Furfural tolerance of mutant Saccharomyces cerevisiae selected via ionizing radiation combined with adaptive laboratory evolution.

BACKGROUND: Lignocellulose is a renewable and sustainable resource used to produce second-generation biofuel ethanol to cope with the resource and energy crisis. Furfural is the most toxic inhibitor of Saccharomyces cerevisiae cells produced during lignocellulose treatment, and can reduce the ability of S. cerevisiae to utilize lignocellulose, resulting in low bioethanol yield. In this study, multiple rounds of progressive ionizing radiation was combined with adaptive laboratory evolution to improve the furfural tolerance of S. cerevisiae and increase the yield of ethanol. RESULTS: In this study, the strategy of multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution significantly improved the furfural tolerance of brewing yeast. After four rounds of experiments, four mutant strains resistant to high concentrations of furfural were obtained (SCF-R1, SCF-R2, SCF-R3, and SCF-R4), with furfural tolerance concentrations of 4.0, 4.2, 4.4, and 4.5 g/L, respectively. Among them, the mutant strain SCF-R4 obtained in the fourth round of radiation had a cellular malondialdehyde content of 49.11 nmol/mg after 3 h of furfural stress, a weakening trend in mitochondrial membrane potential collapse, a decrease in accumulated reactive oxygen species, and a cell death rate of 12.60%, showing better cell membrane integrity, stable mitochondrial function, and an improved ability to limit reactive oxygen species production compared to the other mutant strains and the wild-type strain. In a fermentation medium containing 3.5 g/L furfural, the growth lag phase of the SCF-R4 mutant strain was shortened, and its growth ability significantly improved. After 96 h of fermentation, the ethanol production of the mutant strain SCF-R4 was 1.86 times that of the wild-type, indicating that with an increase in the number of irradiation rounds, the furfural tolerance of the mutant strain SCF-R4 was effectively enhanced. In addition, through genome-transcriptome analysis, potential sites related to furfural detoxification were identified, including GAL7, MAE1, PDC6, HXT1, AUS1, and TPK3. CONCLUSIONS: These results indicate that multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution is an effective mutagenic strategy for obtaining furfural-tolerant mutants and that it has the potential to tap genes related to the furfural detoxification mechanism.

The genome of Vicia sativa ssp. amphicarpa provides insights into the role of terpenoids in antimicrobial resistance within subterranean fruits.

Vicia sativa ssp. amphicarpa is a unique forage crop capable of simultaneously producing fruits above and below ground, representing a typical amphicarpic plant. In this study, we sequenced and assembled seven pseudo-chromosomes of the genome of V. sativa ssp. amphicarpa (n = 7) yielding a genome size of 1.59 Gb, with a total annotation of 48 932 protein-coding genes. Long terminal repeat (LTR) elements constituted 62.28% of the genome, significantly contributing to the expansion of genome size. Phylogenetic analysis revealed that the divergence between V. sativa ssp. amphicarpa and V. sativa was around 0.88 million years ago (MYA). Comparative transcriptomic and metabolomic analysis of aerial and subterranean pod shells showed biosynthesis of terpenoids in the subterranean pod shells indicating a correlation between the antimicrobial activity of subterranean pod shells and the biosynthesis of terpenoids. Furthermore, functional validation indicates that overexpression of VsTPS5 and VsTPS16 enhances terpenoid biosynthesis for antibacterial activity. Metabolomic analysis suggests the involvement of terpenoids in the antimicrobial properties of subterranean pod shells. Deciphering the genome of V. sativa ssp. amphicarpa elucidated the molecular mechanisms behind the antimicrobial properties of subterranean fruits in amphicarpic plants, providing valuable insights for the study of amphicarpic plant biology.

Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

Integration of food raw materials, food microbiology, and food additives: systematic research and comprehensive insights into sweet sorghum juice, Clostridium tyrobutyricum TGL-A236 and bio-butyric acid.

Introduction: Sweet sorghum juice is a typical production feedstock for natural, eco-friendly sweeteners and beverages. Clostridium tyrobutyricum is one of the widely used microorganisms in the food industry, and its principal product, bio-butyric acid is an important food additive. There are no published reports of Clostridium tyrobutyricum producing butyric acid using SSJ as the sole substrate without adding exogenous substances, which could reach a food-additive grade. This study focuses on tailoring a cost-effective, safe, and sustainable process and strategy for their production and application. Methods: This study modeled the enzymolysis of non-reducing sugars via the first/second-order kinetics and added food-grade diatomite to the hydrolysate. Qualitative and quantitative analysis were performed using high-performance liquid chromatography, gas chromatography-mass spectrometer, full-scale laser diffraction method, ultra-performance liquid chromatography-tandem mass spectrometry, the cell double-staining assay, transmission electron microscopy, and Oxford nanopore technology sequencing. Quantitative real-time polymerase chain reaction, pathway and process enrichment analysis, and homology modeling were conducted for mutant genes. Results: The treated sweet sorghum juice showed promising results, containing 70.60 g/L glucose and 63.09 g/L fructose, with a sucrose hydrolysis rate of 98.29% and a minimal sucrose loss rate of 0.87%. Furthermore, 99.62% of the colloidal particles and 82.13% of the starch particles were removed, and the concentrations of hazardous substances were effectively reduced. A food microorganism Clostridium tyrobutyricum TGL-A236 with deep utilization value was developed, which showed superior performance by converting 30.65% glucose and 37.22% fructose to 24.1364 g/L bio-butyric acid in a treated sweet sorghum juice (1:1 dilution) fermentation broth. This titer was 2.12 times higher than that of the original strain, with a butyric acid selectivity of 86.36%. Finally, the Genome atlas view, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and evolutionary genealogy of genes: Non-supervised Orthologous (eggNOG) functional annotations, three-dimensional structure and protein cavity prediction of five non-synonymous variant genes were obtained. Conclusion: This study not only includes a systematic process flow and in-depth elucidation of relevant mechanisms but also provides a new strategy for green processing of food raw materials, improving food microbial performance, and ensuring the safe production of food additives.

Chromosome-level genome assembly of Pedicularis kansuensis illuminates genome evolution of facultative parasitic plant.

Parasitic plants have a heterotrophic lifestyle, in which they withdraw all or part of their nutrients from their host through the haustorium. Despite the release of many draft genomes of parasitic plants, the genome evolution related to the parasitism feature of facultative parasites remains largely unknown. In this study, we present a high-quality chromosomal-level genome assembly for the facultative parasite Pedicularis kansuensis (Orobanchaceae), which invades both legume and grass host species in degraded grasslands on the Qinghai-Tibet Plateau. This species has the largest genome size compared with other parasitic species, and expansions of long terminal repeat retrotransposons accounting for 62.37% of the assembly greatly contributed to the genome size expansion of this species. A total of 42,782 genes were annotated, and the patterns of gene loss in P. kansuensis differed from other parasitic species. We also found many mobile mRNAs between P. kansuensis and one of its host species, but these mobile mRNAs could not compensate for the functional losses of missing genes in P. kansuensis. In addition, we identified nine horizontal gene transfer (HGT) events from rosids and monocots, as well as one single-gene duplication events from HGT genes, which differ distinctly from that of other parasitic species. Furthermore, we found evidence for HGT through transferring genomic fragments from phylogenetically remote host species. Taken together, these findings provide genomic insights into the evolution of facultative parasites and broaden our understanding of the diversified genome evolution in parasitic plants and the molecular mechanisms of plant parasitism.

A Review: Cytochrome P450 in Alcoholic and Non-Alcoholic Fatty Liver Disease.

Alcoholic fatty liver disease (FALD) and non-alcoholic fatty liver disease (NAFLD) have similar pathological spectra, both of which are associated with a series of symptoms, including steatosis, inflammation, and fibrosis. These clinical manifestations are caused by hepatic lipid synthesis and metabolism dysregulation and affect human health. Despite having been studied extensively, targeted therapies remain elusive. The Cytochrome P450 (CYP450) family is the most important drug-metabolising enzyme in the body, primarily in the liver. It is responsible for the metabolism of endogenous and exogenous compounds, completing biological transformation. This process is relevant to the occurrence and development of AFLD and NAFLD. In this review, the correlation between CYP450 and liver lipid metabolic diseases is summarised, providing new insights for the treatment of AFLD and NAFLD.

Physiological and biochemical characteristics of the carbon ion beam irradiation-generated mutant strain Clostridium butyricum FZM 240 in vitro and in vivo.

Clostridium butyricum (C. butyricum) represents a new generation of probiotics, which is beneficial because of its good tolerance and ability to produce beneficial metabolites, such as short-chain fatty acids and enzymes; however, its low enzyme activity limits its probiotic efficacy. In this study, a mutant strain, C. butyricum FZM 240 was obtained using carbon ion beam irradiation, which exhibited greatly improved enzyme production and tolerance. The highest filter paper, endoglucanase, and amylase activities produced by C. butyricum FZM 240 were 125.69 U/mL, 225.82 U/ mL, and 252.28 U/mL, which were 2.58, 1.95, and 2.21-fold higher, respectively, than those of the original strain. The survival rate of the strain increased by 11.40 % and 5.60 % after incubation at 90 °C for 5 min and with simulated gastric fluid at pH 2.5 for 2 h, respectively, compared with that of the original strain. Whole-genome resequencing and quantitative real-time PCR(qRT-PCR) analysis showed that the expression of genes related to enzyme synthesis (GE000348, GE001963 and GE003123) and tolerance (GE001114) was significantly up-regulated, while that of genes related to acid metabolism (GE003450) was significantly down-regulated. On this basis, homology modeling and functional prediction of the proteins encoded by the mutated genes were performed. According to the results, the properties related to the efficacy of C. butyricum as a probiotic were significantly enhanced by carbon ion beam irradiation, which is a novel strategy for the application of Clostridium spp. as feed additives.

Turn Waste Golden Tide into Treasure: Bio-Adsorbent Synthesis for CO2 Capture with K2FeO4 as Catalytic Oxidative Activator.

Converting Sargassum horneri (SH)-a harmful marine stranding that can cause golden tide-to highly porous bio-adsorbent material (via one-step catalytic oxidative pyrolysis with K2FeO4) can be a strategically useful method for obtaining low-cost materials suitable for CO2 capture. In this manuscript, the behavior of different mass ratios of K2FeO4/SH precursor acting on the surface physicochemical properties of carbon materials are reported. The results suggest that specific surface area and total pore volume first increased to the mass ratio of K2FeO4/carbon precursor, then decreased. Among the samples prepared, the highest specific surface area was obtained with a K2FeO4/SH precursor ratio of 1:4 (25%-ASHC), and the CO2 adsorption performance was significantly increased and faster compared with the original biochar. The fitted values of the three kinetic models showed that the double exponential model provided the best description of carbon adsorption, indicating both physical and chemical adsorption; 25%-ASHC also exhibited excellent cyclic stability. The improved CO2 adsorption performance observed after K2FeO4 activation is mainly due to the increase in material porosity, specific surface area, and the enrichment of nitrogen and oxygen functional groups.

San Huang Xiao Yan recipe modulates the HMGB1-mediated abnormal inflammatory microenvironment and ameliorates diabetic foot by activating the AMPK/Nrf2 signalling pathway.

BACKGROUND: Diabetic foot (DF) is one of the serious complications of diabetes and lacks of therapeutic drugs. Abnormal and chronic inflammation promoting foot infection and wound healing delay are the main pathogenesis of DF. The traditional prescription San Huang Xiao Yan Recipe (SHXY) has been used in the clinical treatment of DF for several decades as approved hospital experience prescription and showed remarkable therapeutic effect, but the mechanisms by which SHXY treats DF are still unclear. PURPOSE: Objectives of this study were to investigate SHXY anti-inflammatory effect on DF and explore the molecular mechanism for SHXY. METHODS: We detected the effects of SHXY on DF in C57 mouse and SD rat DF models. Animal blood glucose, weight and wound area were detected every week. Serum inflammatory factors were detected by ELISA. H&E and Masson's trichrome were used to observe tissue pathology. Single-cell sequencing data reanalysis revealed the role of M1 macrophages in DF. Venn analysis showed the co-target genes between DF M1 macrophages and compound-disease network pharmacology. Western blotting was used to explored target protein expression. Meanwhile, RAW264.7 cells were treated with drug-containing serum of SHXY to further unravel the roles of target proteins during high glucose-induced inflammation in vitro. The Nrf2 inhibitor ML385 was used on RAW 264.7 cells to further explore the relationship between Nrf2, AMPK and HMGB1. The main components of SHXY were analysed by HPLC. Finally, the treatment effect of SHXY on DF were detected on rat DF model. RESULTS: In vivo, SHXY can ameliorate inflammatory, accelerate wound healing and upregulate expression of Nrf2, AMPK and downregulate of HMGB1. Bioinformatic analysis showed that M1 macrophages were the main inflammatory cell population in DF. Moreover, the Nrf2 downstream proteins HO-1 and HMGB1 were potential DF therapeutic targets for SHXY. In vitro, we also found that SHXY increased AMPK and Nrf2 protein levels and downregulated HMGB1 expression in RAW264.7 cells. Inhibiting the expression of Nrf2 impaired the inhibition effect of SHXY on HMGB1. SHXY promoted Nrf2 translocation into the nucleus and increased the phosphorylation of Nrf2. SHXY also inhibited HMGB1 extracelluar release under high glucose. In rat DF models, SHXY also exhibited significant anti-inflammatory effect. CONCLUSION: The SHXY activated AMPK/Nrf2 pathway to suppress abnormal inflammation on DF via inhibiting HMGB1 expression. These findings provide novel insight into the mechanisms by which SHXY treats DF.

Lifestyle patterns, genetic susceptibility, and risk of valvular heart disease: a prospective cohort study based on the UK Biobank.

AIMS: Genetic and lifestyle factors are both major contributors to valvular heart disease (VHD). However, it is still uncertain whether genetic susceptibility alters the association between lifestyle and VHD. We aimed to investigate the association between lifestyle and VHD in different genetic risk backgrounds. METHODS AND RESULTS: A prospective cohort study was carried out on 499 341 participants without VHD at baseline. The assessment of lifestyle included smoking, alcohol consumption, diet, activity, and sleep. Genetic susceptibility was separately measured by polygenic risk scores (PRSs) and family history of cardiovascular disease (CVD). Cox regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (95%CIs) between lifestyle and VHD, as well as aortic stenosis (AS). During a median follow-up of 10.8 years, 12 983 incident VHD cases were diagnosed (incidence rate 2.46 per 1000 person-years), including 3527 AS cases (incidence rate 0.66 per 1000 person-years). The risk of VHD and AS decreased with healthier lifestyles (P value for trend <0.001). Compared to individuals with a unhealthy lifestyle, the HRs of VHD in intermediate and healthy lifestyle groups were 0.81 (0.76-0.86) and 0.81 (0.76-0.87). The negative association between healthy lifestyle and VHD events was independent of genetic risk (P for interaction between healthy lifestyle scores and PRSs/family history of CVD was 0.723/0.763). Similar findings were obtained in analyses of AS, and a stronger negative association was found. CONCLUSION: Our study reveals that adherence to a healthy lifestyle is significantly associated with a reduced risk of VHD especially AS, irrespective of genetic susceptibility. SUMMARY: Based on a cohort of around 490 000 participants, the study investigated the association between lifestyle and VHD under different stratifications of genetic risk. The study found that a healthy lifestyle was associated with a lower risk of VHD, particularly AS, independent of genetic risk. Our findings suggest that advance interventions for lifestyle may be an effective way to reduce the global burden of VHD.

Interactions between Candida albicans and the resident microbiota.

Candida albicans is a prevalent, opportunistic human fungal pathogen. It usually dwells in the human body as a commensal, however, once in its pathogenic state, it causes diseases ranging from debilitating superficial to life-threatening systemic infections. The switch from harmless colonizer to virulent pathogen is, in most cases, due to perturbation of the fungus-host-microbiota interplay. In this review, we focused on the interactions between C. albicans and the host microbiota in the mouth, gut, blood, and vagina. We also highlighted important future research directions. We expect that the evaluation of these interplays will help better our understanding of the etiology of fungal infections and shed new light on the therapeutic approaches.

Oxidative stress-triggered pyroptosis mediates Candida albicans susceptibility in diabetic foot.

An accumulating trend of research demonstrates that diabetic patients are susceptible to skin infections with Candida albicans, but the mechanism still remains unclear. The intense oxidative stress (OS) responses were occurred in the lesion of diabetic mice footpads after C. albicans infection. Localised skin infections would lead to more severe complications while the severity of the condition worsens or the inadequate treatment. Notably, in this study, through the investigation of murine diabetic footpad C. albicans infection model and molecular biotechnology, including histopathological staining, immunofluorescence (IF) staining, quantitative real-time PCR (qPCR), western blot (WB), flow cytometry (FCM), sandwich enzyme-linked immunosorbent assay (ELISA) assays, we found that intense OS responses in the footpad tissue not only mediated the activation of NF-κB protein complex, but also triggered downstream pyroptosis and apoptosis through NLRP3 inflammasome, which is one of the potential reasons for the severe condition of infectious skin injuries in diabetic mice. Caspase-1, a classical signal pathway protein in pyroptosis, could promote pore formation on cell membranes and the release of the cytokine after NLRP3 inflammasome activation. With intense immune-inflammatory responses, the organism also stimulates immune organs such as the spleen and lymph nodes to produce negative feedback regulation and generate CD4+CD25+Foxp3+ Treg cells to rectify the process. Therefore, combined with the results of this work, it is possible to design and screen relevant drugs for NLRP3 inflammasomes as core targets to keep the OS response at a low level in the footpad tissues.

Ruan Jian Qing Mai Recipe Inhibits the Inflammatory Response in Acute Lower Limb Ischemic Mice through the JAK2/STAT3 Pathway.

Ruan jian qing mai recipe (RJQM) is an empirical prescription for treating arteriosclerosis obliterans (ASO). However, the mechanism of RJQM recipe-mediated ASO attenuation has not yet been elucidated. Therefore, this study aimed to explore the mechanism by which the RJQM recipe relieves ASO in a mouse model of lower limb ischemia, which was established by ligating and breaking the femoral artery of the left lower limb. The surgical groups were divided into the ischemic group, beraprost sodium group, low-dose RJQM group, medium-dose RJQM group, and high-dose RJQM group. Normal mice were set as the control group. The blood flow of the lower limb was examined on days 7 and 14. At the end of animal procedures, blood samples were collected, and the rectus femoris of the left lower limb were harvested. Results revealed that mice in the ischemic group demonstrated low blood flow. Additionally, hematoxylin and eosin, and Masson staining results showed that inflammation of the rectus femoris was obvious in the ischemia group, and the level of fibrosis was increased. Blood flow was recovered in all treatment groups compared to the ischemic group, and the inflammatory infiltration and fibrosis of the rectus femoris were relieved after RJQM treatment. The serum levels of interleukin (IL)-17A and IL-21 were decreased, and the expression of JAK2/STAT3 proteins was inhibited in all RJQM treatment groups compared to the ischemia group. Furthermore, the improvement of IL-17A, IL-21, and rectus femoris fibrosis was more obvious with increasing treatment time. In conclusion, RJQM can effectively alleviate ASO and promote the recovery of lower limb blood flow by regulating the JAK2/STAT3 signaling pathway to reduce the inflammatory response.

Fisetin Attenuates Diabetic Nephropathy-Induced Podocyte Injury by Inhibiting NLRP3 Inflammasome.

Diabetic nephropathy (DN) is one of the primary complications of diabetes. Fisetin is a flavonoid polyphenol that is present in several vegetables and fruits. The present study investigated the mechanisms of fisetin in DN-induced podocyte injury both in vitro and in vivo. The results revealed that fisetin ameliorated high glucose (HG)-induced podocyte injury and streptozotocin (STZ)-induced DN in mice. CDKN1B mRNA expression in the glomeruli of patients with DN decreased based on the Nephroseq dataset, and fisetin reversed CDKN1B expression at mRNA and protein levels in a dose-dependent manner in podocytes and mice kidney tissues. Furthermore, fisetin suppressed the phosphorylation of P70S6K, a downstream target of CDKN1B, activated autophagosome formation, and inhibited Nod-like receptor protein 3 (NLRP3) inflammasomes. Interfering CDKN1B reduced the protective effects of fisetin against high glucose-induced podocyte injury. Molecular docking results revealed a potential interaction between fisetin and CDKN1B. In summary, the present study revealed that fisetin alleviated high glucose-induced podocyte injury and STZ-induced DN in mice by restoring autophagy-mediated CDKN1B/P70S6K pathway and inhibiting NLRP3 inflammasome.

Recognition of Surface Oxygen Intermediates on NiFe Oxyhydroxide Oxygen-Evolving Catalysts by Homogeneous Oxidation Reactivity.

NiFe oxyhydroxide is one of the most promising oxygen evolution reaction (OER) catalysts for renewable hydrogen production, and deciphering the identity and reactivity of the oxygen intermediates on its surface is a key challenge but is critical to the catalyst design for improving the energy efficiency. Here, we screened and utilized in situ reactive probes that can selectively target specific oxygen intermediates with high rates to investigate the OER intermediates and pathway on NiFe oxyhydroxide. Most importantly, the oxygen atom transfer (OAT) probes (e.g., 4-(diphenylphosphino) benzoic acid) could efficiently inhibit the OER kinetics by scavenging the OER intermediates, exhibiting lower OER currents, larger Tafel slopes, and larger kinetic isotope effect (KIE) values, while probes with other reactivities demonstrated much smaller effects. Combining the OAT reactivity with electrochemical kinetic and operando Raman spectroscopic techniques, we identified a resting Fe═O intermediate in the Ni-O scaffold and a rate-limiting O-O chemical coupling step between a Fe═O moiety and a vicinal bridging O. DFT calculation further revealed a longer Fe═O bond formed on the surface and a large kinetic energy barrier of the O-O chemical coupling step, corroborating the experimental results. These results point to a new direction of liberating lattice O and expediting O-O coupling for optimizing NiFe-based OER electrocatalyst.

MYB transcription factors in alfalfa (Medicago sativa): genome-wide identification and expression analysis under abiotic stresses.

BACKGROUND: Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. Its survival and production are often hampered by environmental changes. However, there are few studies on stress-resistance genes in alfalfa because of its incomplete genomic information and rare expression profile data. The MYB proteins are characterized by a highly conserved DNA-binding domain, which is large, functionally diverse, and represented in all eukaryotes. The role of MYB proteins in plant development is essential; they function in diverse biological processes, including stress and defense responses, and seed and floral development. Studies on the MYB gene family have been reported in several species, but they have not been comprehensively analyzed in alfalfa. METHODS: To identify more comprehensive MYB transcription factor family genes, the sequences of 168 Arabidopsis thaliana, 430 Glycine max, 185 Medicago truncatula, and 130 Oryza sativa MYB proteins were downloaded from the Plant Transcription Factor Database. These sequences were used as queries in a BLAST search against the M. sativa proteome sequences provided by the Noble Research Institute. RESULTS: In the present study, a total of 265 MsMYB proteins were obtained, including 50 R1-MYB, 186 R2R3-MYB, 26 R1R2R3-MYB, and three atypical-MYB proteins. These predicted MsMYB proteins were divided into 12 subgroups by phylogenetic analysis, and gene ontology (GO) analysis indicated that most of the MsMYB genes are involved in various biological processes. The expression profiles and quantitative real-time PCR analysis indicated that some MsMYB genes might play a crucial role in the response to abiotic stresses. Additionally, a total of 170 and 914 predicted protein-protein and protein-DNA interactions were obtained, respectively. The interactions between MsMYB043 and MSAD320162, MsMYB253 and MSAD320162, and MsMYB253 and MSAD308489 were confirmed by a yeast two-hybrid system. This work provides information on the MYB family in alfalfa that was previously lacking and might promote the cultivation of stress-resistant alfalfa.

Baicalin relaxes vascular smooth muscle and lowers blood pressure in spontaneously hypertensive rats.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin is the most abundant flavone compound present in Scutellaria baicalensis Georgi and endothelium-dependent vascular activities of baicalin have been suggested. However, the pharmacological implications and mechanisms of baicalin under hypertensive conditions remain to be investigated. The current study examined the blood pressure-lowering effect of baicalin in a spontaneously hypertensive rat (SHR) model. Moreover, vascular activities and mechanisms of baicalin were investigated under hypertensive conditions. The results demonstrate that baicalin treatment lowers the blood pressure in SHRs in vivo. Ex vivo vascular reactivity assay reveals that baicalin relaxes phenylephrine (PE)-constricted SHR aortas in an endothelium-independent manner. Baicalin attenuates Angiotensin II (Ang II) or potassium chloride (KCl)-induced vasoconstriction in SHR aortas as well. Baicalin also relaxes SHR aortas in the presence of different Ca2+ channel blockers such as nifedipine and SKF96365 in response to PE-induced constriction. Most importantly, ATP-sensitive potassium channel (KATP) blockade partially abrogated the vasorelaxant effect of baicalin. In summary, the current study demonstrates for the first time that intracellular Ca2+ regulation in vascular smooth muscle is mechanistically implicated in the vasorelaxant effect of baicalin under hypertensive conditions. Furthermore, activated KATP channels are in part required for the vasorelaxant effect of baicalin under hypertensive conditions. Thus, the work here sheds novel pharmacological and mechanistic insights into the blood pressure-lowering effect of baicalin, which may help better understand the therapeutic application of Scutellaria baicalensis Georgi in the treatment of hypertension.

Mitochondrial phylogeny, divergence history and high-altitude adaptation of grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) inhabiting the Tibetan Plateau.

Grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) are the most important pests in alpine meadows of the Tibetan Plateau (TP) and have well adapted to high-altitude environments. To further understand the evolutionary history and their adaptation to the TP, we newly determined seven complete TP Gynaephora mitogenomes. Compared to single genes, whole mitogenomes provided the best phylogenetic signals and obtained robust results, supporting the monophyly of the TP Gynaephora species and a phylogeny of Arctiinae + (Aganainae + Lymantriinae). Incongruent phylogenetic signals were found among single mitochondrial genes, none of which recovered the same phylogeny as the whole mitogenome. We identified six best-performing single genes using Shimodaira-Hasegawa tests and found that the combinations of rrnS and either cox1 or cox3 generated the same phylogeny as the whole mitogenome, indicating the phylogenetic potential of these three genes for future evolutionary studies of Gynaephora. The TP Gynaephora species were estimated to radiate on the TP during the Pliocene and Quaternary, supporting an association of the diversification and speciation of the TP Gynaephora species with the TP uplifts and associated climate changes during this time. Selection analyses revealed accelerated evolutionary rates of the mitochondrial protein-coding genes in the TP Gynaephora species, suggesting that they accumulated more nonsynonymous substitutions that may benefit their adaptation to high altitudes. Furthermore, signals of positive selection were detected in nad5 of two Gynaephora species with the highest altitude-distributions, indicating that this gene may contribute to Gynaephora's adaptation to divergent altitudes. This study adds to the understanding of the TP Gynaephora evolutionary relationships and suggests a link between mitogenome evolution and ecological adaptation to high-altitude environments in grassland caterpillars.

A small set of differentially expressed genes was associated with two color morphs in natural populations of the pea aphid Acyrthosiphon pisum.

Color polymorphism is an ecologically important trait, which is related to local adaptation and ecological speciation. The pea aphid Acyrthosiphon pisum shows color polymorphism: the red and green color morphs where differences in ecological adaptation have been observed. Here, we measured genome-wide gene expression profiles of two color morphs in natural populations of A. pisum to explore the genetic basis of differentiated ecological adaptation. The results showed that only 32 genes were significantly differentially expressed between the two morphs, of which 18 had functional annotations. Among them, 13 genes were up-regulated [e.g. genes encoding protoheme IX farnesyltransferase (LOC100570971), carotene dehydrogenase (tor) and V-type proton ATPase subunit B (LOC100169462)] and 5 genes were down-regulated in the red morph (e.g. genes encoding transcription factors and heat shock proteins). To assess the functional importance of these differentially expressed genes (DEGs), we selected three highly expressed DEGs (LOC100169462, LOC100570971 and tor) with functional annotations and analyzed their expression levels in the red morph under three low temperatures (1 °C, 4 °C, and 8 °C) for 24 h. These three DEGs showed an interesting expression response to the cold acclimating conditions which resulted in an obvious phenotypic change of the red individuals to be greenish variants. This study suggests a link between gene expressions and body color polymorphisms in the pea aphid and provides important clues for further studying molecular mechanisms of ecological adaptation in aphids.

Two new phenols from Lysimachia patungensis.

Two new phenols, methyl 3-(2-O-ß-d-glucopyranosyl-3-hydroxy-5-methoxyphenyl) propionate (1) and myricetin-3,3',5'-tri-O-α-l-rhamnopyranoside (2), together with six known phenols compounds (3-8), were isolated from the whole plant of Lysimachia patungensis Hand.-Mazz. Their structures were elucidated on the basis of the interpretation of spectroscopic data, viz., ESI-MS, HR-TOF-MS, UV, IR, and NMR. All the known phenols were isolated from the genus Lysimachia for the first time. A preliminary bioassay revealed that compounds 3 and 7 exhibited significant protective effects against hydrogen peroxide-induced damage in human retinal endothelial cells (HRECs) with the concentration of 10 µM, respectively. Compound 1 showed moderate activity against the HRECs damage at 100 µM.