A taxon-rich and genome-scale phylogeny of Opisthokonta.

Ancient divergences within Opisthokonta-a major lineage that includes organisms in the kingdoms Animalia, Fungi, and their unicellular relatives-remain contentious. To assess progress toward a genome-scale Opisthokonta phylogeny, we conducted the most taxon rich phylogenomic analysis using sets of genes inferred with different orthology inference methods and established the geological timeline of Opisthokonta diversification. We also conducted sensitivity analysis by subsampling genes or taxa from the full data matrix based on filtering criteria previously shown to improve phylogenomic inference. We found that approximately 85% of internal branches were congruent across data matrices and the approaches used. Notably, the use of different orthology inference methods was a substantial contributor to the observed incongruence: analyses using the same set of orthologs showed high congruence of 97% to 98%, whereas different sets of orthologs resulted in somewhat lower congruence (87% to 91%). Examination of unicellular Holozoa relationships suggests that the instability observed across varying gene sets may stem from weak phylogenetic signals. Our results provide a comprehensive Opisthokonta phylogenomic framework that will be useful for illuminating ancient evolutionary episodes concerning the origin and diversification of the 2 major eukaryotic kingdoms and emphasize the importance of investigating effects of orthology inference on phylogenetic analyses to resolve ancient divergences.

Affinity selection of double-click triazole libraries for rapid discovery of allosteric modulators for GLP-1 receptor.

The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

2X-Rhodamine: A Bright and Fluorogenic Scaffold for Developing Near-Infrared Chemigenetic Indicators.

Chemigenetic fusion of synthetic dyes with genetically encoded protein tags presents a promising avenue for in vivo imaging. However, its full potential has been hindered by the lack of bright and fluorogenic dyes operating in the "tissue transparency" near-infrared window (NIR, 700-1700 nm). Here, we report 2X-rhodamine (2XR), a novel bright scaffold that allows for the development of live-cell-compatible, NIR-excited variants with strong fluorogenicity beyond 1000 nm. 2XR utilizes a rigidified π-skeleton featuring dual atomic bridges and functions via a spiro-based fluorogenic mechanism. This design affords longer wavelengths, higher quantum yield (ΦF = 0.11), and enhanced fluorogenicity in water when compared to the phosphine oxide-cored, or sulfone-cored rhodamine, the NIR fluorogenic benchmarks currently used. We showcase their bright performance in video-rate dynamic imaging and targeted deep-tissue molecular imaging in vivo. Notably, we develop a 2XR variant, 2XR715-HTL, an NIR fluorogenic ligand for the HaloTag protein, enabling NIR genetically encoded calcium sensing and the first demonstration of in vivo chemigenetic labeling beyond 1000 nm. Our work expands the library of NIR fluorogenic tools, paving the way for in vivo imaging and sensing with the chemigenetic approach.

Structural Mass Spectrometry Captures Residue-Resolved Comprehensive Conformational Rearrangements of a G Protein-Coupled Receptor.

G protein-coupled receptor (GPCR) structural studies with in-solution spectroscopic approaches have offered distinctive insights into GPCR activation and signaling that highly complement those yielded from structural snapshots by crystallography or cryo-EM. While most current spectroscopic approaches allow for probing structural changes at selected residues or loop regions, they are not suitable for capturing a holistic view of GPCR conformational rearrangements across multiple domains. Herein, we develop an approach based on limited proteolysis mass spectrometry (LiP-MS) to simultaneously monitor conformational alterations of a large number of residues spanning both flexible loops and structured transmembrane domains for a given GPCR. To benchmark LiP-MS for GPCR conformational profiling, we studied the adenosine 2A receptor (A2AR) in response to different ligand binding (agonist/antagonist/allosteric modulators) and G protein coupling. Systematic and residue-resolved profiling of A2AR conformational rearrangements by LiP-MS precisely captures structural mechanisms in multiple domains underlying ligand engagement, receptor activation, and allostery, and may also reflect local conformational flexibility. Furthermore, these residue-resolution structural fingerprints of the A2AR protein allow us to readily classify ligands of different pharmacology and distinguish the G protein-coupled state. Thus, our study provides a new structural MS approach that would be generalizable to characterizing conformational transition and plasticity for challenging integral membrane proteins.

A randomized, double-blind, placebo-controlled study of Cistanche tubulosa and Ginkgo biloba extracts for the improvement of cognitive function in middle-aged and elderly people.

Mild cognitive impairment poses an increasing challenge to middle-aged and elderly populations. Traditional Chinese medicinal herbs like Cistanche tubulosa and Ginkgo biloba (CG) have been proposed as potential agents to improve cognitive and memory functions. A randomized controlled trial involving 100 Chinese middle-aged and elderly participants was conducted to investigate the potential synergistic effects of CG on cognitive function in individuals at risk of neurodegenerative diseases. Over 90 days, both CG group and placebo group received two tablets daily, with each pair of CG tablets containing 72 mg echinacoside and 27 mg flavonol glycosides. Cognitive functions were assessed using multiple scales and blood biomarkers were determined at baseline, Day 45, and Day 90. The CG group exhibited significant improvements in the scores of Mini-Mental State Examination (26.5 at baseline vs. 27.1 at Day 90, p < 0.001), Montreal Cognitive Assessment (23.4 at baseline vs. 25.3 at Day 90, p < 0.001), and World Health Organization Quality of Life (81.6 at baseline vs. 84.2 at Day 90, p < 0.001), all surpassing scores in placebo group. Notably, both the Cognitrax matrix test and the Wechsler Memory Scale-Revised demonstrated enhanced memory functions, including long-term and delayed memory, after CG intervention. Moreover, cognitive-related blood biomarkers, including total tau, pT181, pS199, pT231, pS396, and thyroid-stimulating hormone, significantly decreased, whereas triiodothyronine and free triiodothyronine significantly increased. No treatment-related adverse events were reported, and routine blood and urine tests remained stable. These findings indicated that CG supplementation could potentially serve as an effective supplementary solution for enhancing cognitive and memory functions.

Research on Decoupling Model of Six-Component Force Sensor Based on Artificial Neural Network and Polynomial Regression.

A two-stage decoupling model based on an artificial neural network with polynomial regression is proposed for the six-component force sensor load decoupling problem in the case of multidimensional mixed loading. The six-dimensional load categorization stage model constructed in the first stage combines 63 load category label sets with a deep BP neural network. The six-dimensional load regression stage model was constructed by combining polynomial regression with a BP neural network in the second stage. Meanwhile, the six-component force sensor with a fiber Bragg grating (FBG) sensor as the sensitive element was designed, and the elastomer simulation and calibration experimental dataset was established to realize the validation of the two-stage decoupling model. The results based on the simulation data show that the accuracy of the classification stage is 93.65%. The MAPE for the force channel in the regression stage is 6.29%, and 3.24% for the moment channel. The results based on experimental data show that the accuracy of the classification stage is 87.80%. The MAPE for the force channel in the regression phase is 5.63%, and 4.82% for the moment channel.

Complete Mitochondrial Genomes of Two Toxin-Accumulated Nassariids (Neogastropoda: Nassariidae: Nassarius) and Their Implication for Phylogeny.

The Indo-Pacific nassariids (genus Nassarius) possesses the highest diversity within the family Nassariidae. However, the previous shell or radula-based classification of Nassarius is quite confusing due to the homoplasy of certain morphological characteristics. The toxin accumulators Nassarius glans and Nassarius siquijorensis are widely distributed in the subtidal regions of the Indo-Pacific Ocean. In spite of their biological significance, the phylogenetic positions of N. glans and N. siquijorensis are still undetermined. In the present study, the complete mitochondrial genomes of N. glans and N. siquijorensis were sequenced. The present mitochondrial genomes were 15,296 and 15,337 bp in length, respectively, showing negative AT skews and positive GC skews as well as a bias of AT rich on the heavy strand. They contained 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and several noncoding regions, and their gene order was identical to most caenogastropods. Based on the nucleotide sequences combining 13 protein coding genes and two rRNA genes, a well-supported phylogeny of Nassarius was reconstructed, and several morphological synapomorphies were observed corresponding to the phylogenetic framework. In addition, the sister group relationship between N. variciferus and the remaining toxin-accumulated nassariids was determined, suggesting that the phylogeny might be related to their diet. The divergence time estimation analysis revealed a correlation between speciation events of nassariids and glacial cycles during the Pliocene-Pleistocene epoch.

Human α defensins promote the expression of the inflammatory cytokine interleukin-8 under high-glucose conditions: Novel insights into the poor healing of diabetic foot ulcers.

Sustained infection and chronic inflammation are the most common features and complex mechanisms of diabetic foot disease. In this study, we examined the expression and functional roles of human endogenous α defensins in diabetic foot ulcer. The expression levels of human α defensins HNP1, HNP3, and HNP4 were significantly higher in the wound center than the edge of diabetic foot ulcers. And the inflammatory cytokine interleukin IL-8 (IL-8) was also highly expressed in wound exudates. In human foreskin fibroblasts, these human α defensins were found only slightly to affect IL-8 expression directly. hemoglobin A1C (HbA1c) is the main clinical indicator of diabetic foot disease. Advanced glycation end products of bovine serum albumin (AGE-BSA), as HbA1c analogue, was found to promote IL-8 expression. Human α defensins, in the presence of AGE-BSA, further significantly promoted IL-8 expression. These findings showed that human α defensins aggravated the inflammatory response in diabetic foot ulcers patients, providing new insights in to the poor healing of diabetic foot ulcers.

Translation and validation of the Chinese version of the Cancer Stigma Scale.

PURPOSE: To test the reliability and validity of the Chinese version of the Cancer Stigma Scale (CASS). METHODS: After translation, back-translation and cross-cultural adaptation of the CASS into Chinese (C-CASS), a random online survey of the general population in China was conducted. Reliability was analyzed by internal consistency (Cronbach's α) and construct validity was analyzed by confirmatory factor analysis. The C-CASS was evaluated in a sample of 382 non-cancer patients through online format. RESULTS: The study found that the C-CASS had satisfactory internal reliability (Cronbach's α of the overall scale and six components was 0.88 and 0.70-0.89, respectively). Confirmatory factor analysis confirmed the six-factor structure (χ2/df = 2.2, GFI = 0.91, CFI = 0.94, RMSEA = 0.056, SRMR = 0.065). Younger individuals and those who had less knowledge of cancer showed more negative attitudes towards cancer. CONCLUSION: The C-CASS had adequate internal consistency, reliability and indices of model fit, allowing its feasible use to assess levels of cancer stigma in Chinese populations.

Effect of exogenous abscisic acid on morphology, growth and nutrient uptake of rice (Oryza sativa) roots under simulated acid rain stress.

MAIN CONCLUSION: Application of proper ABA can improve acid tolerance of rice roots by balancing endogenous hormones and promoting nutrient uptake. Abscisic acid (ABA) has an important signaling role in enhancing plant tolerance to environmental stress. To alleviate the inhibition on plant growth and productivity caused by acid rain, it is crucial to clarify the regulating mechanism of ABA on adaptation of plants to acid rain. Here, we studied the effects of exogenously applied ABA on nutrients uptake of rice roots under simulated acid rain (SAR) stress from physiological, biochemical and molecular aspects. Compared to the single SAR treatment (pH 4.5 or 3.5), exogenous 10 µM ABA alleviated the SAR-induced inhibition of root growth by balancing endogenous hormones (abscisic acid, indole-3-acetic acid, gibberellic acid and zeatin), promoting nutrient uptake (nitrate, P, K and Mg) in rice roots, and increasing the activity of the plasma membrane H+-ATPase by up-regulating expression levels of genes (OSA2, OSA4, OSA9 and OSA10). However, exogenous 100 µM ABA exacerbated the SAR-caused inhibition of root growth by disrupting the balance of endogenous hormones, and inhibiting nutrient uptake (nitrate, P, K, Ca and Mg) through decreasing the activity of the plasma membrane H+-ATPase. These results indicate that proper concentration of exogenous ABA could enhance tolerance of rice roots to SAR stress by promoting nutrients uptake and balancing endogenous hormones.

Mouse ß-Defensin 3, A Defensin Inhibitor of Both Its Endogenous and Exogenous Potassium Channels.

The human defensins are recently discovered to inhibit potassium channels, which are classical targets of the animal toxins. Whether other vertebrate defensins are potassium channel inhibitors remains unknown. In this work, we reported that the mouse ß-defensin 3 (mBD3) was a novel inhibitor of both endogenous and exogenous potassium channels. The structural analysis showed that mBD3 is the most identical to human Kv1.3 channel-sensitive human ß-defensin 2 (hBD2). However, the pharmacological profiles indicated that the recombinant mBD3 (rmBD3) weakly inhibited the mouse and human Kv1.3 channels. Different from the pharmacological features of human ß-defensins, mBD3 more selectively inhibited the mouse Kv1.6 and human KCNQ1/KCNE1 channels with IC50 values of 0.6 ± 0.4 µM and 1.2 ± 0.8 µM, respectively. The site directed mutagenesis experiments indicated that the extracellular pore region of mouse Kv1.6 channel was the interaction site of rmBD3. In addition, the minor effect on the channel conductance-voltage relationship curves implied that mBD3 might bind the extracellular transmembrane helices S1-S2 linker and/or S3-S4 linker of mouse Kv1.6 channel. Together, these findings not only revealed mBD3 as a novel inhibitor of both endogenous and exogenous potassium channels, but also provided a clue to investigate the role of mBD3-Kv1.6 channel interaction in the physiological and pathological field in the future.

Urine Metabonomics Reveals Early Biomarkers in Diabetic Cognitive Dysfunction.

Recently, increasing attention has been paid to diabetic encephalopathy, which is a frequent diabetic complication and affects nearly 30% of diabetics. Because cognitive dysfunction from diabetic encephalopathy might develop into irreversible dementia, early diagnosis and detection of this disease is of great significance for its prevention and treatment. This study is to investigate the early specific metabolites biomarkers in urine prior to the onset of diabetic cognitive dysfunction (DCD) by using metabolomics technology. An ultra-high performance liquid-chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-Q/TOF-MS) platform was used to analyze the urine samples from diabetic mice that were associated with mild cognitive impairment (MCI) and nonassociated with MCI in the stage of diabetes (prior to the onset of DCD). We then screened and validated the early biomarkers using OPLS-DA model and support vector machine (SVM) method. Following multivariate statistical and integration analysis, we found that seven metabolites could be accepted as early biomarkers of DCD, and the SVM results showed that the prediction accuracy is as high as 91.66%. The identities of four biomarkers were determined by mass spectrometry. The identified biomarkers were largely involved in nicotinate and nicotinamide metabolism, glutathione metabolism, tryptophan metabolism, and sphingolipid metabolism. The present study first revealed reliable biomarkers for early diagnosis of DCD. It provides new insight and strategy for the early diagnosis and treatment of DCD.

The Application of Quantitative ¹H-NMR for the Determination of Orlistat in Tablets.

A quantitative nuclear magnetic resonance (qNMR) method to measure the content of Orlistat in tablets was studied and found to be efficient, accurate, reliable, and simple. In this paper, phloroglucinolanhydrous and dimethylsulfoxide-d6 (DMSO-d6) served as the internal standard and solvent, respectively. The qNMR methodology, including the linearity, range, the limit of detection (LOD) and quantification (LOQ), stability, precision, and accuracy, was validated seriatim, and the results were very favorable. The content determination results of three batches of Orlistat in tablets were almost identical upon comparing the qNMR method and the high-performance liquid chromatography (HPLC) method. The recommended method authentically compensated the deficiencies of the current HPLC method for determining Orlistat content, and proved to be a method complementary to traditional analysis for the purity measurement of Orlistat in some pharmaceutical preparations.

Quantitative Metabolomic Profiling of Plasma, Urine, and Liver Extracts by 1H NMR Spectroscopy Characterizes Different Stages of Atherosclerosis in Hamsters.

Atherosclerosis (AS) is a progressive disease that contributes to cardiovascular disease and shows a complex etiology, including genetic and environmental factors. To understand systemic metabolic changes and to identify potential biomarkers correlated with the occurrence and perpetuation of diet-induced AS, we applied 1H NMR-based metabolomics to detect the time-related metabolic profiles of plasma, urine, and liver extracts from male hamsters fed a high fat and high cholesterol (HFHC) diet. Conventional biochemical assays and histopathological examinations as well as protein expression analyses were performed to provide complementary information. We found that diet treatment caused obvious aortic lesions, lipid accumulation, and inflammatory infiltration in hamsters. Downregulation of proteins related to cholesterol metabolism, including hepatic SREBP2, LDL-R, CYP7A1, SR-BI, HMGCR, LCAT, and SOAT1 was detected, which elucidated the perturbation of cholesterol homeostasis during the HFHC diet challenge. Using "targeted analysis", we quantified 40 plasma, 80 urine, and 60 liver hydrophilic extract metabolites. Multivariate analyses of the identified metabolites elucidated sophisticated metabolic disturbances in multiple matrices, including energy homeostasis, intestinal microbiota functions, inflammation, and oxidative stress coupled with the metabolisms of cholesterol, fatty acids, saccharides, choline, amino acids, and nucleotides. For the first time, our results demonstrate a time-dependent metabolic progression of multiple biological matrices in hamsters from physiological status to early AS and further to late-stage AS, demonstrating that 1H NMR-based metabolomics is a reliable tool for early diagnosis and monitoring of the process of AS.

The application of HPLC and microprobe NMR spectroscopy in the identification of metabolites in complex biological matrices.

Nuclear magnetic resonance (NMR)-based metabolomics can be used directly to identify a variety of metabolites in biological fluids and tissues. Metabolite analysis is an important part of life science and metabolomics research. However, the identification of some metabolites using NMR spectroscopy remains a big challenge owing to low abundance or signal overlap. It is important to develop a method to measure these compounds accurately. Two-dimensional NMR spectroscopy, metabolite prediction software packages, and spike-in experiments with authentic standards are often used to solve these problems, but they are costly and time-consuming. In this study, methods were developed to identify metabolites in complex biological mixtures using both high-performance liquid chromatography (HPLC) and off-line microprobe NMR spectroscopy. With use of these methods, 83 and 73 metabolites were identified in Sprague Dawley rat urine and feces, respectively. Among them, 40 and 45 metabolites, respectively, could not be identified with traditional NMR methods. Our research revealed that the combination of HPLC and NMR techniques could significantly improve the accuracy of trace and overlapped metabolite identification, while offering an effective and convenient approach to identify potential biomarkers in complex biological systems.

Long-term response of interspecific competition among three typical bloom-forming species to changes in phosphorus and temperature.

Phosphorus and temperature play an important role in the succession of diatom-dinoflagellate blooms. However, there is little long-term research on interspecific competition based on phosphorus source and temperature. Here, interspecific competition among Skeletonema costatum, Prorocentrum donghaiense and Karenia mikimotoi was studied using trialgal laboratory co-cultures under different phosphorus and temperature conditions. These results suggest that S. costatum and P. donghaiense alternated as competing dominant species during the experimental period, which coincides with the different phosphorus conditions. However, K. mikimotoi growth was significantly inhibited throughout the experiment. We suggest that this may be due to different algal requirements for phosphorus, optimal growth temperatures, and possible allelopathic effects. This study provides a comprehensive mechanism of interspecific competition between diatom-dinoflagellate in response to phosphorus and temperature and elucidates the seasonal succession of diatom-dinoflagellate from late spring to early summer in the Changjiang River Estuary and the adjacent East China Sea.

Regulation of tillage on grain matter accumulation in maize.

Introduction: To address issues related to shallow soil tillage, low soil nutrient content, and single tillage method in maize production in the Western Inner Mongolia Region, this study implemented various tillage and straw return techniques, including strip cultivation, subsoiling, deep tillage, no-tillage, straw incorporation with strip cultivation, straw incorporation with subsoiling, straw incorporation with deep tillage, and straw incorporation with no tillage, while using conventional shallow spinning by farmers as the control. Methods: We employed Xianyu 696 (XY696) and Ximeng 6 (XM6) as experimental materials to assess maize 100-grains weight, grain filling rate parameters, and grain nutrient quality. This investigation aimed to elucidate how tillage and straw return influence the accumulation of grain material in different maize varieties. Results and discussion: The results indicated that proper implementation of tillage and straw return had a significant impact on the 100-grains weight of both varieties. In comparison to CK (farmer's rotary rotation), the most notable rise in 100-grains weight was observed under the DPR treatment (straw incorporation with deep tillage), with a maximum increase of 4.84% for XY696 and 6.28% for XM6. The proper implementation of tillage and straw return in the field resulted in discernible differences in the stages of improving the grain filling rates of different maize varieties. Specifically, XY696 showed a predominant increase in the filling rate during the early stage (V1), while XM6 exhibited an increase in the filling rates during the middle and late stages (V2 and V3). In comparison to CK, V1 increased by 1.54% to 27.56% in XY696, and V2 and V3 increased by 0.41% to 10.42% in XM6 under various tillage and straw return practices. The proper implementation of tillage and straw return had a significant impact on the nutritional quality of the grains in each variety. In comparison to CK, the DPR treatment resulted in the most pronounced decrease in the soluble sugar content of grains by 25.43% and the greatest increase in the crude fat content of grains by 9.67%. Conclusion: Ultimately, the proper implementation of soil tillage and straw return facilitated an increase in grain crude fat content and significantly boosted grain weight by improving the grouting rate parameters at all stages for various maize varieties. Additionally, the utilization of DPR treatment proved to be more effective. Overall, DPR is the most promising strategy to improve maize yield and the nutritional quality of grain in the long term in the Western Inner Mongolia Region.

Unique trajectory of gene family evolution from genomic analysis of nearly all known species in an ancient yeast lineage.

Gene gains and losses are a major driver of genome evolution; their precise characterization can provide insights into the origin and diversification of major lineages. Here, we examined gene family evolution of 1,154 genomes from nearly all known species in the medically and technologically important yeast subphylum Saccharomycotina. We found that yeast gene family and genome evolution are distinct from plants, animals, and filamentous ascomycetes and are characterized by small genome sizes and smaller gene numbers but larger gene family sizes. Faster-evolving lineages (FELs) in yeasts experienced significantly higher rates of gene losses-commensurate with a narrowing of metabolic niche breadth-but higher speciation rates than their slower-evolving sister lineages (SELs). Gene families most often lost are those involved in mRNA splicing, carbohydrate metabolism, and cell division and are likely associated with intron loss, metabolic breadth, and non-canonical cell cycle processes. Our results highlight the significant role of gene family contractions in the evolution of yeast metabolism, genome function, and speciation, and suggest that gene family evolutionary trajectories have differed markedly across major eukaryotic lineages.