Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary Electrophoresis-Mass Spectrometry.

Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed "spray-capillary", an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL-nL level). Here, we developed a spray-capillary-CE-MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE-MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE-MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE-MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.

Light Supplementation in Pitaya Orchards Induces Pitaya Flowering in Winter by Promoting Phytohormone Biosynthesis.

The interaction between light and phytohormones is crucial for plant growth and development. The practice of supplementing light at night during winter to promote pitaya flowering and thereby enhance yield has been shown to be crucial and widely used. However, it remains unclear how supplemental winter light regulates phytohormone levels to promote flowering in pitaya. In this study, through analyzing the transcriptome data of pitaya at four different stages (NL, L0, L1, L2), we observed that differentially expressed genes (DEGs) were mainly enriched in the phytohormone biosynthesis pathway. We further analyzed the data and found that cytokinin (CK) content first increased at the L0 stage and then decreased at the L1 and L2 stages after supplemental light treatment compared to the control (NL). Gibberellin (GA), auxin (IAA), salicylic acid (SA), and jasmonic acid (JA) content increased during the formation of flower buds (L1, L2 stages). In addition, the levels of GA, ethylene (ETH), IAA, and abscisic acid (ABA) increased in flower buds after one week of development (L2f). Our results suggest that winter nighttime supplemental light can interact with endogenous hormone signaling in pitaya, particularly CK, to regulate flower bud formation. These results contribute to a better understanding of the mechanism of phytohormone interactions during the induction of flowering in pitaya under supplemental light in winter.

Immunogenicity and safety of an ORF7-deficient skin-attenuated and neuro-attenuated live vaccine for varicella: a randomised, double-blind, controlled, phase 2a trial.

BACKGROUND: The Oka varicella vaccine strain remains neurovirulent and can establish lifelong latent infection, raising safety concerns about vaccine-related herpes zoster. In this study, we aimed to evaluate the immunogenicity and safety of a skin-attenuated and neuro-attenuated varicella vaccine candidate (v7D vaccine). METHODS: We did this randomised, double-blind, controlled, phase 2a clinical trial in Jiangsu, China. Healthy children aged 3-12 years with no history of varicella infection or vaccination were enrolled and randomly assigned (1:1:1:1) to receive a single subcutaneous injection of the v7D vaccine at 3·3 log10 plaque forming units (PFU; low-dose v7D group), 3·9 log10 PFU (medium-dose v7D group), and 4·2 log10 PFU (high-dose v7D group), or the positive control varicella vaccine (vOka vaccine group). All the participants, laboratory personnel, and investigators other than the vaccine preparation and management staff were masked to the vaccine allocation. The primary outcome was assessment of the geometric mean titres (GMTs) and seroconversion rates of anti-varicella zoster virus immunoglobulin G (IgG) induced by different dose groups of v7D vaccine at 0, 42, 60, and 90 days after vaccination in the per-protocol set for humoral immune response analysis. Safety was a secondary outcome, focusing on adverse events within 42 days post-vaccination, and serious adverse events within 6 months after vaccination. This study was registered on Chinese Clinical Trial Registry, ChiCTR2000034434. FINDINGS: On Aug 18-21, 2020, 842 eligible volunteers were enrolled and randomly assigned treatment. After three participants withdrew, 839 received a low dose (n=211), middle dose (n=210), or high dose (n=210) of v7D vaccine, or the vOka vaccine (n=208). In the per-protocol set for humoral immune response analysis, the anti-varicella zoster virus IgG antibody response was highest at day 90. At day 90, the seroconversion rates of the low-dose, medium-dose, and high-dose groups of v7D vaccine and the positive control vOka vaccine group were 100·0% (95% CI 95·8-100·0; 87 of 87 participants), 98·9% (93·8-100·0; 87 of 88 participants), 97·8% (92·4-99·7; 91 of 93 participants), and 96·4% (89·8-99·2; 80 of 83 participants), respectively; the GMTs corresponded to values of 30·8 (95% CI 26·2-36·0), 31·3 (26·7-36·6), 28·2 (23·9-33·2), and 38·5 (31·7-46·7). The v7D vaccine, at low dose and medium dose, elicited a humoral immune response similar to that of the vOka vaccine. However, the high-dose v7D vaccine induced a marginally lower GMT compared with the vOka vaccine at day 90 (p=0·027). In the per-protocol set, the three dose groups of the v7D vaccine induced a similar humoral immune response at each timepoint, with no statistically significant differences. The incidence of adverse reactions in the low-dose, medium-dose, and high-dose groups of v7D vaccine was significantly lower than that in the vOka vaccine group (17% [35 of 211 participants], 20% [41 of 210 participants], and 13% [27 of 210 participants] vs 24% [50 of 208 participants], respectively; p=0·025), especially local adverse reactions (10% [22 of 211 participants], 14% [30 of 210 participants] and 9% [18 of 210 participants] vs 18% [38 of 208 participants], respectively; p=0·016). None of the serious adverse events were vaccine related. INTERPRETATION: The three dose groups of the candidate v7D vaccine exhibit similar humoral immunogenicity to the vOka vaccine and are well tolerated. These findings encourage further investigations on two-dose vaccination schedules, efficacy, and the potential safety benefit of v7D vaccine in the future. FUNDING: The National Natural Science Foundation of China, CAMS Innovation Fund for Medical Sciences, the Fundamental Research Funds for the Central Universities, and Beijing Wantai. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Untargeted metabolomics in Anectocillus roxburghii with habitat heterogeneity and the key abiotic factors affecting its active ingredients.

Introduction: Anoectochilus roxburghii is a rare, endangered herb with diverse pharmacological properties. Understanding the main metabolite types and characteristics of wild A. roxburghii is important for efficiently utilizing resources and examining quality according to origin. Methods: Samples were collected from the main production areas across five regions in Fujian Province, China. An untargeted metabolomics analysis was performed on the entire plants to explore their metabolic profiles. We utilized UPLC-MS/MS to specifically quantify eight targeted flavonoids in these samples. Subsequently, correlation analysis was conducted to investigate the relationships between the flavonoids content and both the biological characteristics and geographical features. Results: A comprehensive analysis identified a total of 3,170 differential metabolites, with terpenoids and flavonoids being the most prevalent classes. A region-specific metabolite analysis revealed that the Yongchun (YC) region showed the highest diversity of unique metabolites, including tangeretin and oleanolic acid. Conversely, the Youxi (YX) region was found to have the smallest number of unique metabolites, with only one distinct compound identified. Further investigation through KEGG pathway enrichment analysis highlighted a significant enrichment in pathways related to flavonoid biosynthesis. Further examination of the flavonoid category showed that flavonols were the most differentially abundant. We quantified eight specific flavonoids, finding that, on average, the YX region exhibited higher levels of these compounds. Correlation analysis highlighted a significant association between flavonoids and habitat, especially temperature and humidity. Discussion: Untargeted metabolomics via LC-MS was suitable for identifying region-specific metabolites and their influence via habitat heterogeneity. The results of this study serve as a new theoretical reference for unique markers exclusively present in a specific sample group.

Functional characterization of terpene synthases SmTPS1 involved in floral scent formation in Salvia miltiorrhiza.

Plants attract beneficial insects and promote pollination by releasing floral scents. Salvia miltiorrhiza, as an insect-pollinated flowering plant, which has been less studied for its floral aroma substances. This study revealed that S. miltiorrhiza flowers produce various volatile terpenoids, including five monoterpenes and ten sesquiterpenes, with the sesquiterpene compound (E)-ß-caryophyllene being the most abundant, accounting for 28.1% of the total volatile terpenoids. Y-tube olfactometer experiments were conducted on the primary pollinator of S. miltiorrhiza, the Apis ceranas. The results indicated that (E)-ß-caryophyllene compound had an attractive effect on the Apis ceranas. By comparing the homologous sequences with the genes of (E)-ß-caryophyllene terpene synthases in other plants, the SmTPS1 gene was selected for further experiment. Subcellular localization experiments showed SmTPS1 localized in the cytoplasm, and its in vitro enzyme assay revealed that it could catalyze FPP into ß-Elemene, (E)-ß-caryophyllene and α-Humulene. Overexpression of SmTPS1 in S. miltiorrhiza resulted in a 5.29-fold increase in gene expression. The GC-MS analysis revealed a significant increase in the concentration of (E)-ß-caryophyllene in the transgenic plants, with levels 2.47-fold higher compared to the empty vector plants. Furthermore, Y-tube olfactometer experiments showed that the transgenic plants were significantly more attractive to Apis ceranas compared to the empty vector plants. Co-expression analysis suggested that four SmMYCs (SmMYC1, SmMYC5, SmMYC10, and SmMYC11) may be involved in the transcriptional regulation of SmTPS1. The yeast one-hybrid screen and the Dual luciferase assay indicated that SmMYC10 positively regulates the expression of SmTPS1. In conclusion, this study lays a foundation for the functional analysis and transcriptional regulation of terpene synthase genes in S. miltiorrhiza.

Aloperine Alleviates Myocardial Injury Induced by Myocardial Ischemia and Reperfusion by Activating the ERK1/2/ß-catenin Signaling Pathway.

OBJECTIVE: Myocardial ischemia/reperfusion (I/R) injury can cause severe cardiac damage. Aloperine is a quinolizidine alkaloid found in the leaves and seeds of Sophora alopecuroides L. It has been recognized that aloperine has organ-protective properties; however, its role in cardioprotection is poorly characterized. This study aimed to evaluate the cardioprotective effects of aloperine against myocardial I/R injury in vivo. METHODS: Adult male Sprague‒Dawley rats were randomly divided into sham-operated, control, and aloperine groups. All rats except for the sham-operated rats were subjected to 45 min of myocardial ischemia (by left anterior descending ligation) followed by 3 h of reperfusion. Aloperine (10 mg/kg) was given intravenously at the onset of reperfusion. The cardioprotective effects of aloperine were evaluated by determining infarct size, hemodynamics, histological changes, cardiac biomarkers, and cardiac apoptosis. RESULTS: Aloperine limited infarct size; improved hemodynamics; attenuated myocardial I/R-induced histological deterioration; decreased serum LDH, CK-MB, and α-HBDH levels; and inhibited apoptosis after myocardial I/R injury. Moreover, aloperine stimulated the phosphorylation of ventricular ERK1/2, which is a major module of MAPK signaling pathways. Furthermore, aloperine increased the ventricular expression levels of ß-catenin. Pharmacological inhibition of ERK1/2 diminished aloperine-induced cardioprotection and blocked ERK1/2/ß-catenin signaling. CONCLUSIONS: These data support the cardioprotective effect of aloperine against myocardial I/R injury, which is mediated, at least in part, by the ERK1/2/ß-catenin signaling pathway.

A densely connected causal convolutional network separating past and future data for filling missing PM2.5 time series data.

Air pollution poses a significant threat to human health and the environment globally. Precise analysis and prediction of pollutant concentrations are essential for monitoring and managing air quality. However, reliable analysis and prediction require comprehensive and high-quality data, which is often compromised due to missing data during collection. Unfortunately, conventional methods for addressing missing data fall short of providing adequate solutions. The missing data for air quality indicators are commonly systematic, with all data points missing for extended periods. This makes it difficult to establish correlations and populate the missing data accurately. To address this problem, we propose a Densely Connected Causal Convolutional Network Separating Past and Future Data (DCCN-SPF), a deep learning-based model that fills in continuous missing PM2.5 concentration data in the original dataset. It extracts features from past and future data separately using densely connected causal convolutional networks and incorporates linear interpolation and deep learning structures to improve prediction accuracy. Using air quality monitoring data from the China Environmental Monitoring Station between 2017 and 2021 in Beijing, we compare our proposed model with baseline models and find that our model outperforms others in predicting PM2.5 concentrations. The evaluation metrics MAE and RMSE are used, revealing significant reductions of 8.7-21.6 % for MAE and 7.1-23.5 % for RMSE in favor of our proposed DCCN-SPF model.

Automatic Quaternion-Domain Color Image Stitching.

Taking advantages of the quaternion representation of the color image, this paper proposes a quaternion perceptual seamline detection model to generate the seamline in the quaternion domain. It considers seamline detection as a quaternion-domain color image labeling problem and minimizes the local-area quaternion perceptual difference cost to obtain the optimal seamline. To assess seamline quality effectively, we develop a quaternion perceptual seamline quality measure. Based on the proposed quaternion perceptual seamline detection model and quality measure, we further propose a general framework for automatic quaternion-domain color image stitching (AQCIS). To the best of our knowledge, this is the first attempt to perform color image stitching completely in the quaternion domain. Meanwhile, AQCIS introduces the joint optimization strategy of local alignment and seamline in an iterative fashion. Extensive experiments on challenging datasets demonstrate that our AQCIS achieves superior performance for color image stitching in comparison with state-of-the-art methods.

Canagliflozin Pretreatment Attenuates Myocardial Dysfunction and Improves Postcardiac Arrest Outcomes After Cardiac Arrest and Cardiopulmonary Resuscitation in Mice.

OBJECTIVE: The SGLT2 inhibitor, canagliflozin, not only reduces glycemia in patients with type 2 diabetes but also exerts cardioprotective effects in individuals without diabetes. However, its potential beneficial effects in cardiac arrest have not been characterized. The purpose of this study was to examine the protective effect of canagliflozin pretreatment on postresuscitation-induced cardiac dysfunction in vivo. METHODS: Male C57/BL6 mice were randomized to vehicle (sham and control) or canagliflozin treatment groups. All mice except for the sham-operated mice were subjected to potassium chloride-induced cardiac arrest followed by chest compressions and intravenous epinephrine for resuscitation. Canagliflozin therapy efficacies were evaluated by electrocardiogram, echocardiography, histological analysis, inflammatory response, serum markers of myocardial injury, protein phosphorylation analysis, and immunohistological assessment. RESULTS: Canagliflozin-pretreated mice exhibited a higher survival rate (P < 0.05), a shorter return of spontaneous circulation (ROSC) time (P < 0.01) and a higher neurological score (P < 0.01 or P < 0.001) than control mice after resuscitation. Canagliflozin was effective at improving cardiac arrest and resuscitation-associated cardiac dysfunction, indicated by increased left ventricular ejection fraction and fractional shortening (P < 0.001). Canagliflozin reduced serum levels of LDH, CK-MB and α-HBDH, ameliorated systemic inflammatory response, and diminished the incidence of early resuscitation-induced arrhythmia. Notably, canagliflozin promoted phosphorylation of cardiac STAT-3 postresuscitation. Furthermore, pharmacological inhibition of STAT-3 by Ag490 blunted STAT-3 phosphorylation and abolished the cardioprotective actions of canagliflozin. CONCLUSIONS: Canagliflozin offered a strong cardioprotective effect against cardiac arrest and resuscitation-induced cardiac dysfunction. This canagliflozin-induced cardioprotection is mediated by the STAT-3-dependent cell-survival signaling pathway.

Active Reconfigurable Intelligent Surface Enhanced Internet of Medical Things.

The incredible potentiality of reconfigurable intelligent surface (RIS) in addressing power supply and obstacle environment of Internet of Medical Things (IoMT) has been capturing our interest. Considering the nettlesome "double-fading" effect introduced by passive RIS, we investigate an active RIS-enhanced IoMT system in this paper, where the wireless power transfer (WPT) from power station (PS) to IoMT devices and the wireless information transfer (WIT) from IoMT devices to the access point (AP) are both implemented with the assistance of active RIS. Aiming to maximize the sum throughput of the considered IoMT system, a joint design of time schedules and reflecting coefficient matrices of the active RIS is proposed. Trapped by the non-convex and obstinate optimization problem, we explore the semi-definite programming (SDP) relaxation and successive convex approximation (SCA) techniques based on alternating optimization (AO) algorithm. Simulation results verify our solution approach to the intractable optimization problem and showcase the boosted spectrum and energy efficiency of the active RIS-enhanced IoMT system.

Global metabolic profile and multiple phytometabolites in the different varieties of Gastrodia elata Blume.

Gastrodia elata Blume (Tianma in Chinese), a myco-heterotrophic orchid, is widely distributed in China. Tubers derived from this orchid are traditionally used as both medicinal and edible materials. At present, five primary varieties of G. elata are recorded in the "Flora of China." Among them, the three main varieties currently in artificial cultivation are G. elata f. elata (GR, red stem), G. elata f. glauca (GB, black stem), and G. elata f. viridis (GG, green stem). In our study, the metabolic profiles and chemical composition of these three varieties were determined via UPLC-MS/MS and HPLC-UV. In total, 11,132 metabolites were detected, from which multiple phytometabolites were identified as aromatic compounds, heteroatomic compounds, furans, carbohydrates, organic acids, and their derivatives. A number of differentially expressed metabolites (DEMs) were annotated as bioactive ingredients. Overall, parishins, vanilloloside, and gastrodin A/B in the GB group were markedly higher, whereas gastrodin, gastrol, and syringic acid were more enriched in the GG or GR groups. Moreover, HPLC fingerprint analysis also found six metabolites used as markers for the identification of Gastrodiae Rhizoma in the Chinese Pharmacopoeia, which were also typical DEMs in metabolomics. Of these, gastrodin, 4-hydroxybenzyl alcohol, citric acid, and adenosine were quantitatively detected, showing a similar result with the metabolomic data. In summary, our findings provide novel insights into the phytochemical ingredients of different G. elata varieties, highlighting diverse biological activities and healthcare value.

Pretreatment with remote ischemic conditioning attenuates testicular damage after testicular ischemia and reperfusion injury in rats.

Testicular torsion is a urological emergency. However, surgical detorsion of the torsed spermatic cord can cause testicular reperfusion injury. Although remote ischemic preconditioning (RIPC) has been convincingly shown to protect organs against ischemia/reperfusion (I/R) injury, little is known regarding the effect of RIPC on testicular torsion/detorsion-induced reperfusion injury. Therefore, we aimed to evaluate the effect of RIPC on testes after testicular I/R injury in a rat model in vivo. Male Sprague-Dawley rats were randomly classified into 4 groups: sham-operated (sham), testicular I/R (TI/R), or remote liver (RIPC liver) and limb (RIPC limb) ischemic preconditioning groups. Testis I/R was induced by 3 h of right spermatic cord torsion (720° clockwise), and reperfusion was allowed for 3 hours. In the RIPC group, four cycles of 5 min of ischemia and 5 min of reperfusion were completed 30 min prior to testicular torsion. The ERK1/2 inhibitor U0126 was administered intravenously at the beginning of reperfusion (1 mg/kg). The testes were taken for the oxidative stress evaluations, histology, apoptosis, immunohistochemical and western blotting analysis. Remote liver and limb ischemic preconditioning attenuated ipsilateral and contralateral testicular damage after testicular I/R injury. For example. RIPC reduced testicular swelling and oxidative stress, lessened structural damage, and inhibited the testicular inflammatory response and apoptosis. Furthermore, RIPC treatment enhanced testicular ERK1/2 phosphorylation postI/R. Inhibition of ERK1/2 activity using U0126 eliminated the protection offered by RIPC. Our data demonstrate for the first time that RIPC protects testes against testicular I/R injury via activation of the ERK1/2 signaling pathway.

IPSO-LSTM hybrid model for predicting online public opinion trends in emergencies.

When emergencies are widely discussed and shared, it may lead to conflicting opinions and negative emotions among internet users. Accurately predicting sudden network public opinion events is of great importance. Therefore, this paper constructs a hybrid forecasting model to solve this problem. First, this model introduces an improved inertia weight and an adaptive variation operation to enhance the Particle Swarm Optimization (PSO) algorithm. Then, the improved PSO (IPSO) algorithm optimizes the parameters of the Long Short-Term Memory (LSTM) neural network. Finally, the IPSO-LSTM hybrid prediction model is constructed to forecast and analyze emergency public opinion dissemination trends. The experimental outcomes indicate that the IPSO-LSTM model surpasses others and has high prediction accuracy. In the four emergency predictions we select, the MAPE value of IPSO-LSTM is 74.27% better than that of BP, 33.96% better than that of LSTM, and 13.59% better than that of PSO-LSTM on average. This study aims to assist authorities in quickly identifying potential public opinion crises, developing effective strategies, and promoting sustainable and positive growth in the network environment.

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.

Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

The plant-derived alkaloid aloperine prevents ischemia/reperfusion injury-induced sudden cardiac death.

Sudden cardiac death (SCD) remains a major cause of global mortality. In addition to modern interventions, botanical folk medicines have long been used to treat cardiovascular disease, although the efficacy and underlying mechanisms are often unresolved. Aloperine, a bioactive quinolizidine alkaloid isolated from Sophora alopecuroides plants, exhibits antioxidant, anti-inflammatory, antitumor, and vasorelaxant properties, but possible antiarrhythmic effects of aloperine in SCD are unclear. Here, we examined whether aloperine protects against ischemia and reperfusion injury-associated lethal ventricular arrhythmia and sudden cardiac death. Rats were divided into sham, control, and aloperine groups, and reperfusion-provoked ventricular arrhythmogenesis, cardiac damage markers, and signaling pathways quantified following left main coronary artery ischemia and reperfusion. In vitro studies of effects of aloperine on hERG and Kv4.3 cardiac voltage-gated potassium (Kv) channels were performed using two-electrode voltage clamp analysis of cloned channels expressed in Xenopus laevis oocytes. Aloperine pretreatment (10 mg/kg) did not affect baseline cardiac electrical stability; yet, it reduced ventricular arrhythmogenesis and susceptibility to SCD (mortality rate: control: 64.3%; aloperine: 0%) induced by reperfusion injury. Aloperine also reduced serum levels of LDH, CK-MB, α-HBDH, and cTnI post-I/R, and stimulated phosphorylation of ventricular ERK1/2 and STAT-3, which are key components of RISK and SAFE signaling pathways. Inhibition of either ERK1/2 (with U0126) or STAT-3 (with Ag490) abolished aloperine-induced anti-arrhythmic effects and ERK1/2 and STAT-3 phosphorylation. Interestingly, while aloperine (100 µM) had no effect on cloned Kv4.3 activity, aloperine (1 µM and up) negative-shifted the voltage dependence of hERG activation by ~10 mV and increased peak hERG current by 35%. Thus, aloperine exerts striking anti-arrhythmic effects against myocardial ischemia and reperfusion injury-induced severe lethal ventricular arrhythmia and sudden cardiac death via the ERK1/2/STAT-3 signaling pathway, with potential additional contribution from increased cardiac myocyte repolarization capacity via augmented hERG activity.

Remote limb ischemic postconditioning attenuates myocardial dysfunction induced by testicular torsion/detorsion in rats.

Torsion of the spermatic cord is a urological emergency that must be treated immediately with surgery, yet detorsion of the testis can cause testicular tissue damage because of ischemia-reperfusion (I/R) injury. I/R injury is a complex pathophysiological process that may affect the functions of distant organs. Here, we examined whether testicular torsion/detorsion (TT) causes myocardial dysfunction. We next investigated the potential beneficial effect and underlying mechanisms of remote ischemic postconditioning (RIPost) on cardiac function after testicular torsion/detorsion. Male Sprague-Dawley rats were assigned to three different sets of experimental groups. Testicular I/R was induced by rotating the right testis to 1080° clockwise for 3 h followed by 3 h of detorsion. RIPost was induced at the onset of testicular detorsion by four cycles of 5-min bilateral femoral artery occlusion with 5-min reperfusion. Cardiac function was determined postdetorsion, and the cardioprotective effect of RIPost was examined. Testicular torsion/detorsion-treated rats had reduced serum testosterone levels, impaired systemic hemodynamics, elevated systemic inflammatory responses, and increased serum levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), α-hydroxybutyrate dehydrogenase (α-HBDH), and cardiac troponin I (cTnI). However, RIPost attenuated remote heart dysfunction induced by testicular torsion/detorsion. Furthermore, RIPost enhanced the phosphorylation of ventricular signal transducer and activator of transcription (STAT)-3, which is a key component of the survivor activating factor enhancement (SAFE) signaling pathways. Inhibition of STAT-3 with Ag490 abolished the RIPost-induced cardioprotection and STAT-3 phosphorylation. Testicular torsion followed by detorsion may cause impaired cardiac function in rats. RIPost effectively attenuates this remote cardiac dysfunction. RIPost-induced protective effects may be mediated by the STAT-3 signaling pathway.

Empagliflozin protects against renal ischemia/reperfusion injury in mice.

Renal ischemia/reperfusion (I/R) can induce acute kidney injury. Empagliflozin is a newly developed inhibitor of sodium-glucose cotransporter-2 (SGLT2) approved as an antidiabetic medication for patients with type 2 diabetes mellitus. Despite the established cardioprotective functions of empagliflozin, its protective role in renal I/R is unclear. Here, the present study evaluated the renoprotective effects of empagliflozin in a mouse model of renal I/R injury. Male C57/BL6 mice were allocated to sham-operated, I/R, and empagliflozin groups. Kidney pedicles on both sides were clamped for 45 min and were reperfused for 24 h. Empagliflozin (1 mg/kg) was administered to the mice for 2 days preischemia. The GSK-3ß inhibitor SB216763 was administered intravenously at the beginning of reperfusion (0.1 mg/kg). Renal function and histological scores were evaluated. The kidneys were taken for immunohistochemical analysis, western blotting and apoptosis measurements. We found that empagliflozin decreased serum levels of creatinine and urea, reduced the average kidney weight-to-tibia length ratio, attenuated tubular damage, reduced renal proinflammatory cytokine expression and inhibited apoptosis in injured kidneys. Furthermore, empagliflozin increased renal glycogen synthase kinase 3ß (GSK-3ß) phosphorylation post I/R. Pharmacological inhibition of GSK-3ß activity mimicked the renal protective effects offered by empagliflozin. In summary, these results support a protective role of empagliflozin against renal I/R injury.

OsSLA1 functions in leaf angle regulation by enhancing the interaction between OsBRI1 and OsBAK1 in rice.

Leaf angle is an important trait in plants. Here, we demonstrate that the leucine-rich repeat receptor-like kinase OsSLA1 plays an important role in leaf angle regulation in rice (Oryza sativa). OsSLA1 mutant plants exhibited a small leaf angle phenotype due to changes of adaxial cells in the lamina joint. GUS staining revealed that OsSLA1 was highly expressed in adaxial cells of the lamina joint. The OsSLA1 mutant plants were insensitive to exogenous epibrassinolide (eBL) and showed upregulated expression of DWARF and CPD, but downregulated expression of BU1, BUL1, and ILI1, indicating that brassinosteroid (BR) signal transduction was blocked. Fluorescence microscopy showed that OsSLA1 was localized to the plasma membrane and nearby periplasmic vesicles. Further study showed that OsSLA1 interacts with OsBRI1 and OsBAK1 via its intracellular domain and promotes the interaction between OsBRI1 and OsBAK1. In addition, phosphorylation experiments revealed that OsSLA1 does not possess kinase activity, but that it can be phosphorylated by OsBRI1 in vitro. Knockout of OsSLA1 in the context of d61 caused exacerbation of the mutant phenotype. These results demonstrate that OsSLA1 regulates leaf angle formation via positive regulation of BR signaling by enhancing the interaction of OsBRI1 with OsBAK1.

Nutritional Characterization and Untargeted Metabolomics of Oyster Mushroom Produced Using Astragalus membranaceus var. mongolicus Stems and Leaves as Substrates.

Astragalus membranaceus var. mongolicus (AMM) is an edible and medicinal material and is commonly used in East Asia. According to the pharmacopeia of China, the dried root of AMM is medicinal. However, the aerial parts of AMM are always directly discarded after harvest. The stems and leaves are also rich in active compounds, including saponins, flavonoids, terpenoids, and polysaccharides. To rationally use resources, waste products from AMM stems and leaves are useful substrates for edible fungus cultivation. Here, oyster mushroom (Pleurotus ostreatus var. florida) was cultivated on a basal substrate supplemented with AMM stems and leaves (AMM group). The nutritional and chemical composition of the fruiting body were analyzed by metabolomics and chemometrics. Our results showed that AMM addition to the substrate affected the fresh weight, moisture, fat, protein, and element concentrations, and amino acid composition of oyster mushroom. Moreover, 2,156 metabolites were detected and annotated based on the metabolomics data, of which 680 were identified as differentially expressed metabolites. Many active phytometabolites previously identified in AMM herbs were also detected in the metabolomics of oyster mushroom from AMM group, including 46 terpenoids, 21 flavonoids, 17 alkaloids, 14 phenylpropanoids, and 3 fatty acids. In summary, our results imply that oyster mushroom cultured with AMM stems and leaves might have very high nutritional therapy health care value.

Utilization of functional agro-waste residues for oyster mushroom production: Nutritions and active ingredients in healthcare.

A large amount of agro-industrial residues are produced from the planting, production and processing of traditional Chinese herbs. As a tonic, edible, and economical herb, Codonopsis pilosula root has been extensively developed into medicine and functional food. However, thousands of tons of aerial parts (stems, leaves, flowers and fruits) have been directly discarded after harvest each year. To utilise agro-wastes, Pleurotus ostreatus was cultivated on a basal substrate supplemented with C. pilosula stems and leaves (CSL). Physicochemical analyses revealed that the basal substrate mixed with CSL was more abundant in cellulose, hemicellulose, and most of micronutrients such as K, Ca, Mg, S, Fe, Zn and Mo. After the first flush, the fruit bodies in CSL group exhibited a higher fresh weight, a wider average pileus diameter and a lower moisture level. Nutrition analyses presented a higher protein content and a lower fat content in mushrooms from CSL group compared with control group. Interestingly, 14 amino acids (glutamine, arginine, valine, leucine, and etc.) and 3 micronutrients (Se, Fe and Zn) were increased after CSL addition to the substrate. Based on untargeted metabolomics, a total of 710 metabolites were annotated. Compared with control group, there were 142 and 117 metabolites significantly increased and decreased in the CSL group. Most of them were grouped into classes of amino acids and peptids, fatty acids, carbohydrates, terpenoids, and etc. Moreover, an abundance of phytometabolites from Codonopsis were detected in P. ostreatus from CSL group, including polyacetylenes or polyenes, flavonoids, alkaloids, terpenoids, organic acids, and etc. UPLC-MS/MS results demonstrated that lobetyolin content in the CSL group samples was 0.0058%. In summary, the aerial parts of C. pilosula processed for use in the production of edible mushroom is an emerging strategy to converting agricultural waste into functional foods.