Early identification of stroke through deep learning with multi-modal human speech and movement data.

JOURNAL/nrgr/04.03/01300535-202501000-00031/figure1/v/2024-05-14T021156Z/r/image-tiff Early identification and treatment of stroke can greatly improve patient outcomes and quality of life. Although clinical tests such as the Cincinnati Pre-hospital Stroke Scale (CPSS) and the Face Arm Speech Test (FAST) are commonly used for stroke screening, accurate administration is dependent on specialized training. In this study, we proposed a novel multimodal deep learning approach, based on the FAST, for assessing suspected stroke patients exhibiting symptoms such as limb weakness, facial paresis, and speech disorders in acute settings. We collected a dataset comprising videos and audio recordings of emergency room patients performing designated limb movements, facial expressions, and speech tests based on the FAST. We compared the constructed deep learning model, which was designed to process multi-modal datasets, with six prior models that achieved good action classification performance, including the I3D, SlowFast, X3D, TPN, TimeSformer, and MViT. We found that the findings of our deep learning model had a higher clinical value compared with the other approaches. Moreover, the multi-modal model outperformed its single-module variants, highlighting the benefit of utilizing multiple types of patient data, such as action videos and speech audio. These results indicate that a multi-modal deep learning model combined with the FAST could greatly improve the accuracy and sensitivity of early stroke identification of stroke, thus providing a practical and powerful tool for assessing stroke patients in an emergency clinical setting.

The interaction between lipid oxidation and the Maillard reaction model of lysine-glucose on aroma formation in fragrant sesame oil.

The formation mechanism behind the sophisticated aromas of sesame oil (SO) has not been elucidated. The interaction effects of the Maillard reaction (MR) and lipid oxidation on the aroma formation of fragrant sesame oil were investigated in model reaction systems made of l-lysine (Lys) and d-glucose (Glc) with or without fresh SO (FSO) or oxidized SO (OSO). The addition of OSO to the Lys-Glc model increased the MR browning at 294 nm and 420 nm and enhanced the DPPH radical scavenging activity greater than the addition of FSO (p < 0.05). The presence of lysine and glucose inhibited the oxidation of sesame oil, reduced the loss of γ-tocopherol, and facilitated the formation of sesamol (p < 0.05). The Maillard-lipid interaction led to the increased concentrations of some of the alkylpyrazines, alkylfurans, and MR-derived ketones and acids (p < 0.05) while reducing the concentrations of other pyrazines, lipid-derived furans, aliphatic aldehydes, ketones, alcohols, and acids (p < 0.05). The addition of FSO to the MR model enhanced the characteristic roasted, nutty, sweet, and fatty aromas in sesame oil (p < 0.05), while excessive lipid oxidation (OSO) brought about an unpleasant oxidized odor and reduced the characteristic aromas. This study helps to understand the sophisticated aroma formation mechanism in sesame oil and provides scientific instruction for precise flavor control in the production of sesame oil.

Concentration-QTc Modeling of the DPP-4 Inhibitor HSK7653 in a First-in-Human Study of Chinese Healthy Volunteers.

Cofrogliptin (HSK7653) is a long-acting dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes mellitus with a twice-monthly dosing regimen. This study included 62 participants (48 without food effect, 14 with food effect) receiving single doses of HSK7653 (5, 10, 25, 50, 100, and 150 mg) or placebo. Pharmacokinetic samples were collected over 24 hours postdosing and sampling times are aligned with 12-lead electrocardiograms (ECGs) which were derived from continuous ECG recordings. For the concentration-QT interval corrected for heart rate (C-QTc) analysis, we used linear mixed-effects modeling to characterize the correlation between plasma concentrations of HSK7653 and the change from baseline in the QT interval which was corrected by Fridericia's formula (ΔQTcF). The result showed that a placebo-corrected Fridericia corrected QT interval (ΔΔQTcF) prolongation higher than 10 milliseconds is unlikely at the mean maximum observed concentration (Cmax) (411 ng/mL) associated with the recommended therapeutic doses (25 mg twice-monthly), even at the highest supratherapeutic concentration (2425 ng/mL). Thus, HSK7653 does not significantly affect QT prolongation at either recommended doses or the highest supratherapeutic concentration.

The CNS Relapse in T-Cell Lymphoma Index Predicts CNS Relapse in Patients with T- and NK-Cell Lymphomas.

Little is known about risk factors for central nervous system (CNS) relapse in mature T- and NK-cell neoplasms (MTNKN). We aimed to describe the clinical epidemiology of CNS relapse in patients with MTNKN and developed the CNS relapse In T-cell lymphoma Index (CITI) to predict patients at highest risk of CNS relapse. We reviewed data from 135 patients with MTNKN and CNS relapse from 19 North American institutions. After exclusion of leukemic and most cutaneous forms of MTNKN, patients were pooled with non-CNS relapse control patients from a single institution to create a CNS relapse-enriched training set. Using a complete case analysis (N=182), of whom 91 had CNS relapse, we applied a LASSO Cox regression model to select weighted clinicopathologic variables for the CITI score, which we validated in an external cohort from the Swedish Lymphoma Registry (N=566). CNS relapse was most frequently observed in patients with PTCL, NOS (25%). Median time to CNS relapse and median overall survival after CNS relapse was 8.0 months and 4.7 months, respectively. We calculated unique CITI risk scores for individual training set patients and stratified them into risk terciles. Validation set patients with low-risk (N=158) and high-risk (N=188) CITI scores had a 10-year cumulative risk of CNS relapse of 2.2% and 13.4%, respectively (HR 5.24, 95%CI 1.50-18.26, P=0.018). We developed an open-access web-based CITI calculator (https://redcap.link/citicalc) to provide an easy tool for clinical practice. The CITI score is a validated model to predict patients with MTNKN at highest risk of developing CNS relapse.

Prognostic significance of myeloid-derived suppressor cells and systemic inflammation in newly diagnosed diffuse large B cell lymphoma treated with chemoimmunotherapy.

The revised International Prognostic Index (R-IPI) is an important prognostic tool in diffuse large B cell lymphoma (DLBCL); however, outcomes can vary markedly within R-IPI groups, and additional prognostic markers are needed. We conducted a prospective observational study to evaluate the circulating immature myeloid (IM) cell subsets and cytokine profiles of 31 patients with newly diagnosed DLBCL before and after chemoimmunotherapy. Among circulating IM cells, myeloid-derived suppressor cells (MDSCs) were the predominant cell type (73.8% ± 26%). At baseline, circulating monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) were predominantly mutually exclusive. Patients with DLBCL clustered into three distinct immunotypes according to MDSC levels and subtype predominance: M-MDSChigh, PMN-MDSChigh, and MDSClow. The M-MDSChigh immunotype was associated with the germinal center B cell-like (GCB) subtype and elevated serum IL-8 and MIP-1α levels. PMN-MDSChigh was associated with the non-GCB subtype and elevated IL-8, MCP-1, IP-10, TNFα, and IL-1Ra levels. Standard chemoimmunotherapy partially reduced M-MDSC distribution across the MDSClow and M-MDSChigh groups. By contrast, among the MDSClow and PMN-MDSChigh groups, PMN-MDSCs persisted after treatment. Two high-risk patients with non-GCB DLBCL and MDSClow immunotype experienced early disease recurrence within 12 months of treatment completion. This study demonstrates that distinct types of MDSCs are associated with subtypes of DLBCL. MDSC levels are dynamic and may be associated with disease status. Persistence of PMN-MDSCs among high-risk patients with DLBCL may be associated with early relapse.

Exosomal miR-9-5p derived from iPSC-MSCs ameliorates doxorubicin-induced cardiomyopathy by inhibiting cardiomyocyte senescence.

Doxorubicin (DOX) is a chemotherapeutic agent widely used for tumor treatment. Nonetheless its clinical application is heavily limited by its cardiotoxicity. There is accumulated evidence that transplantation of mesenchymal stem cell-derived exosomes (MSC-EXOs) can protect against Dox-induced cardiomyopathy (DIC). This study aimed to examine the cardioprotective effects of EXOs isolated from human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) against DIC and explore the potential mechanisms. EXOs were isolated from the cultural supernatant of human BM-MSCs (BM-MSC-EXOs) and iPSC-MSCs (iPSC-MSC-EXOs) by ultracentrifugation. A mouse model of DIC was induced by intraperitoneal injection of Dox followed by tail vein injection of PBS, BM-MSC-EXOs, or iPSC-MSC-EXOs. Cardiac function, cardiomyocyte senescence and mitochondrial dynamics in each group were assessed. In vitro, neonatal mouse cardiomyocytes (NMCMs) were subjected to Dox and treated with BM-MSC-EXOs or iPSC-MSC-EXOs. The mitochondrial morphology and cellular senescence of NMCMs were examined by Mitotracker staining and senescence-associated-ß-galactosidase assay, respectively. Compared with BM-MSC-EXOs, mice treated with iPSC-MSC-EXOs displayed improved cardiac function and decreased cardiomyocyte mitochondrial fragmentation and senescence. In vitro, iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuation of cardiomyocyte mitochondrial fragmentation and senescence caused by DOX. MicroRNA sequencing revealed a higher level of miR-9-5p in iPSC-MSC-EXOs than BM-MSC-EXOs. Mechanistically, iPSC-MSC-EXOs transported miR-9-5p into DOX-treated cardiomyocytes, thereby suppressing cardiomyocyte mitochondrial fragmentation and senescence via regulation of the VPO1/ERK signal pathway. These protective effects and cardioprotection against DIC were largely reversed by knockdown of miR-9-5p in iPSC-MSC-EXOs. Our results showed that miR-9-5p transferred by iPSC-MSC-EXOs protected against DIC by alleviating cardiomyocyte senescence via inhibition of the VPO1/ERK pathway. This study offers new insight into the application of iPSC-MSC-EXOs as a novel therapeutic strategy for DIC treatment.

Cirrhotic-extracellular matrix attenuates aPD-1 treatment response by initiating immunosuppressive neutrophil extracellular traps formation in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is closely associatedwith chronic liver diseases, particularly liver cirrhosis, which has an altered extracellular matrix (ECM) composition. The influence and its mechanism of the cirrhotic-ECM on the response of HCC to immune checkpoint inhibitor (ICI) remains less clarified. METHODS: In silico, proteomic and pathological assessment of alteration of cirrhotic-ECM were applied in clinical cohort. Multiple pre-clinical models with ECM manipulation were used to evaluate cirrhotic-ECM's effect on ICI treatment. In silico, flow cytometry and IHC were applied to explore how cirrhotic-ECM affect HCC microenvironment. In vitro and in vivo experiments were carried out to identify the mechanism of how cirrhotic-ECM undermined ICI treatment. RESULTS: We defined "a pro-tumor cirrhotic-ECM" which was featured as the up-regulation of collagen type 1 (Col1). Cirrhotic-ECM/Col1 was closely related to impaired T cell function and limited anti PD-1 (aPD-1) response of HCC patients from the TCGA pan cancer cohort and the authors' institution, as well as in multiple pre-clinical models. Mechanically, cirrhotic-ECM/Col1 orchestrated an immunosuppressive microenvironment (TME) by triggering Col1-DDR1-NFκB-CXCL8 axis, which initiated neutrophil extracellular traps (NETs) formation to shield HCC cells from attacking T cells and impede approaching T cells. Nilotinib, an inhibitor of DDR1, reversed the neutrophils/NETs dominant TME and efficiently enhanced the response of HCC to aPD-1. CONCLUSIONS: Cirrhotic-ECM modulated a NETs enriched TME in HCC, produced an immune suppressive TME and weakened ICI efficiency. Col1 receptor DDR1 could be a potential target synergically used with ICI to overcome ECM mediated ICI resistance. These provide a mechanical insight and novel strategy to overcome the ICI resistance of HCC.

Apelin ameliorates sepsis-induced myocardial dysfunction via inhibition of NLRP3-mediated pyroptosis of cardiomyocytes.

Sepsis-induced myocardial dysfunction (SMD) is the major cause of death in sepsis. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3)-mediated pyroptosis contributes to the occurrence and development of SMD. Although Apelin confers direct protection against SMD, the potential mechanisms remain unclear. This study aimed to determine whether Apelin protects against SMD via regulation of NLRP3-mediated pyroptosis of cardiomyocytes. Experimental SMD was induced in wild-type (WT) control mice and Apelin knockout (Apelin-/-) mice by cecal ligation and puncture (CLP). Neonatal mouse cardiomyocytes (NMCs) were treated with lipopolysaccharide (LPS) to simulate the physiological environment of SMD in vitro. The expression of Apelin was greatly decreased in the plasma from septic patients and septic mouse heart. Knockout of Apelin aggravated SMD, evidenced by decreased cardiac function, and increased cardiac fibrosis and NLRP3 inflammasome and pyroptosis levels in CLP-treated Apelin-/- mice compared with WT mice. Overexpression of Apelin activated the AMPK pathway and thereby inhibited NLRP3 inflammasome-mediated pyroptosis of NMCs induced by LPS in vitro These protective effects were partially abrogated by AMPK inhibitor. In conclusion, Apelin attenuated SMD by inhibiting NLRP3-mediated pyroptosis via activation of the AMPK pathway. Apelin may serve as a promising therapeutic target for SMD.

Evaluating short-term effects of rice straw management on carbon fractions, composition and stability of soil aggregates in an acidic red soil with a vegetable planting history.

Red soils are characterised by acidic pH and limitations in carbon, nitrogen, water, and soil structure. To overcome such limitations, improved soil aggregation is the key to improving the physical and chemical properties of soil. Applying organic amendments such as straw can lead to corresponding soil aggregation and stability changes. Therefore, we explored the short-term effects of rice straw amendment, either alone or in combination with biochar, on improving the carbon fractions, stability, and composition of soil aggregates in red soil with a history of vegetable planting. The study consisted of four treatments: control (no organic material, CK), biochar alone (5% homemade straw biochar, B), straw alone (12% rice straw, S), and biochar with straw (5% homemade straw biochar + 12% rice straw, BS). Our results showed that equal amounts of straw and biochar substantially reduced the number of mechanically stable aggregates (MSA), mean weight diameter (MWD), and geometric mean diameter (GMD) of the soil. BS treatment reduced >0.25 mm aggregate content (R0.25), MWD and GMD by 24.06%, 56.81%, and 62.19%, respectively, compared with that of the control. The addition of straw greatly enhanced the water-stable macromolecular content and stability coefficient of the soil, but treatment B had no obvious effect. The S treatment had the greatest effect on R0.25, MWD and GMD, increasing them by 143.94%, 246.67%, and 181.82%, respectively, compared with that of the control. Soil organic carbon (SOC) was significantly increased by straw addition and carbonisation treatment, and the effect of the BS treatment was the best, with an increase of 325.63% compared with that of the control. The organic carbon content in the aggregates of different particle sizes treated with different organic materials also increased significantly. In the soil reactive organic carbon fraction, applying biochar alone did not affect microbial biomass carbon (MBC), dissolved organic carbon (DOC), or easily oxidized organic carbon (EOC) but could increase the particulate organic carbon (POC) content. All the treatments with straw application significantly increased the MBC, DOC, EOC, and POC content, and the highest effect was obtained by applying both straw and biochar in an integrated form, i.e., the BS treatment. In conclusion, the co-application of biochar and straw sequestered more carbon and revamped soil C pools than either biochar or straw alone and could be a promising option for the sustainable use of red soils to ameliorate the aforementioned limitations associated with this soil type.

[Rapid determination of 87 prohibited ingredients in cosmetics by ultra performance liquid chromatography-tandem mass spectrometry].

The methods of detecting numerous prohibited components are not included in the Technical Specifications for Cosmetic Safety (2015 Edition). Recently, owing to its high speed, sensitivity, and anti-interference properties, ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) became the preferred method of detecting banned substances in cosmetics. In this study, a UPLC-MS/MS method was developed for use in determining 87 prohibited ingredients in cosmetics, including 33 sex hormones, 20 anti-infective drugs, 15 antihistamines, 7 coumarins, 4 sedative-hypnotic drugs, 4 antipyretic and analgesic drugs, 2 allergenic fragrances, and 2 drugs with vasoconstriction effects. The main factors affecting the response, recovery, and sensitivity of the method, such as the type of extraction solvent, extraction time, ratio of the mobile phases, and MS conditions, were optimized during sample pretreatment and instrumental analysis. Accordingly, approximately 0.2 g of the toner or cream sample was dispersed in 2 mL acetonitrile in a 10 mL colorimetric tube. After diluting to 10 mL with 50% acetonitrile aqueous solution, the sample was ultrasonically extracted for 20 min and centrifuged, and the mixture was then filtered through a 0.22 µm membrane. Approximately 0.2 g of the oil sample was dispersed in 2 mL n-hexane in a 15 mL polypropylene centrifuge tube and extracted twice with 3 mL 70% acetonitrile aqueous solution. The extracts were transferred into a 10 mL colorimetric tube and diluted to 10 mL with 50% acetonitrile aqueous solution, and the mixture was then filtered through a 0.22 µm membrane. The samples were separated using a CORTECS C18 column (150 mm×2.1 mm, 2.7 µm), employing a gradient elution program with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The flow rate, column temperature, and injection volume were respectively set at 0.3 mL/min, 40 ℃, and 2 µL. The 87 compounds were monitored in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) under positive and negative conditions. Matrix-matched external standard calibration was used for quantification, and the analysis was completed within 33 min. The prohibited compounds exhibited good linear relationships, with r values of >0.99, and the limits of detection (LODs) and quantification (LOQs) for the 87 compounds were 0.07-0.38 and 0.21-1.15 µg/g, respectively. Three types of cosmetic matrices were selected to verify the recovery and precision of the method at LOQ, 2 LOQ, and 10 LOQ levels. The average recoveries of the 87 prohibited compounds were in the range of 81.7%-115.4%, and the relative standard deviations (RSDs, n=6) were 0.4%-9.9%. The reliability of the developed method was demonstrated by applying it to 349 commercial cosmetics obtained from the market, and 8 positive samples were identified. The positive components included trimethoprim, terbinafine, naphazoline, 7-methoxycoumarin, and 7-methylcoumarin. The established method displays the advantages of simple operation and rapidness and a high sensitivity and good recovery. And, this method provides technical support for rapid risk screening and the revision of national standards for cosmetics.

Allocholic acid protects against α-naphthylisothiocyanate-induced cholestasis in mice by ameliorating disordered bile acid homeostasis.

Cholestasis is a pathological condition characterized by disruptions in bile flow, leading to the accumulation of bile acids (BAs) in hepatocytes. Allocholic acid (ACA), a unique fetal BA known for its potent choleretic effects, reappears during liver regeneration and carcinogenesis. In this research, we investigated the protective effects and underlying mechanisms of ACA against mice with cholestasis brought on by α-naphthylisothiocyanate (ANIT). To achieve this, we combined network pharmacology, targeted BA metabolomics, and molecular biology approaches. The results demonstrated that ACA treatment effectively reduced levels of serum AST, ALP, and DBIL, and ameliorated the pathological injury caused by cholestasis. Network pharmacology analysis suggested that ACA primarily regulated BA and salt transport, along with the signaling pathway associated with bile secretion, to improve cholestasis. Subsequently, we examined changes in BA metabolism using UPLC-MS/MS. The findings indicated that ACA pretreatment induced alterations in the size, distribution, and composition of the liver BA pool. Specifically, it reduced the excessive accumulation of BAs, especially cholic acid (CA), taurocholic acid (TCA), and ß-muricholic acid (ß-MCA), facilitating the restoration of BA homeostasis. Furthermore, ACA pretreatment significantly downregulated the expression of hepatic BA synthase Cyp8b1, while enhancing the expression of hepatic efflux transporter Mrp4, as well as the renal efflux transporters Mdr1 and Mrp2. These changes collectively contributed to improved BA efflux from the liver and enhanced renal elimination of BAs. In conclusion, ACA demonstrated its potential to ameliorate ANIT-induced liver damage by inhibiting BA synthesis and promoting both BA efflux and renal elimination pathways, thus, restoring BA homeostasis.

Highly-efficient selection of aptamers for detecting various HPV subtypes in clinical samples.

Nucleic acid aptamers are of great potentials in diagnostic and therapeutic applications because of their unique molecular recognition capabilities. However, satisfactory aptamers with high affinity and specificity are still in short supply. Herein, we have developed new selection methods allowing the free interactions between the targets and potential aptamers in solution. In our selection system, the protein targets (biotinylated randomly or site-specifically) were first incubated with the random DNA library, followed by the pull-down with the streptavidin magnetic beads or biolayer-interferometry (BLI) sensors. By comparing the two biotinylation strategies (random or site-specific) and two states of the targets (free or immobilized), we have found that the combination of the site-specific biotinylation and free-target strategies was most successful. Based on these highly-efficient selection strategies, HPV L1 aptamers were obtained. By designing the sandwich aptasensor assisted with RCA and CRISPR/Cas12a, we have diagnosed various HPV subtypes in clinical samples, such as easily-collected urine samples. In summary, our new strategy can allow efficient selection of aptamers with high affinity and specificity for clinical applications.

Rotundic acid improves nonalcoholic steatohepatitis in mice by regulating glycolysis and the TLR4/AP1 signaling pathway.

BACKGROUND: Steatosis and inflammation are the hallmarks of nonalcoholic steatohepatitis (NASH). Rotundic acid (RA) is among the key triterpenes of Ilicis Rotundae Cortex and has exhibited multipronged effects in terms of lowering the lipid content and alleviating inflammation. The study objective is to systematically evaluate the potential mechanisms through which RA affects the development and progression of NASH. METHODS: Transcriptomic and proteomic analyses of primary hepatocytes isolated from the control, high-fat diet-induced NASH, and RA treatment groups were performed through Gene Ontology analysis and pathway enrichment. Hub genes were identified through network analysis. Integrative analysis revealed key RA-regulated pathways, which were verified by gene and protein expression studies and cell assays. RESULTS: Hub genes were identified and enriched in the Toll-like receptor 4 (TLR4)/activator protein-1 (AP1) signaling pathway and glycolysis pathway. RA reversed glycolysis and attenuated the TLR4/AP1 pathway, thereby reducing lipid accumulation and inflammation. Additionally, lactate release in L-02 cells increased with NaAsO2-treated and significantly decreased with RA treatment, thus revealing that RA had a major impact on glycolysis. CONCLUSIONS: RA is effective in lowering the lipid content and reducing inflammation in mice with NASH by ameliorating glycolysis and TLR4/AP1 pathways, which contributes to the existing knowledge and potentially sheds light on the development of therapeutic interventions for patients with NASH.

Structural Insight into Polymerase Mechanism via a Chiral Center Generated with a Single Selenium Atom.

DNA synthesis catalyzed by DNA polymerase is essential for all life forms, and phosphodiester bond formation with phosphorus center inversion is a key step in this process. Herein, by using a single-selenium-atom-modified dNTP probe, we report a novel strategy to visualize the reaction stereochemistry and catalysis. We capture the before- and after-reaction states and provide explicit evidence of the center inversion and in-line attacking SN2 mechanism of DNA polymerization, while solving the diastereomer absolute configurations. Further, our kinetic and thermodynamic studies demonstrate that in the presence of Mg2+ ions (or Mn2+), the binding affinity (Km) and reaction selectivity (kcat/Km) of dGTPαSe-Rp were 51.1-fold (or 19.5-fold) stronger and 21.8-fold (or 11.3-fold) higher than those of dGTPαSe-Sp, respectively, indicating that the diastereomeric Se-Sp atom was quite disruptive of the binding and catalysis. Our findings reveal that the third metal ion is much more critical than the other two metal ions in both substrate recognition and bond formation, providing insights into how to better design the polymerase inhibitors and discover the therapeutics.

Trial of the Pluslife SARS-CoV-2 Nucleic Acid Rapid Test Kit: Prospective Cohort Study.

BACKGROUND: In response to the SARS-CoV-2 epidemic, a convenient, rapid, and sensitive diagnostic method for detecting COVID-19 is crucial for patient control and timely treatment. OBJECTIVE: This study aimed to validate the detection of SARS-CoV-2 with the Pluslife SARS-CoV-2 rapid test kit developed based on a novel thermostatic amplification technique called RNase hybridization-assisted amplification. METHODS: From November 25 to December 8, 2022, patients with suspected or confirmed COVID-19, close contacts, and health care workers at high risk of exposure were recruited from 3 hospitals and 1 university. Respiratory specimens were collected for testing with the Pluslife SARS-CoV-2 rapid test kit and compared with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a commercial antigen assay kit. Samples from 1447 cases were obtained from 3 "ready-to-test" scenarios in which samples were collected on site and tested immediately, and samples from 503 cases were obtained from a "freeze-thaw test" scenario in which samples were collected, frozen, and thawed for testing. RESULTS: Pluslife SARS-CoV-2 rapid testing of samples from the "ready-to-test" scenario was found to be accurate (overall sensitivity and specificity of 98.3% and 99.3%, respectively) and diagnostically useful (positive and negative likelihood ratios of 145.45 and 0.02, respectively). Pluslife SARS-CoV-2 rapid testing of samples from the "freeze-thaw test" scenario was also found to be accurate (overall sensitivity and specificity of 71.2% and 98.6%, respectively) and diagnostically useful (positive and negative likelihood ratios of 51.01 and 0.67, respectively). Our findings demonstrated that the time efficiency and accuracy of the results in a "ready-to-test" scenario were better. The time required from sample preparation to the seeing the result of the Pluslife SARS-CoV-2 rapid test was 10 to 38 minutes, which was substantially shorter than that of RT-qPCR (at least 90 minutes). In addition, the diagnostic efficacy of the Pluslife SARS-CoV-2 rapid test was better than that of a commercial antigen assay kit. CONCLUSIONS: The developed RNase hybridization-assisted amplification assay provided rapid, sensitive, and convenient detection of SARS-CoV-2 infection and may be useful for enhanced detection of COVID-19 in homes, high-risk industries, and hospitals.

Downregulation of ALKBH5 rejuvenates aged human mesenchymal stem cells and enhances their therapeutic efficacy in myocardial infarction.

Despite promising results in myocardial infarction (MI), mesenchymal stem cell (MSC)-based therapy is limited by cell senescence. N6-methyladenosine (m6A) messenger RNA methylation has been reported to be closely associated with cell senescence. Nonetheless, its role in the regulation of MSC senescence remains unclear. We examined the role of ALKB homolog 5 (ALKBH5) in regulating MSC senescence and determined whether ALKBH5 downregulation could rejuvenate aged MSCs (AMSCs) to improve their therapeutic efficacy for MI. RNA methylation was determined by m6A dot blotting assay. MSC senescence was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. A mouse model of acute MI was established by ligation of the left anterior decedent coronary artery (LAD). Compared with young MSCs (YMSCs), m6A level was significantly reduced but ALKBH5 was greatly increased in AMSCs. Overexpression of ALKBH5 reduced m6A modification and accelerated YMSC senescence. Conversely, ALKBH5 knockdown increased m6A modifications and alleviated AMSC senescence. Mechanistically, ALKBH5 regulated the m6A modification and stability of CDKN1C mRNA, which further upregulated CDKN1C expression, leading to MSC senescence. CDKN1C overexpression ameliorated the inhibition of cellular senescence of ALKBH5 siRNA-treated AMSCs. More importantly, compared with AMSCs, shALKBH5-AMSCs transplantation provided a superior cardioprotective effect against MI in mice by improving MSC survival and angiogenesis. We determined that ALKBH5 accelerated MSC senescence through m6A modification-dependent stabilization of the CDKN1C transcript, providing a potential target for MSC rejuvenation. ALKBH5 knockdown rejuvenated AMSCs and enhanced cardiac function when transplanted into the mouse heart following infarction.

TRAF3IP2 drives mesenchymal stem cell senescence via regulation of NAMPT-mediated NAD biosynthesis.

The cellular senescence of mesenchymal stem cells (MSCs) limits their application in regenerative medicine. This study aimed to clarify the role of TNF receptor-associated factor 3 interacting protein 2 (TRAF3IP2), a pro-inflammatory cytoplasmic adaptor protein, in regulating MSC senescence and to explore the potential mechanisms. Methods: MSC senescence was determined by senescence-associated ß-galactosidase (SA-ß-gal) staining. The expression of TRAF3IP2 and senescence-related proteins was detected by Western blotting. The nicotinamide adenine dinucleotide (NAD+) level and nicotinamide phosphoribosyl transferase (NAMPT) expression in MSCs was measured. Results: Compared with that in MSCs isolated from young donors (YMSCs), the expression of TRAF3IP2 was greatly increased in MSCs derived from aged donors (AMSCs). Overexpression of TRAF3IP2 accelerated YMSC senescence whereas downregulation significantly rescued cellular senescence. The protein level of NAMPT and the level of NAD+ were significantly decreased in AMSCs compared with YMSCs. Mechanistically, TRAF3IP2 induced MSC senescence via downregulation of NAMPT expression and NAD + level by inhibiting the AMPK signaling pathway. These effects were partially reversed by treatment with an AMPK or NAMPT activator. Conclusion: We revealed that TRAF3IP2 accelerated MSC senescence via downregulation of NAMPT-mediated NAD biosynthesis by mediation of the AMPK pathway, highlighting a novel means to rejuvenate senescent MSCs.

Genetic Predictors of Ibrutinib-related Cardiovascular Side Effects in Patients with Chronic Lymphocytic Leukemia.

PURPOSE: Patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib are at risk of developing cardiovascular side effects (CVSE). The molecular determinants of CVSEs have not been fully elucidated. We interrogated genetic polymorphisms in the Bruton tyrosine kinase (BTK) signaling pathway for their association with ibrutinib-related CVSEs. EXPERIMENTAL DESIGN: We conducted a retrospective/prospective observational pharmacogenetic study of 50 patients with newly diagnosed or relapsed CLL who received ibrutinib at a starting daily dose of 420 mg for at least 6 months. CVSEs, primarily atrial fibrillation and hypertension, occurred in 10 patients (20%), of whom 4 discontinued therapy. DNA was isolated from buccal swabs of all 50 patients and genotyped for 40 SNPs in GATA4, SGK1, KCNQ1, KCNA4, NPPA, and SCN5A using a customized next-generation sequencing panel. Univariate and multivariate logistic regression analysis were performed to determine genetic and clinical factors associated with the incidence of ibrutinib-related CVSEs. RESULTS: GATA4 rs804280 AA (P = 0.043), KCNQ1 rs163182 GG (P = 0.036), and KCNQ1 rs2237895 AA (P = 0.023) genotypes were univariately associated with ibrutinib-related CVSEs. On the basis of multivariate analysis, a high genetic risk score, defined as the presence of at least two of these genotypes, was associated with 11.5-fold increased odds of CVSEs (P = 0.019; 95% confidence interval, 1.79-119.73). CONCLUSIONS: Our findings suggest possible genetic determinants of ibrutinib-related CVSEs in CLL. If replicated in a larger study, pretreatment pharmacogenetic testing for GATA4 and KCNQ1 polymorphisms may be a useful clinical tool for personalizing treatment selection for CLL and/or instituting early risk mitigation strategies.

Safety and efficacy of zanubrutinib in relapsed/refractory marginal zone lymphoma: final analysis of the MAGNOLIA study.

The primary analysis of MAGNOLIA, an open-label, single-arm, multicenter, phase 2 study, demonstrated that the next-generation Bruton tyrosine kinase (BTK) inhibitor zanubrutinib provided a high overall response rate (ORR) in patients with relapsed/refractory marginal zone lymphoma (R/R MZL), with a favorable safety/tolerability profile. Presented here, is the final analysis of MAGNOLIA, performed to characterize the durability of response and longer-term safety and tolerability. Zanubrutinib (160 mg twice daily) was evaluated in 68 patients with R/R MZL who had received at least 1 anti-CD20-directed regimen. The primary end point was independent review committee (IRC)-assessed ORR. Secondary end points included investigator-assessed ORR, duration of response (DOR), progression-free survival (PFS), overall survival (OS), health-related quality of life, safety, and tolerability. With a median follow-up of 27.4 months, the IRC-assessed ORR was 68.2% (95% confidence interval [CI], 55.6-79.1), with a 24-month DOR event-free rate of 72.9% (95% CI, 54.4-84.9). PFS and OS at 24 months were 70.9% (95% CI, 57.2-81.0) and 85.9% (95% CI, 74.7-92.4), respectively. The zanubrutinib safety profile was consistent with the primary analysis, with no new safety signals observed. Atrial fibrillation/flutter (n = 2 [2.9%]) and hypertension (n = 3 [4.4%]) were uncommon. Neutropenia (n = 8 [11.8%]) was the most common grade ≥3 adverse event. In this final analysis of MAGNOLIA, zanubrutinib demonstrated sustained clinical responses beyond 2 years, with 73% of responders alive and progression free. Zanubrutinib continued to demonstrate a favorable safety/tolerability profile with the additional time on treatment. This trial was registered at www.clinicaltrials.gov as #NCT03846427.

Single-cell mapping reveals several immune subsets associated with liver metastasis of pancreatic ductal adenocarcinoma.

BACKGROUND: Identifying a metastasis-correlated immune cell composition within the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) will help to develop promising and innovative therapeutic strategies. However, the dynamics of immune cell lineages in the TME of advanced PDAC remains elusive. METHODS: Twenty-six samples from 11 patients (including 11 primary tumor tissues, 10 blood, and 5 lymph nodes) with different stages were used to develop a multiscale immune profile. High-dimensional single-cell analysis with mass cytometry was performed to search for metastasis-correlated immune changes in the microenvironment. The findings were further validated by published single-cell RNA sequencing (scRNA-seq) data and multiplex fluorescent immunohistochemistry. FINDINGS: High-dimensional single-cell profiling revealed that the three immune-relevant sites formed a distinct immune atlas. Interestingly, the PDAC microenvironment with the potential for metastatic spread to the liver was characterized by a decreased proportion of CD103+PD-1+CD39+ T cells with cytotoxic and exhausted functional status and an increased proportion of CD73+ macrophages. Analysis of scRNA-seq data of PDAC further confirmed the identified subsets and revealed strong potential interactions via various ligand-receptor pairs between the identified T subsets and the macrophages. Moreover, stratified patients with different immune compositions correlated with clinical outcomes of PDAC. CONCLUSIONS: Our study uncovered metastasis-correlated immune changes, suggesting that ecosystem-based patient classification in PDAC will facilitate the identification of candidates likely to benefit from immunotherapy. FUNDING: This work was supported by the National Key Research and Development Program of China, the Shanghai International Science and Technology Collaboration Program, the Shanghai Sailing Program, and the Key Laboratory of diagnosis and treatment of severe hepato-pancreatic diseases of Zhejiang Province.