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Extracellular vesicles (EVs) are membrane vesicles secreted by cells, playing critical roles in mediating intercellular communications for various physiological and pathological processes. Most of the EV analysis is currently performed at the bulk level, obscuring the origin of the EVs and diverse characteristics of the individual extracellular vesicle. Technologies to analyze the extracellular vesicles at the single-cell and single-vesicle levels are needed to evaluate EV comprehensively and decode the heterogeneity underlying EV secretion. Microfluidic platforms that could control and manipulate fluids at the microscale provide an efficient way to achieve the aims. Various microfluidics-based technologies are emerging to realize single-cell EV secretion analysis and single EV analysis, which would be summarized in this mini-review.

Rare earth-doped upconversion nanoparticles (UCNPs) have the advantages of a narrow emission peak, large anti-Stokes displacement, negligible autofluorescence and photobleaching, which make them popular in biomedical fields. With the development of biological technology and nanotechnology, great progress has been achieved in use of rare earth-doped UCNPs in the treatment and diagnosis of various diseases. To date, based on the light conversion characteristics of upconverted rare earth nanocrystals, their medical applications are mainly focused on the detection and treatment of tumors. In view of the above situation, this paper focuses on reviewing UCNPs applications for the treatment and detection of various diseases, including atherosclerosis, inflammation, bacterial infections, viruses, and tumors. Finally, challenges and opportunities regarding the use of UCNPs in diseases treatment and detection are discussed. Overall, our goal is to provide readers with a fresh perspective on recent developments in the treatment and detection of various diseases with rare earth-doped upconversion materials.

Blood vessel dysfunction causes several retinal diseases, including diabetic retinopathy, familial exudative vitreoretinopathy, macular degeneration and choroidal neovascularization in pathological myopia. Vascular endothelial growth factor (VEGF)-neutralizing proteins provide benefits in most of those diseases, yet unsolved haemorrhage and frequent intraocular injections still bothered patients. Here, we identified endothelial CD146 as a new target for retinal diseases. CD146 expression was activated in two ocular pathological angiogenesis models, a laser-induced choroid neovascularization model and an oxygen-induced retinopathy model. The absence of CD146 impaired hypoxia-induced cell migration and angiogenesis both in cell lines and animal model. Preventive or therapeutic treatment with anti-CD146 antibody AA98 significantly inhibited hypoxia-induced aberrant retinal angiogenesis in two retinal disease models. Mechanistically, under hypoxia condition, CD146 was involved in the activation of NFκB, Erk and Akt signalling pathways, which are partially independent of VEGF. Consistently, anti-CD146 therapy combined with anti-VEGF therapy showed enhanced impairment effect of hypoxia-induced angiogenesis in vitro and in vivo. Given the critical role of abnormal angiogenesis in retinal and choroidal diseases, our results provide novel insights into combinatorial therapy for neovascular fundus diseases.

The objective of this experiment was to investigate the potential benefits of active dry yeast (ADY) on the growth performance, rumen fermentation, nutrient digestibility, and serum parameters of weaned beef calves. Thirty Simmental crossbred male calves (body weight = 86.47 ± 4.41 kg and 70 ± 4 d of age) were randomly divided into 2 groups: control (CON) (fed basal ration) and ADY (fed basal ration and 5 g/d ADY per calf). The dietary concentrate-to-roughage ratio was 35:65. All the calves were regularly provided rations 3 times a day at 07:00, 13:00, and 19:00 and had free access to water. The experiment lasted for 60 d. The average daily gain of ADY group was higher (P = 0.007) than that of the CON group, and the ratio of feed intake to average daily gain in the ADY group was reduced (P = 0.022) as compared to the CON group. The concentration of ruminal ammonia-N was higher (P = 0.023) in the CON group than that in the ADY group, but an opposite trend of microbial protein was found between the 2 groups. Also, the ruminal concentrations of propionate and butyrate were higher (P

Recent evidence has shown that methionine (Met) supplementation can improve milk protein synthesis under hyperthermia (which reduces milk production). To explore the mechanism by which milk protein synthesis is affected by Met supplementation under hyperthermia, mammary alveolar (MAC-T) cells were incubated at a hyperthermic temperature of 42°C for 6 h in media with different concentrations of Met. While the control group (CON) contained a normal amino acid concentration profile (60 μg/mL of Met), the three treatment groups were supplemented with Met at concentrations of 10 μg/mL (MET70, 70 μg/mL of Met), 20 μg/mL (MET80, 80 μg/mL of Met), and 30 μg/mL (MET90,90 μg/mL of Met). Our results show that additional Met supplementation increases the mRNA and protein levels of BCL2 (B-cell lymphoma-2, an anti-apoptosis agent), and decreases the mRNA and protein levels of BAX (Bcl-2-associated X protein, a pro-apoptosis agent), especially at an additional supplementary concentration of 20 μg/mL (group Met80). Supplementation with higher concentrations of Met decreased the mRNA levels of Caspase-3 and Caspase-9, and increased protein levels of heat shock protein (HSP70). The total protein levels of the mechanistic target of rapamycin (mTOR) and the mTOR signalling pathway-related proteins, AKT, ribosomal protein S6 kinase B1 (RPS6KB1), and ribosomal protein S6 (RPS6), increased with increasing Met supplementation, and peaked at 80 μg/mL Met (group Met80). In addition, we also found that additional Met supplementation upregulated the gene expression of αS1-casein (CSN1S1), β-casein (CSN2), and the amino acid transporter genes SLC38A2, SLC38A3 which are known to be mTOR targets. Additional Met supplementation, however, had no effect on the gene expression of κ-casein (CSN3) and solute carrier family 34 member 2 (SLC34A2). Our results suggest that additional Met supplementation with 20 μg/mL may promote the synthesis of milk proteins in bovine mammary epithelial cells under hyperthermia by inhibiting apoptosis, activating the AKT-mTOR-RPS6KB1 signalling pathway, and regulating the entry of amino acids into these cells.

This study was conducted to examine the influence of uni and bilateral castration on growth performance and lipid metabolism in yellow cattle. Eighteen 9-month-old healthy yellow cattle (average body weight 184.03 ± 4.09 kg) were selected and divided into three groups: The uncastrated cattle (C), half castrated cattle (HC) and full castrated cattle (FC). The results showed that the growth rate of FC group was significantly reduced as compared to HC and C group, while the feed to gain ratio exhibited an opposite trend. The concentrations of triglycerides (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein (HDL) were increased significantly in FC group from day 60 to the end of the trial compared to HC and control groups. Serum testosterone concentration of FC group cattle was decreased from day 60 to 120 d of the trial compared to HC and control groups. The concentration of the lauric acid in FC cattle was significantly increased from the HC and control groups. In the FC group, the acetyl-CoA carboxylase alpha (ACACA), ACC and fatty acid synthase (FAS) gene expression levels were significantly higher compared to control and HC groups. Our results of this study suggest that bilateral castration increased the lipid metabolism and fatty acid composition compared to unilateral castrated and un-castrated cattle.HighlightsBilateral castration alters the growth performance in yellow cattle.Bilateral castration alters hormones levels and lipid metabolites levels in serum.Bilateral castration improves the lipid metabolism and fatty acid profile.

Background Night-break (NB) has been proven to repress flowering of short-day plants (SDPs). Long-noncoding RNAs (lncRNAs) play key roles in plant flowering. However, investigation of the relationship between lncRNAs and NB responses is still limited, especially in Chenopodium quinoa, an important short-day coarse cereal. Results In this study, we performed strand-specific RNA-seq of leaf samples collected from quinoa seedlings treated by SD and NB. A total of 4914 high-confidence lncRNAs were identified, out of which 91 lncRNAs showed specific responses to SD and NB. Based on the expression profiles, we identified 17 positive- and 7 negative-flowering lncRNAs. Co-expression network analysis indicated that 1653 mRNAs were the common targets of both types of flowering lncRNAs. By mapping these targets to the known flowering pathways in model plants, we found some pivotal flowering homologs, including 2 florigen encoding genes (FT (FLOWERING LOCUS T) and TSF (TWIN SISTER of FT) homologs), 3 circadian clock related genes (EARLY FLOWERING 3 (ELF3), LATE ELONGATED HYPOCOTYL (LHY) and ELONGATED HYPOCOTYL 5 (HY5) homologs), 2 photoreceptor genes (PHYTOCHROME A (PHYA) and CRYPTOCHROME1 (CRY1) homologs), 1 B-BOX type CONSTANS (CO) homolog and 1 RELATED TO ABI3/VP1 (RAV1) homolog, were specifically affected by NB and competed by the positive and negative-flowering lncRNAs. We speculated that these potential flowering lncRNAs may mediate quinoa NB responses by modifying the expression of the floral homologous genes. Conclusions Together, the findings in this study will deepen our understanding of the roles of lncRNAs in NB responses, and provide valuable information for functional characterization in future.

It is increasingly recognized that the cellular microenvironment plays critical roles in regulating the fate and physiology of cells. Despite recent advancements in single-cell analysis technologies, engineering and integration of the microenvironment for single-cell analysis platforms remain limited. Here, we report a single-cell cytokine secretion analysis platform that integrated both the three-dimensional cell culture and the primary oral squamous cell carcinoma tumor cell co-culture to provide both physical and physiological cues for single cells to be analyzed. We apply the platform to investigate the immune responses of human macrophages stimulated with the ligand of toll-like receptor 4 lipopolysaccharide. Notably, we observe the differential modulation effect in cytokine secretions by the tumor microenvironment, in which antitumor cytokine TNF-a secretion was attenuated, and protumor cytokine IL-6 would increase. The differential modulation effect is conserved from cell line-derived macrophages to primary macrophages derived from healthy donors. Immunofluorescence staining further reveals that ∼50% of macrophage cells could be polarized from M1 to the M2 phenotype within 12 h in the engineered tumor microenvironment. This work demonstrates the significance of the cell microenvironment toward single-cell analysis, which could help to evaluate how immune cells will respond in the complex microenvironment more accurately.

A new trend is emerging that flexible batteries will play an indispensable role in the progress of social science and technology. However, flexibility exists only in a single direction for the existing electrode material. Searching for flexible battery materials has attracted more and more attention from researchers. In this article, the lattice structural stability, electronic structure modulation, and the Li adsorption properties of the heterostructures designed by assembling GeP3 and NbX2 (X = S, Se) together were methodically explored based on van der Waals. We found that diffusion barrier of the GeP3/NbS2 heterostructure with metallic properties is 0.21 eV for Li. It greatly improves the charge and discharge performance of the battery. The predicted heterostructure shows quite high theoretical specific capacity with 540.24 mA h/g, which is higher than the traditional graphite anode (372 mA h/g). It demonstrates superior isotropic flexibility with a considerable small Young's modulus (151.98-159.02 N/m), which has promising application as flexible electrodes for rechargeable battery equipment.

High thermal resistance frequently occurred at both filler-matrix and filler-filler interfaces lays down paramount bottlenecks for thermal management materials (TMMs). Herein, pony-size silver (Ag,...

The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol co-production from straw feedstocks.

The growth and ethanol fermentation of Saccharomyces cerevisiae are vulnerable to weak acid that coexists with sugar in lignocellulosic hydrolysate. In this study, the molecular basis of S. cerevisiae in response to acetic and formic acids was investigated when fermented with glucose and xylose at pH 5.0. Transcriptional profiling revealed that 294 (657 in total) differentially expressed genes (DEGs) were exclusive to mixed acetic and formic acids (Aa_Fa) group, the DEGs in acetic acid (Aa) and formic acid (Fa) group were 308 and 326, respectively. The general responses to Aa, Fa, and Aa_Fa mainly involved carbohydrate metabolism, amino acid metabolism, transcriptional regulation, and stress response. In Aa_Fa group, the enrichment ratio of each pathway was much higher than being exposed to Aa or Fa stress individually, and some enrichments not existed in Aa or Fa group were observed, suggesting the synergistic anti-inhibition responses of S. cerevisiae was triggered. In yeast cells, bioconversion, stress response, and transmembrane transport could be activated to resist acetic and formic acids accompanying with energy consumption. Except the putative transcriptional factor (TF) - Haa1p, the other three TFs, Hap4p, Yox1p, and Mag1p, might as well be considered to participate in acetic and formic acids resistance responses.

This experiment was conducted to investigate the effects of live yeast and yeast cell wall polysaccharides on growth performance, rumen function and plasma lipopolysaccharides (LPS) content and immunity parameters of beef cattle. Forty Qinchuan cattle were randomly assigned to one of four treatments with 10 replicates in each treatment. The dietary treatments were: control diet (CTR), CTR supplemented with 1 g live yeast (2×1010 live cell g−1 per cattle per day (YST1), CTR supplemented with 2 g live yeast per cattle per day (YST2) and CTR supplemented with 20 g of yeast cell wall polysaccharides (30.0%≤β-glucan≤35.0%, and 28.0%≤mannanoligosaccharide≤32.0%) per cattle per day (YCW). The average daily gain was higher (P=0.023) and feed conversion ratio was lower (P=0.042) for the YST2 than the CTR. The digestibility of neutral detergent fiber (P=0.039) and acid detergent fiber (P=0.016) were higher in yeast supplemented groups. The acetic acid:propionic acid of the YST2 was lower compared with the CTR (P=0.033). Plasma LPS (P=0.032), acute phase protein haptoglobin (P=0.033), plasma amyloid A (P=0.015) and histamine (P=0.038) were lower in the YST2 compared with the CTR. The copies of fibrolytic microbial populations such as Fibrobacter succinogenes S85, Ruminococcus albus 7 and Ruminococcus flavefaciens FD-1 of the YST2 were higher (P

The development of non-noble, efficient, and durable catalysts to replace expensive Pt-based catalysts for boosting the oxygen reduction reaction (ORR) is always an imperative and challenging topic. Herein, a triazine-linked covalent framework was prepared by using a cobalt-centered porphyrin derivative via a facile ionothermal approach, which was readily converted to a series of porous carbons (termed Co/N-PCs) with high surface areas upon controlled thermal treatment. The cobalt atoms coordinated with nitrogen were uniformly dispersed within the resulting porous structures. Accordingly, such as-prepared Co/N-PCs exhibited high activity for the ORR with a positive onset potential of 0.91 V (vs. RHE) and half-wave potential of 0.83 V, as well as good methanol tolerance and long-time durability. When used as air cathodes for Zn–air batteries, high power density (158.9 mW cm−2) and specific capacity (797 mA h g−1) were achieved, outperforming commercial Pt/C.

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

Anaplastic thyroid carcinoma (ATC), as one of the most aggressive human malignancies, cannot be cured by 131iodine (131I) internal radiotherapy (RT) because the tumor cells cannot effectively take up 131I and are resistant to radiotherapy. In this study, a facile and simple method was proposed to synthesize mesoporous polydopamine nanoparticles (MPDA) and tailor their morphologies by component-adjusting Pluronic micelle-guided polymerization. Then, MPDA were used not only as nanocarriers to radiolabel 131I, but also as photothermal conversion agents for photothermal therapy (PTT) to promote RT. The iodine-labeling capacity and photothermal conversion efficiency of MPDA can be enhanced by optimizing their morphologies. It was found that MPDA NPs with a cerebroid pore channel structure (CPDA) showed the highest iodine-carrying capacity and a higher photothermal conversion efficiency as a result of their maximum specific surface area and unique morphology. In subsequent experiments in vitro and in vivo, our ATC animal models showed impressive therapeutic responses to CPDA-131I NPs because of the synergistic effect of PTT and RT. Additionally, CPDA-125I NPs can be utilized to obtain high-quality SPETC/CT images of tumors, which can guide clinical therapy for ATC. Considering their great biosafety, these radioiodine-labeled CPDA NPs may serve as a promising tool in combined therapy and diagnosis in ATC.

This study aimed to investigate the effects of N-carbamylglutamate (NCG) supplementation on the follicular development of yaks to identify potential mechanisms essential for fertility in yaks. Twelve multiparous anoestrous female yaks were randomly assigned to two groups—Control (fed with a basal diet, n = 6) and NCG (basal diet supplemented with 6.0 g day−1 NCG, n = 6). Yaks in the NCG group had higher numbers of large follicles (&gt, 5 mm in diameter) than those in the Control group. An RNA-sequencing analysis of yak ovaries revealed a total of 765 genes were differentially expressed between experimental groups, of which 181 genes were upregulated and 584 genes were downregulated following NCG supplementation. The results of a transcriptome functional analysis, qRT-PCR validation, and immunohistochemistry revealed that NCG supplementation increased angiogenesis and de novo synthesis of cholesterol in yak ovaries. NCG was also found to upregulate the gene expression of steroidogenic enzymes. Based on this, it was concluded that NCG supplementation promotes the follicular development of yaks mainly by affecting cholesterol metabolism to initiate steroidogenesis in ovaries. The results provide evidence for understanding the mechanisms responsible for NCG promoting follicular development of female yaks, which may contribute to the development and application of NCG in animal reproduction.

Proteinase K (PRK) is a proteolytic enzyme that has been widely used in industrial applications. However, poor stability has severely limited the uses of PRK. In this work, we used two structure-guided rational design methods, Rosetta and FoldX, to modify PRK thermostability. Fifty-two single amino acid conversion mutants were constructed based on software predictions of residues that could affect protein stability. Experimental characterization revealed that 46% (21 mutants) exhibited enhanced thermostability. The top four variants, D260V, T4Y, S216Q, and S219Q, showed improved half-lives at 69°C by 12.4-, 2.6-, 2.3-, and 2.2-fold that of the parent enzyme, respectively. We also found that selecting mutations predicted by both methods could increase the predictive accuracy over that of either method alone, with 73% of the shared predicted mutations resulting in higher thermostability. In addition to providing promising new variants of PRK in industrial applications, our findings also show that combining these programs may synergistically improve their predictive accuracy.

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

Bacillus thuringiensis (Bt) are entomopathogenic bacteria that produce different kinds of insecticidal proteins. However, studies on Bt exopolysaccharides are lacking. Here, we aimed to explore the characteristics and insecticidal synergism of EPS, an exopolysaccharide from Bt strain 4D19. The molecular weight of EPS-2 was 58.0 kDa, which consisted of mannose (44.2%), GlcN (35.5%), D-GalN (8.0%), glucose (5.5%), arabinose (5.1%), galactose (0.9%), Man-UA (0.3%) and Glc-UA (0.2%). The toxicity of insecticidal proteins against Plutella xylostella was increased by adding EPS. EPS-2 bound to Cry1Ac protoxin and promoted the binding of Cry1Ac protoxin to the gut membrane of P. xylostella, but did not bind to activated toxins. These results suggested that EPS-2 may bind to the protoxin C-terminal region to enhance insecticidal activity. Our findings indicated that Bt strains produce exopolysaccharide to enhance the toxicity of insecticidal crystal proteins, which could be applied in biopesticide research and product development.

This paper details a new method for characterizing the interfacial bonding performance of short-fiber reinforced rubber composites and its correlation with fatigue properties. Meanwhile, the experimental study of interfacial damage during the fatigue process is presented. Aramid fiber (AF) was modified with an aqueous solution of lithium chloride and then coated with butadiene styrene vinyl-pyridine rubber latex (VPL) or maleated polybutadiene liquid rubber (MLPB). Subsequently, the coated AF was introduced into a butadiene styrene rubber (SBR) matrix to fabricate reinforced butadiene styrene rubber/carbon black/aramid fiber (SBR/CB/AF) composites. Atomic force microscopy (AFM) was employed to identify the flexible interface layer of the SBR composites. After 30,000 fatigue cycles, the modulus of the interface layer was obviously reduced to below that of the SBR matrix, which produced hysteresis of the reinforcing effect of AF due to concentrated damage in the interface layer. The relative debonding energy (RDE) is innovatively introduced to characterize the interfacial adhesion performance. A higher RDE value can lead to the interface layer absorbing more deformation work and a longer fatigue life of the composite. SBR reinforced by T-AF/MLPB, with its high modulus interface, has the longest fatigue life, corresponding to the highest RDE value.

A heterologous xylose utilization pathway, either xylose reductase-xylitol dehydrogenase (XR-XDH) or xylose isomerase (XI), is usually introduced into Saccharomyces cerevisiae to construct a xylose-fermenting strain for lignocellulosic ethanol production. To investigate the molecular basis underlying the effect of different xylose utilization pathways on the xylose metabolism and ethanol fermentation, transcriptomes of flocculating industrial strains with the same genetic background harboring different xylose utilization pathways were studied. A different source of xylA did not obviously affect the change of the strains transcriptome, but compared with the XR-XDH strain, several key genes in the central carbon pathway were downregulated in the XI strains, suggesting a lower carbon flow to ethanol. The carbon starvation caused by lower xylose metabolism in XI strains further influenced the stress response and cell metabolism of amino acid, nucleobase, and vitamin. Besides, the downregulated genes mostly included those involved in mitotic cell cycle and the cell division-related process. Moreover, the transcriptomes analysis indicated that the after integrate xylA in the δ region, the DNA and chromosome stability and cell wall integrity of the strains were affected to some extent. The aim of this was to provide some reference for constructing efficient xylose-fermenting strains.

Molecular simulations in the canonical ensemble were performed to probe a variety of thermophysical properties of both homogeneously stretched and bubbly water systems at a temperature of 298 K. Tw...

Transcriptional regulation involves a series of sophisticated protein-protein and protein-DNA interactions (PPI and PDI). Some transcriptional complexes, such as c-Fos/c-Jun and their binding DNA fragments, have been conserved over the past one billion years. Considering the thermodynamic principle for transcriptional complex formation, we hypothesized that the c-Fos/c-Jun complex may represent a thermodynamic summit in the evolutionary space. To test this, we invented a new method, termed One-Pot-seq, which combines cDNA display and proximity ligation to analyse PPI/PDI complexes simultaneously. We found that the wild-type c-Fos/c-Jun complex is indeed the most thermodynamically stable relative to various mutants of c-Fos/c-Jun and binding DNA fragments. Our method also provides a universal approach to detect transcriptional complexes and explore transcriptional regulation mechanisms.

Supramolecular chemistry provides a means to integrate multi-type molecules leading to a dynamic organization. The study of functional nanoscale drug-delivery systems based on supramolecular interactions is a recent trend. Much work has focused on the design of supramolecular building blocks and the engineering of supramolecular integration, with the goal of optimized delivery behavior and enhanced therapeutic effect. This review introduces recent advances in supramolecular designs of nanoscale drug delivery. Supramolecular affinity can act as a main driving force either in the self-assembly of carriers or in the loading of drugs. It is also possible to employ strong recognitions to achieve self-delivery of drugs. Due to dynamic controllable drug-release properties, the supramolecular nanoscale drug-delivery system provides a promising platform for precision medicine.

Hydrophobic PLLA porous materials exhibits excellent oil-removal performance, fabricated by a simple slow solvent-evaporation induced precipitation method, which is of significance for curbing oil-pollution environment. Therefore, porous PLLA materials with tunable microstructures (fibrous networks, microsphere and multiple structures) were conveniently manufactured by adjusting the preparation parameters, solution concentration and precipitation temperature. The results show the wettability of the prepared porous materials could be controlled by the tailorable hierarchical structures. The apparent contact angle of porous PLLA materials was 121°, significantly improved in contrast with 67° of the solvent-casting bulk PLLA film. Moreover, the PLLA materials with high porosity up to 48.4% were obtained from 5 wt% PLLA chloroform solution at the precipitating temperature of 30 °C. Predictably, due to the favorable porosity and the excellent surface hydrophobicity, the porous PLLA materials have great potential in the wild application of oil-water separation.

Using boron powder as additive, the preparation of zirconium diboride (ZrB2) by carbothermal reduction was investigated. The results show that the carbothermal reduction cannot be completely done until the temperature is more than 1900 °C. The ZrB2 particles prepared without boron (B) additive at 1900 °C for 3 h are rodlike and show a preferential grain growth along [001] direction. B additive changes the heat effect of the raw materials. With B additive, the morphology of ZrB2 particles turns to be regular shape. The average particle size is about 3.6 μm with 2.5 wt% B additives. With more B additive, the shape of particles turns to be round like and the average particle size is decreased to 2.3 μm when 5 wt% B is added. The existence of oxides in grain boundary is a key factor to keep ZrB2 ceramic from deep densification. Using ZrB2 powder prepared with 5 wt% B additives, by controlling carbon content in ZrB2 powder, ZrB2 ceramic with 93% relative density is hot-pressed.

Monte Carlo simulations are performed to predict the interfacial tension (IFT) for water/ n -dodecane, water/toluene, and water/(50 wt% n -dodecane + 50 wt% toluene) mixtures at a pressure of 1.83 MPa and temperatures ranging from 383.15 to 443.15 K. Simulations for the binary mixtures are performed in the N 1 N 2 p N A T ensemble and those for the ternary mixtures are performed in the N 1 μ 2 μ 3 p N A T ensemble. In order to control the composition of the organic phase for ternary mixture simulations, separate reservoirs for toluene and n -dodecane molecules are utilized, and the minimum and maximum numbers of these molecules in the interfacial box are monitored. Identity switch moves are applied to facilitate the sampling of the spatial distributions of the organic molecules. Calculations of the IFT for water/ n -decane and water/benzene mixtures are used to benchmark the force fields and to develop mixing rules. The combination of the TraPPE–UA force field and the TIP4P/2005 water model with Lorentz-Berthelot combining rules yields fairly accurate predictions at moderate temperatures for water/ n -decane, but appears to slightly overestimate the IFT at elevated temperatures. For the water/benzene mixtures, the TraPPE–EH//TIP4P/2005 combination significantly overestimates the IFT over the entire temperature range. Subsequent modifications of the energetic Lennard-Jones cross-interaction parameters yield good agreement between experimental and simulated IFT data of water/ n -decane and water/benzene. Using a preliminary set of modified cross-interaction parameters for the TraPPE–UA/EH//TIP4P/2005 combination, our predictions for the Industrial Fluid Properties Simulation Challenge yield satisfactory agreement with the benchmark data (mean signed errors of − 2.2 , − 5.1 , and − 0.1 dyn/cm for water/ n -dodecane, water/toluene, and water/(50 wt% n -dodecane + 50 wt% toluene), respectively). A new set of modified cross-interaction parameters developed later based on longer simulation trajectories and a larger set of experimental data is found to reduce the mean signed errors to − 0.1 , − 0.6 , and + 2.5 dyn/cm, respectively, for these three systems. Although a significant enrichment of toluene molecules at the interface is found for the ternary mixture, this enrichment is not sufficient to reproduce the experimental IFT data.

Three ruminally fistulated Xuanhan steers weighting 312.5 (±23.85) kg were used to determine the kinetics of ruminal degradation of nutrients using in situ nylon bag technique, and a modified 3-step in vitro procedure was adopted to estimate intestinal digestibility of 16-h rumen undegradable protein (RUP) of maize cob (MC), distillers grains (DG), spent mushroom substrate (SMS), starch residue of sweet potato (SRSP), citrus pulp (CPP), and rice straw (RS). Samples were incubated for 0, 2, 6, 16, 24, 36, 48 and 72 h. Additional samples were incubated for 16 h in the rumen, and the residues from these bags were transferred to the nitrogen-free polyester bags for determination of intestinal digestibility in vitro. The highest DM disappearance at 6-h incubation was in SRSP (P 0.07) on CP disappearance between DG and MC at all the time points except for 16 and 24 h. NDF and ADF disappearance for SRSP was significantly higher (P < 0.01) than other roughages at all the time points except for ADF at 72 h. The lowest NDF and ADF disappearance was in DG at all the time points (P < 0.01) except 2 and 6 h. The effective degradability (ED) of DM was the highest in CPP (P < 0.01) and the lowest in MC and RS (P < 0.01). The highest and lowest ED of CP was in DG and in RS (P < 0.01), respectively. The ED of NDF was the highest in SRSP (P < 0.01), followed by CPP and RS, and the lowest in DG (P < 0.01). The ED of ADF was the highest in SRSP and CPP (P < 0.05), and the lowest in DG (P < 0.01). For MC, DG SMS, SRSP, CPP, and RS, the intestinal digestibility of RUP was 95.28%, 37.23%, 38.72%, 48.06%, 54.49%, and 37.88%, respectively, and the content of intestinal digestible crude protein (IDCP) was 23.65, 83.63, 35.63, 15.03, 25.60, and 12.03 g/kg, respectively. Distillers grain was considered to be of good quality for the greatest content of IDCP. Although not readily degraded in rumen, CP in MC may be digested well in small intestine.

Monte Carlo simulations in the Gibbs ensemble were carried out to determine the mutual solubilities for water/n-alkane mixtures over a wide range of temperatures, pressures, and alkane chain length...

Supplementary feeding has a significant effect on the growth performance of grazing yaks. However, as far as is known, little information is available concerning how energy or protein feed supplementation affects the serum metabolome of grazing yaks during the warm season. We investigated the effects of supplementation with two different concentrates on the serum metabolome in grazing yaks using nuclear magnetic resonance spectroscopy in conjunction with multivariate data analysis. Twenty-four 2-year-old female yaks (133.04 ± 6.52 kg BW) were randomly divided into three groups and fed three different regimes (n = 8 per group): (1) grazing plus hull-less barley (HLB) supplementation, (2) grazing plus rapeseed meal (RSM) supplementation, and (3) grazing without supplementation. Both HLB and RSM supplementation significantly increased the average daily gain (ADG), and ADG under HLB supplementation was 11.9% higher (P < 0.05) than that of the RSM group. Supplementation markedly altered glucose, lipid, and protein metabolism, with the difference manifested as increased levels of some amino acids, acetyl-glycoproteins, low-density lipoproteins, and very low-density lipoproteins . Furthermore, the levels of 3-hydroxybutyrate, acetoacetate, and lactate metabolism were decreased. Serum metabolite changes in yaks in the HLB supplementation treatment differed from those in the RSM supplementation treatment; the difference was primarily manifested in lipid- and protein-related metabolites. We conclude that both the energy supplementation (HLB) and the protein supplementation (RSM) could remarkably promote the growth of yak heifers during the warm season, and the effect of energy supplementation was superior. Supplementary feeding changed the serum metabolite levels of yak heifers, indicating that such feeding could improve glucose's energy-supply efficiency and increase the metabolic intensity of lipids and proteins. Supplementation of yaks with HLB was more efficient in the promotion of yak glucose and protein anabolism compared to supplementation with RSM, while having a lesser effect on lipid metabolism.

Despite recent advances in single-cell analysis technologies, lacking simple methods to keep the live single-cells traceable for longitudinal detection reliably poses a significant obstacle in single-cell secretion analysis. Here we reported the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of ≥97% of trapped single cells in dedicated spatial locations during repetitive detection procedures, verified with both adherent and suspension cells by two researchers independently. We applied it in monitoring single-cell protein secretions sequentially from the same single cells, and we found the digital protein secretion patterns dominate the protein secretion. We also demonstrated the microchip for longitudinally tracking IL-8 and the CD81+EV secretions from the same single-cells over days, which revealed the presence of “super secretors” within the cell population be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for tracking sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.Table of Contents Graphic

Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of Saccharomyces cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) were revealed during mixed glucose and xylose fermentation. Carbohydrate metabolism pathways were significantly enriched in response to Aa, while pathways of xenobiotic biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, other pathways were activated in response to Aa_Fur, i.e., cofactor and vitamin metabolism and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rates by nearly 50%, while the ethanol yield was enhanced by nearly 8% under acetic acid and furfural stress conditions. Co-overexpression of Haa1p and Tye7p resulted in a 59% increase in xylose consumption rate and a 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed acetic acid and furfural. IMPORTANCE Inhibitor tolerance is essential for S. cerevisiae when fermenting lignocellulosic hydrolysate with various inhibitors, including weak acids, furans, and phenols. The details regarding how xylose-fermenting S. cerevisiae strains respond to multiple inhibitors during fermenting mixed glucose and xylose are still unknown. This study revealed the transcriptional regulation mechanism of an industrial xylose-fermenting S. cerevisiae strain in response to acetic acid and furfural. The transcription factor Haa1p was found to be involved in both acetic acid and furfural tolerance. In addition to Haa1p, four other transcription factors, Hap4p, Yox1p, Tye7p, and Mga1p, were identified as able to improve the resistance of yeast to these two inhibitors. This study underscores the feasibility of uncovering effective transcription factors for constructing robust strains for lignocellulosic bioethanol production.

International audience; Conversion-type batteries with electrode materials partially dissolved in a liquid electrolyte exhibit high specific capacity and excellent redox kinetics, but currently poor stability due to the shuttle effect. Using a solid-electrolyte separator to block the mass exchange between the cathode and the anode can eliminate the shuttle effect. A stable interface between the solid-electrolyte separator and the liquid electrolyte is essential for the battery performance. Here, we demonstrate that a stable interface with low interfacial resistance and limited side reactions can be formed between the sulfide solid-electrolyte β-Li(3)PS(4) and the widely used ether-based liquid electrolytes, under both reduction and oxidation conditions, due to the rapid formation of an effective protective layer of ether-solvated Li(3)PS(4) at the sulfide/liquid electrolyte interface. This discovery has inspired the design of a β-Li(3)PS(4)-coated solid-electrolyte Li(7)P(3)S(11) separator with a simultaneously high ion-conduction ability and good interfacial stability with the liquid electrolyte, so that hybrid lithium-sulfur (Li-S) batteries with this composite separator conserve a high discharge capacity of 1047 mA h g(-1) and a high second discharge plateau of 2.06 V after 150 cycles.

BackgroundXylitol accumulation is a major barrier for efficient ethanol production through heterologous xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in recombinantSaccharomyces cerevisiae. Mutated NADH-preferring XR is usually employed to alleviate xylitol accumulation. However, it remains unclear how mutated XR affects the metabolic network for xylose metabolism. In this study, haploid and diploid strains were employed to investigate the transcriptional responses to changes in cofactor preference of XR through RNA-seq analysis during xylose fermentation.ResultsFor the haploid strains, genes involved in xylose-assimilation (XYL1,XYL2,XKS1), glycolysis, and alcohol fermentation had higher transcript levels in response to mutated XR, which was consistent with the improved xylose consumption rate and ethanol yield. For the diploid strains, genes related to protein biosynthesis were upregulated while genes involved in glyoxylate shunt were downregulated in response to mutated XR, which might contribute to the improved yields of biomass and ethanol. When comparing the diploids with the haploids, genes involved in glycolysis and MAPK signaling pathway were significantly downregulated, while oxidative stress related transcription factors (TFs) were significantly upregulated, irrespective of the cofactor preference of XR.ConclusionsOur results not only revealed the differences in transcriptional responses of the diploid and haploid strains to mutated XR, but also provided underlying basis for better understanding the differences in xylose metabolism between the diploid and haploid strains.

With the increasing application of nanomaterials, evaluation of the phytotoxicity of nanoparticles has attracted considerable interest. Buckwheat is an economically pseudocereal crop, which is a potential model for investigating the response of plants to hazardous materials. In this study, the response of buckwheat to graphene oxide (GO) was investigated by integrating physiological and transcriptome analysis. GO can penetrate into buckwheat root and stem, and high concentrations of GO inhibited seedlings growth. High concentration of GO improved ROS production and regulated the activities and gene expression of oxidative enzymes, which implying GO may affect plant growth via regulating ROS detoxification. Root and stem exhibit distinct transcriptomic responses to GO, and the GO-responsive genes in stem are more enriched in cell cycle and epigenetic regulation. GO inhibited plant hormone biosynthesis and signaling by analyzing the expression data. Additionally, 97 small secreted peptides (SSPs) encoding genes were found to be involved in GO response. The gene expression of 111 transcription factor (TFs) and 43 receptor-like protein kinases (RLKs) were regulated by GO, and their expression showed high correlation with SSPs. Finally, the TFs-SSPs-RLKs signaling networks in regulating GO response were proposed. This study provides insights into the molecular responses of plants to GO.

In this study, chelating surfactant N-lauroyl ethylenediamine triacetate (N-LED3A) was used as strengthening agent for electrokinetic (EK) remediation of copper (Cu) and decabromodiphenyl ether (BDE209) co-contaminated low permeability soil. The results indicated that negligible amount of N-LED3A would be adsorbed on the experimental soil. The synchronous elution efficiencies (SEEs) of Cu and BDE209 had reached 65.4% and 49.9%, respectively, when the concentration of N-LED3A was 4000 mg/L, and they kept almost unchanged as the concentration of N-LED3A further increased. Meanwhile, the optimal SEEs were obtained at the pH condition within 6–8. The removal efficiencies of Cu (55.3%–65.8%) and BDE209 (31.4%–46.4%) would be increased with the applied voltage gradient and concentration of N-LED3A. In addition, BDE209 and Cu contaminants were also detected in the catholyte and anolyte, respectively, and their concentrations still showed an uptrend by the end of the experiments. While in the control experiments, the removal efficiency of Cu was in the range of 18.2%–23.6%, and almost no BDE209 was migrated out. The electric current would be increased with N-LED3A concentration increased, further resulting in the enhancement of cumulative electro-osmotic flow (EOF). However, the increment of EOF was limited after an 8-day treatment due to the declined capacity of the soil water supply, and the removal efficiency of BDE209 did not change proportionally to the cumulative EOF as a consequence. The accumulated (21 days) energy consumption under the optimal operation conditions (voltage gradient 1 V/cm, N-LED3A 1 g/L) was 377.28 KWh/m3. Efficiently synchronous removal of BDE209 and Cu could be achieved by the N-LED3A enhanced EK technique, exhibiting a promising application potential in the organic pollutant and heavy metal co-contaminated soil remediation.

In the present experiment, we investigated the impact of heat stress (HS) on physiological parameters, dry matter intake, milk production, the metabolome of milk, and blood plasma in lactating Holstein dairy cows. For this purpose, 20 Holstein lactating cows were distributed in two groups in such a way that each group had 10 cows. A group of 10 cows was reared in HS conditions, while the other group of 10 cows was reared in the thermoneutral zone. The results of the experiment showed that cows subjected to HS had higher respiration rates (p &lt, 0.01) and greater rectal temperature (p &lt, 0.01). Results of milk production and composition explored that HS lowered milk production (p &lt, 0.01) and milk protein percentage (p &lt, 0.05) than cows raised in a thermoneutral place. Furthermore, HS increased the concentrations of N-acetyl glycoprotein, scyllo-inositol, choline, and pyridoxamine in milk, while HS decreased the concentrations of O-acetyl glycoprotein, glycerophosphorylcholine, citrate, and methyl phosphate in milk. Moreover, HS enhanced plasma concentrations of alanine, glucose, glutamate, urea, 1-methylhistidine, histidine, and formate in cows, while the plasma concentration of low-density lipoprotein, very-low-density lipoprotein, leucine, lipid, and 3-hydroxybutyrate decreased due to HS. Based on the findings of the current research, it is concluded that HS alters the milk and blood plasma metabolites of lactating Holstein dairy cows. Overall, in the current experiment, HS altered eight metabolites in milk and twelve metabolites in the plasma of lactating Holstein dairy cows. Furthermore, the current study explored that these metabolites were mainly involved in proteolysis, gluconeogenesis, and milk fatty acid synthesis and could be potential biomarkers for dairy cows undergoing HS.

Background Hyperandrogenism (HA), inflammation, and oxidative damage play key roles in the pathophysiology of polycystic ovary syndrome (PCOS). Whether vitamin D adjuvant therapy improves hormonal, inflammation, and oxidative damage in PCOS patients has aroused widespread interest, but the results are controversial. To evaluate the effect of vitamin D supplementation on hormonal, oxidative stress, and inflammatory parameters in patients with PCOS. Methods A literature search was conducted in Medline, the Cochrane Library, EMBASE, and Web of Science for studies related to PCOS and vitamin D supplementation. All reports of randomized controlled trials (RCTs) published before December 2019 were identified. The fixed-effects model or random-effects model was used to calculate pooled estimates of standardized differences in means (SMD) with 95% confidence intervals (CI). Results A total of 956 identified studies were retrieved, and eighteen RCTs involving 1,060 participants were ultimately included in the current meta-analysis. The pooled results demonstrated that vitamin D supplementation in patients with PCOS resulted in a significant improvement in serum total testosterone (TT), high sensitivity C-reactive protein (hs-CRP), total antioxidant capacity (TAC), and malondialdehyde (MDA). No significant effect on free testosterone (FT), dehydroepiandrosterone sulfate (DHEA-S), sex hormone-binding globulin (SHBG), free androgen index (FAI), nitric oxide (NO), and total glutathione (GSH) levels was found. Subgroup analysis showed that vitamin D supplementation reduced hs-CRP and MDA irrespective of the treatment course, type of vitamin D intervention, supplementation frequency, and dosage. Twelve weeks of vitamin D supplementation improved TT and TAC while low-dose vitamin D supplementation (≤1,000 IU/day) improved TT and DHEA-S. Vitamin D co-supplementation reduced TT, FT, and DHEA-S, while a daily supplementation regime improved TT, DHEA-S, and TAC in patients with PCOS. Conclusions The current meta-analysis demonstrates that vitamin D supplementation in patients with PCOS resulted in an improvement in the levels of TT, hs-CRP, TAC, and MDA, but did not affect FT, DHEA-S, SHBG, FAI, NO, and GSH levels. Vitamin D co-supplementation, low-dose vitamin D supplementation (≤1,000 IU/day), and daily supplementation frequency appeared to be more conducive to improving hormones, inflammation, and oxidative stress in PCOS patients.

Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of S. cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) while fermenting with glucose and xylose were revealed. The pathways classified as carbohydrate metabolism were significantly enriched in response to Aa, while the pathways belonged to xenobiotics biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, some new pathways were activated in response to Aa_Fur, i.e., cofactors and vitamins metabolism, and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rate by nearly 50%, while the ethanol yield enhanced by nearly 8%. Further co-overexpression of Haa1p and Tye7 resulted in 59% increase in xylose consumption rate and 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed fermentation inhibitors.

Supplementation plays an important role in reversing the weight loss of grazing yaks during cold season. However, little is known about the effect of supplementation on the serum metabolites of grazing yaks. The objective of this study was to explore the effects of supplementary feeding on average daily gain (ADG) and serum metabolites with nuclear magnetic resonance (NMR)-based metabolomics method in growing yaks during cold season on the Qinghai-Tibetan plateau. Twenty 1.5-year-old female yaks (91.38 &plusmn, 10.43 kg LW) were evenly divided into three treatment groups and a control group (CON) (n = 5 per group). All the yaks were released to graze during daytime, whereas the yaks in the treatment groups were supplemented with highland barley (HLB), rapeseed meal (RSM), and highland barley plus rapeseed meal (HLB + RSM) at night. The whole experiment lasted for 120 days. Results indicated that the ADG of growing yak heifers was increased by concentrate supplementations, and ADG under HLB and HLB + RSM group was 37.5% higher (p &lt, 0.05) than that with RSM supplementation. Supplementary feeding increased the plasma concentrations of total protein (TP), albumin (ALB), and blood urea nitrogen (BUN) of those in the CON group, and concentrations of BUN were higher in the RSM group than in the HLB and HLB + RSM group. Compared with the CON group, serum levels of glutamine, glycine, &beta, glucose were lower and that of choline was higher in the HLB group, serum levels of lactate were lower and that of choline, glutamate were higher in the HLB + RSM group. Compared with the HLB + RSM group, serum levels of glycerophosphoryl choline (GPC) and lactate were higher, and those of choline, glutamine, glutamate, leucine, N-acetyaspartate, &alpha, glucose, and &beta, glucose were lower in the HLB group, serum levels of citrate, GPC and lactate were higher, and those of 3-Hydroxybutyrate, betaine, choline, glutamate, glutamine, N-acetylglycoprotein, N-acetyaspartate, &alpha, glucose were lower in the RSM group. It could be concluded that concentrate supplementations significantly improved the growth performance of growing yaks and supplementation with HBL or HLB plus RSM was better than RSM during the cold season. Supplementation with HBL or HLB plus RSM affected the serum metabolites of grazing yaks, and both treatments promoted lipid synthesis. Supplementation of yaks with HBL plus RSM could improve energy-supply efficiency, protein and lipid deposition compared with HLB and RSM.

The yak rumen microflora has more efficient fiber-degrading and energy-harvesting abilities than that of low-altitude cattle, however, few studies have investigated the effects of dietary energy levels on the rumen bacterial populations and the relationship between rumen bacteria and the intramuscular fatty acid profile of fattening yaks. In this study, thirty yaks were randomly assigned to three groups. Each group received one of the three isonitrogenous diets with low (3.72 MJ/kg), medium (4.52 MJ/kg), and high (5.32 MJ/kg) levels of net energy for maintenance and fattening. After 120 days of feeding, results showed that increasing dietary energy significantly increased ruminal propionate fermentation and reduced ammonia concentration. The 16S rDNA sequencing results showed that increasing dietary energy significantly increased the ratio of Firmicutes to Bacteroidetes and stimulated the relative abundance of Succiniclasticum, Saccharofermentans, Ruminococcus, and Blautia populations. The quantitative real-time PCR analysis showed that high dietary energy increased the abundances of Streptococcus bovis, Prevotella ruminicola, and Ruminobacter amylophilus at the species level. Association analysis showed that ruminal acetate was positively related to some intramuscular saturated fatty acid (SFA) contents, and Prevotella was significantly positively related to intramuscular total polyunsaturated fatty acid content and negatively related to intramuscular total SFA content. This study showed that high dietary energy mainly increased ruminal amylolytic and propionate-producing bacteria populations, which gave insights into how the effects of dietary energy on rumen bacteria are related to intramuscular fat fatty acids of fattening yaks.

Background Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR. Results We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability. Conclusions Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.

The ocean harbors a variety of bacteria that contain huge protease resources and offer a great potential for industrial and biotechnological applications. Here, we isolated a protease-secreting bacterium Vibrio pomeroyi strain 12613 from Atlantic seawater and purified a protease VP9 from strain 12613. VP9 was identified as a metalloprotease of the M4 family. VP9 could hydrolyze casein and gelatin but not elastin and collagen. With gelatin as the substrate, VP9 showed the highest activity at 40°C and pH 6.0–8.0. It was stable at temperatures of 50°C and less and in the range of pH 5.0–11.0. VP9 also had good tolerance to NaCl, non-ionic detergents, and organic solvent methanol. Unlike other M4 metalloproteases, VP9 has distinct collagen-swelling ability, and its collagen-swelling effect was concentration dependent. The relative expansion volume of collagen increased by approximately eightfold after treatment with 10 μM VP9 at 37°C for 12 h. The collagen-swelling mechanism of VP9 on bovine-insoluble type I collagen was further studied. Atomic force microscopy observation and biochemical analyses showed that VP9 can degrade proteoglycans in collagen fibers, resulting in the release of collagen fibrils from collagen fibers and the swelling of the latter. In addition, VP9 can degrade glycoproteins, a non-collagenous constituent interacting with collagen in the skin. The characteristics of VP9, such as sufficient specificity toward proteoglycans and glycoproteins but no activity toward collagen, suggest its promising potential in the unhairing and fiber-opening processing in leather industry.

Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass.