteries at Low Temperatures.

High-rate and stable Zn-ion batteries working at low temperatures are highly desirable for practical applications, but are challenged by sluggish kinetics and severe corrosion. Herein, inspired by frost-resistant plants, we report trace hydroxyl-rich electrolyte additives that implement a dual remodeling effect for high-performance low-temperature Zn-ion batteries. The additive with high Zn absorbability not only remodels Zn2+ primary solvent shell by alternating H2 O molecules, but also forms a shielding layer thus remodeling the Zn surface, which effectively enhances fast Zn2+ de-solvation reaction kinetics and prohibits Zn anode corrosion. Taking trace α-D-glucose (αDG) as a demonstration, the electrolyte obtains a low freezing point of -55.3 °C, and the Zn//Zn cell can stably cycle for 2000 h at 5 mA cm-2 under -25 °C, with a high cumulative capacity of 5000 mAh cm-2 . A full battery that stably operates for 10000 cycles at -50 °C is also demonstrated.

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.

Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.

Association between life's essential 8 and biological ageing among US adults.

BACKGROUND: Biological ageing is tightly linked to cardiovascular disease (CVD). We aimed to investigate the relationship between Life's Essential 8 (LE8), a currently updated measure of cardiovascular health (CVH), and biological ageing. METHODS: This cross-sectional study selected adults ≥ 20 years of age from the 2005-2010 National Health and Nutrition Examination Survey. LE8 scores (range 0-100) were obtained from measurements based on American Heart Association definitions, divided into health behavior and health factor scores. Biological ageing was assessed by different methods including phenotypic age, phenotypic age acceleration (PhenoAgeAccel), biological age and biological age acceleration (BioAgeAccel). Correlations were analyzed by weighted linear regression and restricted cubic spline models. RESULTS: Of the 11,729 participants included, the mean age was 47.41 ± 0.36 years and 5983 (51.01%) were female. The mean phenotypic and biological ages were 42.96 ± 0.41 and 46.75 ± 0.39 years, respectively, and the mean LE8 score was 67.71 ± 0.35. After adjusting for potential confounders, higher LE8 scores were associated with lower phenotypic age, biological age, PhenoAgeAccel, and BioAgeAccel, with nonlinear dose-response relationships. Negative associations were also found between health behavior and health factor scores and biological ageing, and were stronger for health factors. In health factor-specific analyses, the ß negativity was greater for blood glucose and blood pressure. The inverse correlations of LE8 scores with phenotypic age and biological age in the stratified analyses remained solid across strata. CONCLUSIONS: LE8 and its subscale scores were strongly negatively related to biological ageing. Encouraging optimal CVH levels may be advantageous in preventing and slowing down ageing.

Mechanisms of SGLT2 Inhibitors in Heart Failure and Their Clinical Value.

ABSTRACT: Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used to treat diabetes mellitus. Abundant evidence has shown that SGLT2 inhibitors can reduce hospitalization for heart failure (HF) in patients with or without diabetes. An increasing number of studies are being conducted on the mechanisms of action of SGLT2 inhibitors in HF. Our review summarizes a series of clinical trials on the cardioprotective effects of SGLT2 inhibitors in the treatment of HF. We have summarized several classical SGLT2 inhibitors in cardioprotection research, including empagliflozin, dapagliflozin, canagliflozin, ertugliflozin, and sotagliflozin. In addition, we provided a brief overview of the safety and benefits of SGLT2 inhibitors. Finally, we focused on the mechanisms of SGLT2 inhibitors in the treatment of HF, including ion-exchange regulation, volume regulation, ventricular remodeling, and cardiac energy metabolism. Exploring the mechanisms of SGLT2 inhibitors has provided insight into repurposing these diabetic drugs for the treatment of HF.

Understanding the Excited-State Relaxation Mechanisms of Xanthophyll Lutein by Multi-configurational Electronic Structure Calculations.

The contradictory behaviors in light harvesting and non-photochemical quenching make xanthophyll lutein the most attractive functional molecule in photosynthesis. Despite several theoretical simulations on the spectral properties and excited-state dynamics, the atomic-level photophysical mechanisms need to be further studied and established, especially for an accurate description of geometric and electronic structures of conical intersections for the lowest several electronic states of lutein. In the present work, semiempirical OM2/MRCI and multi-configurational restricted active space self-consistent field methods were performed to optimize the minima and conical intersections in and between the 1Ag-, 2Ag-, 1Bu+, and 1Bu- states. Meanwhile, the relative energies were refined by MS-CASPT2(10,8)/6-31G*, which can reproduce correct electronic state properties as those in the spectroscopic experiments. Based on the above calculation results, we proposed a possible excited-state relaxation mechanism for lutein from its initially populated 1Bu+ state. Once excited to the optically bright 1Bu+ state, the system will propagate along the key reaction coordinate, i.e., the stretching vibration of the conjugated carbon chain. During this period of time, the 1Bu- state will participate in and forms a resonance state between the 1Bu- and 1Bu+ states. Later, the system will rapidly hop to the 2Ag- state via the 1Bu+/2Ag- conical intersection. Finally, the lutein molecule will survive in the 2Ag- state for a relatively long time before it internally converts to the ground state directly or via a twisted S1/S0 conical intersection. Notably, though the photophysical picture may be very different in solvents and proteins, the current theoretical study proposed a promising calculation protocol and also provided many valuable mechanistic insights for lutein and similar carotenoids.

Delayed intubation associated with in-hospital mortality in patients with COVID-19 respiratory failure who fail heated and humified high flow nasal canula.

BACKGROUND: Advanced respiratory support modalities such as non-invasive positive pressure ventilation (NiPPV) and heated and humidified high flow nasal canula (HFNC) served as useful alternatives to invasive mechanical ventilatory support for acute respiratory failure (ARF) during the peak of the SARS-CoV-2/COVID-19 pandemic. Unlike NiPPV, HFNC is a newer modality and its role in the treatment of patients with severe ARF is not yet clearly defined. Furthermore, the characteristics of responders versus non-responders to HFNC have not been determined. Although recent evidence indicates that many patients with ARF treated with HFNC survive without needing intubation, those who fail and are subsequently intubated have worse outcomes. Given that prolonged use of HFNC in patients with ARF might exacerbate patient self-inflicted lung injury, we hypothesized that among those patients with ARF due to COVID-19 pneumonia, prolonged HFNC beyond 24 h before intubation would be associated with increased in-hospital mortality. METHODS: This was a retrospective, multicenter, observational cohort study of 2720 patients treated for ARF secondary to SARS-CoV-2/COVID-19 pneumonia and initially managed with HFNC within the Banner Health system during the period from March 1st, 2020, to July 31st, 2021. In the subgroup of patients for went from HFNC to IMV, we assessed the effect of the duration of HFNC prior to intubation on mortality. RESULTS: 1392 (51%) were successfully treated with HFNC alone and 1328 (49%) failed HFNC and were intubated (HFNC to IMV). When adjusted for the covariates, HFNC duration less than 24 h prior to intubation was significantly associated with reduced mortality. CONCLUSIONS: Among patients with ARF due to COVID-19 pneumonia who fail HFNC, delay of intubation beyond 24 h is associated with increased mortality.

[Nursery Services for Children Under 3 in Sichuan Province].

Objective: To study the current status of nursery services for children under 3 in Sichuan Province, to analyze the problems and causes, and to provide policy recommendations for the high-quality development of nursery services in Sichuan Province for children under the age of 3. Methods: Using a combination of qualitative and quantitative methods, we analyzed the current status of nursery services in Sichuan Province and the problems encountered in the development of nursery services in Sichuan Province by collecting information on relevant government policies of Sichuan and the status quo of nursery services for children under 3 in the 21 cities/prefectures of Sichuan through policy research, interviews conducted at nursery service institutions, questionnaire surveys, and expert panel discussions. Results: The supply of nursery services, or nursery enrollment capacity, in Sichuan Province reached 3 nursery enrollment opportunities per thousand people, with the enrollment utilization rate reaching 48.2%. Among all the nursery service institutions in the province, 55.4% were concentrated in the Chengdu, Mianyang, and Meishan regions. There were 1667 pilot nursery service institutions providing government-subsidized affordable nursery services and supplying 72971 subsidized affordable enrollment opportunities, accounting for 28.7% of the total number of all nursery service institutions and 29.1% of their total enrollment. There were a total of 37995 nursery service professionals, including 28468 caregivers (including teachers), with an average of 6 employees per institution and 5 caregivers per institution. 49.6% of the nursery service institutions hired healthcare workers on a part-time basis and 11.3% of them did not have healthcare workers on their staffs. Conclusion: Nursery services for children under 3 years old have developed rapidly in Sichuan Province, but there are a number of problems, including uneven distribution of resources in different regions, inadequate supply of government-subsidized affordable nursery services, mismatch between supply and demand, insufficient utilization of services, lack of professionals, and insufficient driving force for sustainable development in the industry. We have suggested that actions be taken to improve the policy and regulatory system for nursery services, actively develop care facilites that are government-subsidized affordable services, integrated daycare service for all preschool children, and integrated medical and education facilites, provide support for home care, establish an information system for nursery services, optimize the professional training and development system, strengthen scientific research and international exchanges on nursery services, and comprehensively promote the high-quality development of nursery services and the construction of a childrearing-friendly environment in Sichuan Province.

Janus Hollow Nanofiber with Bifunctional Oxygen Electrocatalyst for Rechargeable Zn-Air Battery.

Zn-air battery technologies have received increasing attention, while the application is hindered by the sluggish kinetics of the oxygen evolution reaction (OER) and oxygen reduction reaction (ORR). In order to explore an efficient method to fabricate a high-performance electrocatalyst via modification of advanced nanostructure, a coaxial electrospinning method with in-situ synthesis and subsequent carbonization to construct 3D flexible Janus-like electrocatalysts is developed. The resulting Janus nanofibers have a unique core-shell hollow fiber structure, where NiFe alloy electrocatalysts supported by N-doped carbon nanobelt are located on the inner wall of the carbon layer, and leaf-like Co-N nanosheets are anchored on the outer wall of the carbon layer. As a result, the electrocatalyst exhibits excellent bifunctional catalytic performance for ORR and OER, demonstrating the small potential gap value of 0.73 V between the ORR half-wave potential and the OER potential at 10 mA cm-2 , which is even comparable to the mixed commercial noble catalyst with 20% Pt/C and RuO2 . The rechargeable Zn-air battery is constructed and displays a large open-circuit voltage of 1.44 V, high power density (130 mW cm-2 ) and energy density (874 Wh kg-1 ). This study provides a concept to synthesize and construct high performance bifunctional electrocatalysts.

Factors predicting self-report adherence (SRA) behaviours among DS-TB patients under the "Integrated model": a survey in Southwest China.

BACKGROUND: China is one of 30 countries with a high tuberculosis (TB) burden, and poor adherence to TB treatment is one of the biggest challenges for TB control. We aimed to explore the barriers and facilitators of treatment adherence among drug-sensitive tuberculosis (DS-TB) patients under the "Integrated model" in Western China, to provide evidence-based treatment and control regimens for DS-TB patients to improve adherence behaviours. METHODS: Both qualitative and quantitative research methods were used to explore the factors associated with self-reported adherence (SRA) behaviours. Questionnaire surveys with DS-TB patients and in-depth interviews with leaders from the Centers for Disease Control and Prevention (CDC) and community health sectors (CHCs), healthcare workers (HCWs) from CHCs, and DS-TB patients were conducted. RESULTS: A total of 459 eligible patients were included in the quantitative survey, and two patients and 13 healthcare providers were included in the in-depth interviews. The percentage of patients who experienced a missed dose, lack of follow-up sputum examination, and interrupted treatment were 19.0%, 11.3%, and 9.2%, respectively. Patients aged 20-39 had a higher risk of missed dose [OR (95% CI): 2.302 (1.001-5.305)] and a lower risk of interrupted treatment [OR (95% CI): 0.278 (0.077-0.982)] than patients more than 60 years. Patients who were of Han ethnicity (OR [95% CI]: 0.524 [0.301-0.912]) received psychological support (OR [95% CI]: 0.379 [0.144-0.998]) from their family and had a lower risk of missed doses. Patients who had drug side effects had a higher risk of interrupted treatment (OR [95% CI]: 2.587 [1.237-5.412]). Patients who possessed higher knowledge had a lower risk of lack of follow-up sputum examination [OR (95% CI): 0.817 (0.673-0.991)]. The results of the qualitative study also reported that patients' poor TB knowledge was the main reason for their non-SRA behaviours. CONCLUSIONS: Patient-centred strategies should be implemented to improve health literacy and strengthen psychological support. More effective case management should be designed and implemented based on different patient characteristics to improve adherence behaviours in further studies.

Failure of High Flow Nasal Cannula and Subsequent Intubation Is Associated With Increased Mortality as Compared to Failure of Non-Invasive Ventilation and Mechanical Ventilation Alone: A Real-World Retrospective Analysis.

BACKGROUND: Despite the increasing use of high flow nasal cannula oxygenation systems (HFNC) in clinical practice, little is known about its role in all cause respiratory failure as compared to traditional non-invasive ventilation (BiPAP). Furthermore, the effect of HFNC on mortality is unknown. METHODS: We conducted a retrospective analysis of 49,853 patients with respiratory failure treated with non-invasive respiratory support (HFNC or BiPAP) and/or invasive mechanical ventilation (IMV) between 2017 and 2018. RESULTS: Patients initially treated with HFNC who underwent subsequent intubation and IMV had a higher mortality rate as compared to patients who were initially treated with BiPAP and underwent subsequent intubation and IMV (34.8% vs 26.3%, p < 0.0001, OR 1.49, 95% CI 1.26,1.76). Patients first treated with HFNC who underwent subsequent intubation and IMV had a significantly increased mortality compared to patients who underwent immediate intubation and IMV (34.8% vs. 21.5%, p ≤ 0.0001, OR 1.94, 95% CI 1.67, 2.27). Stratified based on ICD-10 diagnosis, patients with a diagnosis of COPD exacerbation or heart failure treated with HFNC and subsequent intubation and IMV had higher mortality as compared to those treated with immediate IMV alone. This trend did not hold true for patients with a diagnosis of pneumonia. CONCLUSION: In a real-world retrospective analysis, use of HFNC was associated with increased mortality as compared to BiPAP and IMV alone. Further study is needed to confirm these associations.

Clinician Practice Patterns That Result in the Diagnosis of Coccidioidomycosis Before or During Hospitalization.

BACKGROUND: Coccidioidomycosis (CM) is common and important within endemic regions, requiring specific testing for diagnosis. Long delays in diagnosis have been ascribed to ambulatory clinicians. However, how their testing practices have impacted patient care has not been systematically unexplored. METHODS: We analyzed practice patterns for CM diagnoses over 3 years within a large Arizona healthcare system, including diagnosis location, patient characteristics, and care-seeking patterns associated with missed diagnosis. RESULTS: For 2043 CM diagnoses, 72.9% were made during hospital admission, 21.7% in ambulatory clinics, 3.2% in emergency units, and only 0.5% in urgent care units. A 40.6% subgroup of hospitalized patients required neither intensive care unit or hospital-requiring procedures, had a median length of stay of only 3 days, but still incurred both substantial costs ($27.0 million) and unnecessary antibiotic administrations. Prior to hospital diagnosis (median of 32 days), 45.1% of patients had 1 or more visits with symptoms consistent with CM. During those visits, 71.3% were not tested for CM. Diagnoses were delayed a median of 27 days. CONCLUSIONS: Lack of testing for CM in ambulatory care settings within a region endemic for CM resulted in a large number of hospital admissions, attendant costs, and unneeded antibacterial drug use, much of which would otherwise be unnecessary. Improving this practice is challenging since many clinicians did not train where CM is common, resulting in significant inertia to change. Determining the best way to retrain clinicians to diagnose CM earlier is an opportunity to explore which strategies might be the most effective.

Effect of Endovascular Treatment Alone vs Intravenous Alteplase Plus Endovascular Treatment on Functional Independence in Patients With Acute Ischemic Stroke: The DEVT Randomized Clinical Trial.

Importance: For patients with large vessel occlusion strokes, it is unknown whether endovascular treatment alone compared with intravenous thrombolysis plus endovascular treatment (standard treatment) can achieve similar functional outcomes. Objective: To investigate whether endovascular thrombectomy alone is noninferior to intravenous alteplase followed by endovascular thrombectomy for achieving functional independence at 90 days among patients with large vessel occlusion stroke. Design, Setting, and Participants: Multicenter, randomized, noninferiority trial conducted at 33 stroke centers in China. Patients (n = 234) were 18 years or older with proximal anterior circulation intracranial occlusion strokes within 4.5 hours from symptoms onset and eligible for intravenous thrombolysis. Enrollment took place from May 20, 2018, to May 2, 2020. Patients were enrolled and followed up for 90 days (final follow-up was July 22, 2020). Interventions: A total of 116 patients were randomized to the endovascular thrombectomy alone group and 118 patients to combined intravenous thrombolysis and endovascular thrombectomy group. Main Outcomes and Measures: The primary end point was the proportion of patients achieving functional independence at 90 days (defined as score 0-2 on the modified Rankin Scale; range, 0 [no symptoms] to 6 [death]). The noninferiority margin was -10%. Safety outcomes included the incidence of symptomatic intracerebral hemorrhage within 48 hours and 90-day mortality. Results: The trial was stopped early because of efficacy when 234 of a planned 970 patients had undergone randomization. All 234 patients who were randomized (mean age, 68 years; 102 women [43.6%]) completed the trial. At the 90-day follow-up, 63 patients (54.3%) in the endovascular thrombectomy alone group vs 55 (46.6%) in the combined treatment group achieved functional independence at the 90-day follow-up (difference, 7.7%, 1-sided 97.5% CI, -5.1% to ∞)P for noninferiority = .003). No significant between-group differences were detected in symptomatic intracerebral hemorrhage (6.1% vs 6.8%; difference, -0.8%; 95% CI, -7.1% to 5.6%) and 90-day mortality (17.2% vs 17.8%; difference, -0.5%; 95% CI, -10.3% to 9.2%). Conclusions and Relevance: Among patients with ischemic stroke due to proximal anterior circulation occlusion within 4.5 hours from onset, endovascular treatment alone, compared with intravenous alteplase plus endovascular treatment, met the prespecified statistical threshold for noninferiority for the outcome of 90-day functional independence. These findings should be interpreted in the context of the clinical acceptability of the selected noninferiority threshold. Trial Registration: Chinese Clinical Trial Registry: ChiCTR-IOR-17013568.

Toward Accurate and Robust Liquid Chromatography-Mass Spectrometry-Based Quantification of Antibody Biotherapeutics in Tissues.

Liquid chromatography-mass spectrometry (LC-MS) affords a highly promising solution for absolute quantification of biotherapeutics/targets in tissues, which is critical for drug development. Nonetheless, accurate/robust tissue quantification remains challenging largely owing to the lack of optimal approaches to address the following fundamental prerequisites: (i) efficient removal of residual blood without losing tissue-associated biotherapeutics; (ii) an optimal method to exhaustively/quantitatively recover target proteins from tissues; and (iii) an appropriate strategy to prepare calibration/quality-control samples to ensure accurate tissue analysis. Here, we devised novel analytical procedures enabling extensive and systematic investigation of the above issues and thereby development of optimal strategies for accurate tissue analysis. Key discoveries include: first, using a novel procedure of sequential administration of nonlabeled and then stable-isotope-labeled monoclonal antibody (mAb); it was determined that perfusion with three blood volumes of heparinized saline is optimal, achieving efficient blood removal (95-99%) and low quantitative bias (0.5-13%); second, a reference sample set established by mass-balanced, exhaustive extraction, permitted accurate measurement of absolute protein recovery from tissues of dosed animals; with this method, we found mAb biotherapeutics present in free-(49.3-75.4%) and bound-forms (24.6-50.7%) in tissues, even without a target; therefore, a denaturing detergent buffer is necessary for exhaustive extraction (recovery>90%); third, overnight-incubation of calibration samples after spiking mAb to tissue was found to improve quantitative accuracy, especially for nondenaturing buffer extraction. These investigations established the critical parameters and optimal protocols that can be universally applied to achieve accurate and robust quantification of biotherapeutics/targets in tissues. As a proof of concept, we conducted the first-ever extensive pharmacokinetics measurement of mAb in major tissues with a LC-MS-based method, where interesting features of mAb tissue disposition were observed.

Clinical characteristics of a group of deaths with COVID-19 pneumonia in Wuhan, China: a retrospective case series.

BACKGROUND: With the widespread outbreak of novel coronavirus diseases 2019(COVID-19), more and more death cases were reported, however, limited data are available for the patients who died. We aimed to explore the clinical characteristics of deaths with COVID-19 pneumonia. METHODS: We abstracted and analyzed epidemiological, demographic, clinical, and laboratory data from 83 death cases with COVID-19 pneumonia in East Hospital of Wuhan University Renmin Hospital, between January 26, 2020, and February 28, 2020. RESULTS: Of the 83 deaths, none was the medical staff. The mean age was 71.8 years (SD 13.2; range, 34-97 years) and 53(63.9%) were male. The median from onset to admission was 10 days (IQR 7-14: range, 2-43 days), to death was 17 days (IQR 14-21: range, 6-54 days). Most deaths (66[80%]) had underlying comorbid diseases, the most of which was hypertension [47(57%)]. The main initial symptoms of these 83 deaths were shortness of breath(98.8%), fever(94%), and myalgia or fatigue(90.4%). Laboratory analyses showed the lymphocytopenia in 69(83%) deaths, hypoalbuminemia in 77(93%) deaths, the elevation of lactate dehydrogenase in 79(95%) deaths, procalcitonin in 69(83%) deaths and C-reactive protein in 79(95%) deaths. All 83 patients received antiviral treatment, 81(97.6%) deaths received antibiotic therapy, 54(65.1%) deaths received glucocorticoid therapy, and 20(24.1%) patients received invasive mechanical ventilation. CONCLUSION: Most of the deaths with COVID-19 pneumonia were elderly patients with underlying comorbid diseases, especially those over 70 years of age. The time of death after the onset of the disease was mostly 15-21 days. More care should be given to the elderly in further prevention and control strategies of COVID-19.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.

Competency building for lay health workers is an intangible force driving basic public health services in Southwest China.

BACKGROUND: Providing universal basic public health services (BPHS) for residents is the main goal of the new health reform in China. Lay health workers (LHWs) in primary health care (PHC) sectors play key roles in BPHS delivery. The competency of LHWs is critical to quality BPHS. This study assessed LHWs' competency to deliver BPHS and related training in resource-limited Southwest China. METHODS: A mixed research method combining in-depth interviews with secondary data collection was used to collect data in this cross-sectional study. Fifty-four LHWs and 16 leaders in 16 PHC sectors were recruited for in-depth interviews. Secondary data on 198 LHWs were collected through standard forms. RESULTS: Both the interviews and secondary data suggested that all PHC sectors did not have sufficient LHWs and lacked qualified LHWs to deliver BPHS overall, particularly in relatively low economic rural areas in Guizhou province. Furthermore, PHC sectors had difficulties retaining existing LHWs due to low incomes and fewer opportunities for self-development. In-depth interviews discovered that, although numerous training opportunities have been provided for LHWs since 2009, the trainings did not achieve the expected outcome in LHW competency building, as LHWs actually did not have access to the trainings and the training design was unresponsive to the actual needs of LHWs. Both LHWs and leaders expressed an urgent need for effective training for LHWs based on systematic needs assessments and the use of qualified trainers and materials. CONCLUSIONS: The shortage of qualified LHWs in PHC sectors became the bottleneck for BPHS delivery in Southwest China. Recent trainings for LHWs were less effective with regard to LHW competency building. A need-based professional training programme for LHWs by qualified trainers was expected by both LHWs and leaders in PHC sectors.