Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine.

Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.

Dissecting and improving gene regulatory network inference using single-cell transcriptome data.

Single-cell transcriptome data has been widely used to reconstruct gene regulatory networks (GRNs) controlling critical biological processes such as development and differentiation. Although a growing list of algorithms has been developed to infer GRNs using such data, achieving an inference accuracy consistently higher than random guessing has remained challenging. To address this, it is essential to delineate how the accuracy of regulatory inference is limited. Here, we systematically characterized factors limiting the accuracy of inferred GRNs and demonstrated that using pre-mRNA information can help improve regulatory inference compared to the typically used information (i.e., mature mRNA). Using kinetic modeling and simulated single-cell data sets, we showed that target genes' mature mRNA levels often fail to accurately report upstream regulatory activities because of gene-level and network-level factors, which can be improved by using pre-mRNA levels. We tested this finding on public single-cell RNA-seq data sets using intronic reads as proxies of pre-mRNA levels and can indeed achieve a higher inference accuracy compared to using exonic reads (corresponding to mature mRNAs). Using experimental data sets, we further validated findings from the simulated data sets and identified factors such as transcription factor activity dynamics influencing the accuracy of pre-mRNA-based inference. This work delineates the fundamental limitations of gene regulatory inference and helps improve GRN inference using single-cell RNA-seq data.

Profiling transcription factor activity dynamics using intronic reads in time-series transcriptome data.

Activities of transcription factors (TFs) are temporally modulated to regulate dynamic cellular processes, including development, homeostasis, and disease. Recent developments of bioinformatic tools have enabled the analysis of TF activities using transcriptome data. However, because these methods typically use exon-based target expression levels, the estimated TF activities have limited temporal accuracy. To address this, we proposed a TF activity measure based on intron-level information in time-series RNA-seq data, and implemented it to decode the temporal control of TF activities during dynamic processes. We showed that TF activities inferred from intronic reads can better recapitulate instantaneous TF activities compared to the exon-based measure. By analyzing public and our own time-series transcriptome data, we found that intron-based TF activities improve the characterization of temporal phasing of cycling TFs during circadian rhythm, and facilitate the discovery of two temporally opposing TF modules during T cell activation. Collectively, we anticipate that the proposed approach would be broadly applicable for decoding global transcriptional architecture during dynamic processes.

Cognitive improvement in late-life depression treated with vortioxetine and duloxetine in an eight-week randomized controlled trial: The role of age at first onset and change in depressive symptoms.

BACKGROUND: Age at first onset of depression as a clinical factor affecting cognitive improvement in late life depression was investigated. METHODS: This is a secondary analysis of an eight-week randomized controlled trial involving 452 elderly patients treated by vortioxetine, duloxetine or placebo (1:1:1). Patients were subcategorized into early-onset (LLD-EO) and late-onset (LLD-LO) groups divided by onset age of 50. Cognitive performance was assessed by composite score of Digit Symbol Substitution Test (DSST) and the Rey Auditory Verbal Learning Test (RAVLT) tasks, while depressive symptoms were assessed by Montgomery-Åsberg Depression Rating Scale (MADRS). RESULTS: Vortioxetine and duloxetine exhibited advantages versus placebo in improving cognitive performance in the LLD-LO group, yet not in the LLD-EO group after eight weeks. Patients in the LLD-EO group showed overall advantage to placebo in depressive symptoms before endpoint (week 8) of treatment, while patients in the LLO-LO group showed no advantage until endpoint. Path analysis suggested a direct effect of vortioxetine (B = 0.656, p = .036) and duloxetine (B = 0.726, p = .028) on improving cognition in the LLD-LO group, yet in all-patients treated set both medications improved cognition indirectly through changes of depressive symptoms. LIMITATION: Reliability of clinical history could raise caution as it was collected by subjective recall of patients. CONCLUSION: Age at first onset might affect cognitive improvement as well as change in depressive symptoms and its mediation towards cognitive improvement in late life depression treated with vortioxetine and duloxetine.

Multi-instance learning for identifying high-risk subregions associated with synchronous distant metastasis in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most common histological subtypes of renal tumors. PURPOSE: To identify high-risk subregions associated with synchronous distant metastasis. METHODS: This study enrolled a total of 277 patients with ccRCC. Voxel intensity and local entropy values were compiled within the region of interest for all patients. Unsupervised k-means clustering yielded three subregions per tumor. Radiomic features were extracted, and random forest-based feature selection was conducted. The selected features were used in a multi-instance support vector machine (mi-SVM) model for training, and predictions were made on the validation cohort. Model performance was evaluated using five-fold cross-validation. The subregion with the highest score for patients with synchronous distant metastasis was identified across all cohorts. RESULTS: The mi-SVM model yielded an average area under the curve (AUC) of 0.812 in the training cohort and 0.805 in the validation cohort. In the entire cohort of patients with synchronous distant metastasis, subregion 2, characterized by tumor periphery and intratumoral transitional components, accounted for the highest proportion (48.57%, 30.6/63) among all subregions. It represents a high-risk subregion for synchronous distant metastasis of clear cell renal cell carcinoma. CONCLUSION: The peripheral and intratumoral transition zones of clear cell renal cell carcinoma are high-risk subregions associated with synchronous distant metastasis.

The Effect of Dietary Protein Hydrolysate from Black Soldier Fly Larvae and Schizochytrium on Palatability, Nutrient Metabolites and Health Status in Beagle Dogs.

Protein hydrolysate from black soldier fly larvae (BSFP) has garnered great attention with its lower allergenicity, high amount of essential amino acids, and small bioactive peptides. Schizochytrium is a promising alternative source of n-3 FUFA because it has enriched docosahexaenoic acid (DHA, C22: 6). The aim of this study was to assess palatability, the presence of diarrhea, plasma biochemistry panels, anti-oxidative and anti-inflammatory effects, and immune function in beagle dogs when supplementing a mixture of protein hydrolysate from black soldier fly larvae and schizochytrium (BSFPs) into their diets. Experiment I: 24 young beagle dogs (16 males and 8 females; 4-5 months; BW: 6.40 ± 0.15 kg) were randomly divided into four groups: (1) control (CON), (2) 5% BSFPs, (3) 10% BSFPs, (4) 15% BSFPs. Their body weights and fecal scores were recorded, and blood samples were collected for analysis. Experiment II: three diets containing 5%, 10%, and 15% BSFPs were evaluated by comparing them with a basal diet (CON) to evaluate palatability. These results suggested that a lower presence of diarrhea existed in the BSFP diet than the CON diet (p < 0.05). Three treatment groups remarkably increased their total protein (TP) and albumin (ALB) contents and decreased their concentrations of triglyceride (TG) and total cholesterol (TC) in plasma (p < 0.05). Moreover, the 5% and 15% BSFPs groups had a higher calcium (CA) content in plasma, and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and contents of creatinine (CREA) and urea nitrogen (BUN) were significantly reduced by supplementing BSFP in their diets (p < 0.05). Their anti-oxidative enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were dramatically enhanced, and their malondialdehyde (MDA) concentrations were remarkably reduced (p < 0.05). Immunoglobulin A and G (IgA and IgG) concentrations in the plasma in the 10% and 15% BSFPs groups were significantly increased (p < 0.05). Furthermore, lower interleukin-8 (IL-8) contents were shown in the BSFP diets than the CON diet (p < 0.05). Similarly, the diets supplemented with BSFPs exhibited a positive effect on palatability (p < 0.05). To sum up, the diets supplemented with BSFPs significantly enhanced palatability, immune function, and anti-oxidative and anti-inflammatory capacity to alleviate diarrhea and improve the general health of the beagle dogs.

Calcium binding and permeation in TRPV channels: Insights from molecular dynamics simulations.

Some calcium channels selectively permeate Ca2+, despite the high concentration of monovalent ions in the surrounding environment, which is essential for many physiological processes. Without atomistic and dynamical ion permeation details, the underlying mechanism of Ca2+ selectivity has long been an intensively studied, yet controversial, topic. This study takes advantage of the homologous Ca2+-selective TRPV6 and non-selective TRPV1 and utilizes the recently solved open-state structures and a newly developed multisite calcium model to investigate the ion binding and permeation features in TRPV channels by molecular dynamics simulations. Our results revealed that the open-state TRPV6 and TRPV1 show distinct ion binding patterns in the selectivity filter, which lead to different ion permeation features. Two Ca2+ ions simultaneously bind to the selectivity filter of TRPV6 compared with only one Ca2+ in the case of TRPV1. Multiple Ca2+ binding at the selectivity filter of TRPV6 permeated in a concerted manner, which could efficiently block the permeation of Na+. Cations of various valences differentiate between the binding sites at the entrance of the selectivity filter in TRPV6. Ca2+ preferentially binds to the central site with a higher probability of permeation, repelling Na+ to a peripheral site. Therefore, we believe that ion binding competition at the selectivity filter of calcium channels, including the binding strength and number of binding sites, determines Ca2+ selectivity under physiological conditions.

Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health.

The objective of this study was to evaluate the effects of adding essential oils to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. A total of 96 weaning pigs (8.37±1.58 kg) were allotted to one of three dietary treatments. The treatments consisted of an unsupplemented basal diet (negative control, NC) or similar diets supplemented with 0.01% of an essential oil product which contained 18% thymol and cinnamaldehyde (EOD) as well as a diet supplemented with 0.19% of an antibiotic mixture which provided 150 ppm chlortetracycline, 80 ppm colistin sulfate and 50 ppm kitasamycin (positive control, PC). Each treatment was provided to eight pens of pigs with four pigs per pen. Over the entire 35 d experiment, ADG and fecal score were improved (p<0.05) for pigs fed the PC and EOD compared with the NC. Dry matter and crude protein digestibility as well as lymphocyte proliferation for pigs fed the PC and EOD diets were increased significantly compared with NC (p<0.05). IGF-I levels in plasma were significantly increased (p<0.05) in pigs fed the PC diet compared with pigs fed the NC diet. Interleukin-6 concentration was lower (p<0.05) and the tumor necrosis factor-α level was higher (p<0.05) in the plasma of pigs fed the EOD diet than the NC diet. Plasma total antioxidant capacity level increased (p<0.05) in pigs fed the EOD diet compared with pigs fed the NC. Villus height to crypt depth ratio in the jejunum was greater (p<0.05) in pigs fed the PC and EOD diets than the NC. The numbers of E. coli in the cecum, colon and rectum were reduced (p<0.05) in pigs fed the PC and EOD diets compared with the control. In the colon, the ratio of Lactobacilli to E. coli was increased (p<0.05) in pigs fed the EOD diet compared with NC diet. Total aerobe numbers in the rectum were decreased (p<0.05) in pigs fed the PC and EOD diets compared with the control. Collectively, these results indicate that blends of essential oils could be a candidate for use as an alternative to traditional antibiotics in weaning pig diets.

Improving visual perspective-taking performance in children with autism spectrum conditions: Effects of embodied self-rotation and object-based mental rotation strategies.

LAY ABSTRACT: When answering how the same object might appear to others in different locations, people can provide answers by mentally putting themselves into another person's location using the embodied self-rotation strategy or by rotating the target object toward themselves using the object-based mental rotation strategy. In this study, after learning the embodied self-rotation or object-based mental rotation strategies, autistic children improved their visual perspective-taking performance, which is believed to be impaired or delayed in autistic individuals. We recruited 34 autistic children and an equal number of ability-matched typical children and examined their visual perspective-taking performance at baseline and after learning the embodied self-rotation and object-based mental rotation strategies. As previous visual perspective-taking and other social cognition interventions for autistic individuals have primarily focused on the embodied self-rotation strategy, showing moderate effectiveness and limited generalizability, we explored the effects of both embodied self-rotation and object-based mental rotation strategies for improving perspective-taking performance and discussed their implications in this study. The results showed that autistic children had a lower performance at baseline compared with typical children; however, they were still sensitive to both embodied self-rotation and object-based mental rotation strategies. Unlike typical children, who gained more from the embodied self-rotation strategy, autistic children benefited similarly from the two strategies. This suggests that there are multiple ways to helping autistic children overcome their difficulty in perspective-taking tasks. Future interventions for autistic children could consider combining various strategies that better suit their autistic traits.

Effects of the standardized ileal digestible lysine to metabolizable energy ratio on performance and carcass characteristics of growing-finishing pigs.

A total of 2,121 growing-finishing pigs (Duroc × Landrace × Large White) were utilized in six experiments conducted to determine the effects of different ratios of standardized ileal digestible lysine (SID-Lys) to metabolizable energy (ME) on the performance and carcass characteristics of growing-finishing pigs. Exps. 1 (30 to 50 kg), 2 (52 to 70 kg) and 3 (81 to 104 kg) were conducted to find an optimum ME level and then this level was used in Exps. 4 (29 to 47 kg), 5 (54 to 76 kg) and 6 (84 to 109 kg) to test the response of pigs to different ratios of SID-Lys:ME. In Exps.1 to 3, four treatments were used consisting of diets with a formulated ME content of 3.1, 3.2, 3.3 or 3.4 in Exps. 1 and 2 while Exp. 3 used 3.05, 3.15, 3.25 or 3.35 Mcal/kg. A constant SID-Lys:ME ratio of 2.6, 2.3 or 2.0 g/Mcal was used in Exps. 1, 2 and 3, respectively. Weight gain was significantly increased with increasing energy level in Exp.1 while weight gain was unaltered in Exps. 2 and 3. For all three experiments, feed intake was decreased (P < 0.05) and feed efficiency was improved (P < 0.05) with increasing energy level. Tenth rib back fat thickness linearly increased (P < 0.05) with increasing energy level. In Exps. 4 to 6, five treatments were used consisting of diets with a SID-Lys:ME ratio of 2.4, 2.6, 2.8, 3.0 or 3.2 in Exp. 1, 2.1, 2.3, 2.5, 2.7, 2.9 or 3.2 in Exp. 2 and 1.8, 2.0, 2.2, 2.4, or 2.6 in Exp. 3. A constant ME level 3.2, 3.2 and 3.05 Mcal/kg was used in Exps. 1, 2 and 3, respectively (selected based on the results of weight gain). For all three experiments, weight gain increased (P < 0.05) and feed efficiency improved linearly (P < 0.05) as the SID-Lys:ME ratio increased. Tenth rib back fat thickness linearly decreased (P < 0.05) as the SID-Lys:ME ratio increased. Based on a straight broken-line model, the estimated SID-Lys:ME ratio to maximize weight gain was 3.0, 2.43 and 2.2 for 29 to 47, 54 to76 and 84 to 109 kg of pigs, respectively.

The effect of the ratio of standardized ileal digestible lysine to metabolizable energy on growth performance, blood metabolites and hormones of lactating sows.

A total of 335 lactating sows (Landrace × Large White) were used in two experiments to determine the optimum ratio of standardized ileal digestible lysine (SID-Lys) to metabolizable energy (ME) for mixed parity sows during lactation. In Exp. 1, 185 sows (weighing an average of 256.2 ± 6.5 kg and having an average parity of 3.4 ± 0.3) were allocated to one of six experimental diets in a completely randomized block design within parity groups (1, 2, and 3+). The experimental diets were formulated to contain 3.06, 3.16, 3.20, 3.25, 3.30 or 3.40 Mcal/kg of ME and each diet was fed to the sows throughout a 28 day lactation. All diets provided a similar SID-lysine level (0.86%). As a result, the diets provided a SID-Lys:ME ratio of 2.81, 2.72, 2.69, 2.65, 2.61 or 2.53 g/Mcal ME. Sow feed intake was significantly (P < 0.01) affected by the energy content of the diet as well as by sow parity. Using regression analysis, feed intake was shown to be maximized at 3.25, 3.21, 3.21 and 3.21 Mcal/kg of ME for parity 1, 2, 3+ sows and the entire cohort of sows respectively (quadratic; P < 0.01). In addition, the result of feed intake can be expressed as 2.65, 2.69, 2.69 and 2.68 g/Mcal based on analysis of SID-Lys:ME ratio. Litter weight gain was affected by dietary treatment for parity 3+ sows and the entire cohort (P < 0.01). Based on regression analysis, litter weight gain was maximized at 3.25 and 3.24 Mcal/kg of ME for parity 3+ (quadratic; P < 0.01) and the entire cohort (quadratic; P < 0.01). Similarly, the result of litter weight gain could be expressed as 2.65 and 2.66 g/Mcal of SID-Lys:ME ratio. Therefore, 3.25 Mcal/kg of ME was selected for Exp. 2 in which 150 sows (weighing 254.6 ± 7.3 kg and having an average parity of 3.4 ± 0.4) were allocated to one of five treatments in a completely randomized block design within parity (1, 2, and 3+). The experimental diets were formulated to contain 2.1, 2.4, 2.7, 3.0 or 3.3 g/Mcal of SID-Lys:ME ratio with all diets providing 3.25 Mcal/kg of ME. The diets were fed to the sows throughout a 28 day lactation. Sow body weight loss was affected by dietary treatment (parity 3+ sows, P = 0.02; entire cohort, P < 0.01) and by sow parity (P < 0.01). Litter weight at weaning and litter weight gain were affected by dietary treatment for parity 1, 2, 3+ sows and the entire cohort (P < 0.01) as well as by sow parity (P < 0.01). Plasma urea nitrogen (P < 0.01), creatinine (P < 0.01) and non-esterifide fatty acids (P = 0.04) were decreased as the SID-Lys:ME ratio of the diet increased. Insulin-like growth factor-1 (P = 0.02), estradiol (P < 0.01) and luteinizing hormone (P = 0.02) were increased as the SID-Lys:ME ratio in diet increased. Based on a broken-line model, the estimated SID-Lys:ME ratio to maximize litter weight gain was estimated to be 3.05 g/Mcal.

Effects of keratinase supplementation of corn-soybean meal based diets on apparent ileal amino acid digestibility in growing pigs and serum amino acids, cytokines, immunoglobulin levels and loin muscle area in nursery pigs.

Two experiments were conducted to evaluate effects of keratinase for growing and nursery pigs. In Exp. 1, six pigs (32.3 +/- 2.8 kg body weight), fitted with a simple T-cannula at the distal ileum, were assigned to one of two 3 x 3 Latin squares involving three periods and three diets including a basal diet and the same diets supplemented with 0, 0.05 or 0.1% keratinase. Dietary keratinase supplementation increased the apparent ileal digestibility of crude protein (CP), arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, alanine, glutamic acid and proline (p < 0.05). Digestibility coefficients did not differ between pigs fed 0.05 and 0.1% keratinase. In Exp. 2, 24 piglets weaned at 30 +/- 2 d of age were used in a 2 x 2 factorial design experiment with two CP concentrations (19 vs. 22%) and two levels of keratinase supplementation (0 vs. 0.05%). Keratinase supplementation increased (p < 0.05) average daily gain, serum arginine concentration and loin muscle area but decreased (p < 0.05) serum interleukin-10 concentrations. The reduction in dietary CP level decreased (p < 0.05) serum urea nitrogen concentrations, isoleucine, serine and proline concentrations, but increased serum arginine concentrations. Few interactions between keratinase supplementation and dietary CP concentration were observed. This study indicated that dietary keratinase supplementation improved apparent ileal amino acid digestibility for growing pigs and had a positive effect on weight gain, immune response and loin muscle area for nursery pigs.