Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents.

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from ß-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Light irradiation changes the regulation pattern of BtCrgA on carotenogenesis in Blakeslea trispora.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

Effect of Astragalus membranaceus on left ventricular remodeling in HFrEF: a systematic review and meta-analysis.

Background: Left ventricular remodeling (LVR) is a key factor leading to the onset and progression of heart failure with reduced ejection fraction (HFrEF). Improving LVR can delay the progression of HFrEF and improve quality of life. Objective: To evaluate the improvement effect of Astragalus membranaceus (A. membranaceus) on LVR in patients with HFrEF. Method: We retrieved randomized controlled trials (RCTs) of A. membranaceus in treating HFrEF from eight Chinese and English databases, up until 31 October 2023. To assess the quality of the literature, we utilized the bias risk tool from the Cochrane Handbook. For meta-analysis, we employed Review Manager 5.4.1 software. Additionally, we performed sensitivity analysis and publication bias assessment using Stata 17.0 software. Result: Totally 1,565 patients were included in 19 RCTs. Compared to conventional treatment (CT), the combination therapy of A. membranaceus with CT demonstrated significant improvements in LVR, specifically increasing left ventricular ejection fraction (LVEF, MD = 5.82, 95% CI: 4.61 to 7.03, p < 0.00001), decreasing left ventricular end-diastolic diameter (LVEDD, MD = -4.05, 95% CI: -6.09 to -2.01, p = 0.0001), and left ventricular end-systolic diameter (LVESD, MD = -12.24, 95% CI: -15.24 to -9.24, p < 0.00001). The combination therapy of A. membranaceus with CT also improved clinical efficacy (RR = 4.81, 95% CI: 3.31 to 7.00, p < 0.00001), reduced brain natriuretic peptide (BNP, MD = -113.57, 95% CI: -146.91 to -81.22, p < 0.00001) level, and increased 6-min walking distance (6-MWD, MD = 67.62, 95% CI: 41.63 to 93.60, p < 0.00001). In addition, the combination therapy of A. membranaceus with CT mitigated inflammatory responses by reducing tumor necrosis factor-alpha (TNF-α, MD = -16.83, 95% CI: -22.96 to -10.71, p < 0.00001), interleukin-6 (IL-6, MD = -29.19, 95% CI: -36.08 to -22.30, p < 0.00001), and high-sensitivity C-reactive protein (hs-CRP, MD = -0.98, 95% CI: -1.43 to -0.52, p < 0.0001). Notably, the combination therapy of A. membranaceus with CT did not increase the incidence of adverse reactions (RR = 0.86, 95% CI: 0.25 to 2.96, p = 0.81). Conclusion: This systematic review and meta-analysis revealed that the combination therapy of A. membranaceus with CT has more advantages than CT alone in improving LVR and clinical efficacy in HFrEF patients, without increasing the incidence of adverse reactions. However, due to the limited quality of included studies, more high-quality investigations are required to provide reliable evidence for clinical use. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=397571, Identifier: CRD42023397571.

Discovery of a brain-permeable bromodomain and extra terminal domain (BET) inhibitor with selectivity for BD1 for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease and lacks effective therapeutic agents. Dysregulation of transcription mediated by bromodomain and extra-terminal domain (BET) proteins containing two different bromodomains (BD1 and BD2) is an important factor in multiple diseases, including MS. Herein, we identified a series of BD1-biased inhibitors, in which compound 16 showed nanomolar potency for BD1 (Kd = 230 nM) and a 60-fold selectivity for BRD4 BD1 over BD2. The co-crystal structure of BRD4 BD1 with 16 indicated that the hydrogen bond interaction of 16 with BD1-specific Asp145 is important for BD1 selectivity. 16 showed favorable brain distribution in mice and PK properties in rats. 16 was able to inhibit microglia activation and had significant therapeutic effects on EAE mice including improvement of spinal cord inflammatory conditions and demyelination protection. Overall, these results suggest that brain-permeable BD1 inhibitors have the potential to be further investigated as therapeutic agents for MS.

Alternative Splicing, An Overlooked Defense Frontier of Plants with Respect to Bacterial Infection.

Disease represents a major problem in sustainable agricultural development. Plants interact closely with various microorganisms during their development and in response to the prevailing environment. In particular, pathogenic microorganisms can cause plant diseases, affecting the fertility, yield, and longevity of plants. During the long coevolution of plants and their pathogens, plants have evolved both molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) signaling networks in order to regulate host cells in response to pathogen infestation. Additionally, in the postgenomic era, alternative splicing (AS) has become uncovered as one of the major drivers of proteome diversity, and abnormal RNA splicing is closely associated with bacterial infections. Currently, the complexity of host-bacteria interactions is a much studied area of research that has shown steady progress over the past decade. Although the development of high-throughput sequencing technologies and their application in transcriptomes have revolutionized our understanding of AS, many mechanisms related to host-bacteria interactions remain still unclear. To this end, this review summarizes the changes observed in AS during host-bacteria interactions and outlines potential therapeutics for bacterial diseases based on existing studies. In doing so, we hope to provide guidelines for plant disease management in agriculture.

Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.

AaABI5 transcription factor mediates light and abscisic acid signaling to promote anti-malarial drug artemisinin biosynthesis in Artemisia annua.

Artemisia annua, a member of the Asteraceae family, remains the primary source of artemisinin. However, the artemisinin content in the existing varieties of this plant is very low. In this study, we found that the environmental factors light and phytohormone abscisic acid (ABA) could synergistically promote the expression of artemisinin biosynthetic genes. Notably, the increased expression levels of those genes regulated by ABA depended on light. Gene expression analysis found that AaABI5, a transcription factor belonging to the basic leucine zipper (bZIP) family, was inducible by the light and ABA treatment. Analysis of AaABI5-overexpressing and -suppressing lines suggested that AaABI5 could enhance artemisinin biosynthesis and activate the expression of four core biosynthetic genes. In addition, the key regulator of light-induced artemisinin biosynthesis, AaHY5, could bind to the promoter of AaABI5 and activate its expression. In conclusion, our results demonstrated that AaABI5 acts as an important molecular junction for the synergistic promotion of artemisinin biosynthesis by light and ABA signals, which provides a candidate gene for developing new germplasms of high-quality A. annua.

Application of different proportions of sweet sorghum silage as a substitute for corn silage in dairy cows.

This experiment explored the effects of different proportions of sweet sorghum silage as a substitute for corn silage on dry matter intake (DMI), milk yield, milk quality, apparent digestibility, rumen fermentation parameters, serum amino acid profile, and rumen microbial composition of dairy cows. A total of 32 mid-lactation Holstein dairy cows with similar body weights and parities were randomly divided into four treatments: 100% corn silage +0% sorghum silage (CON), 75% corn silage +25% sorghum silage (CS1), 50% corn silage +50% sorghum silage (CS2), and 25% corn silage +75% sorghum silage (CS3). The milk yield was increased (linear, p = .048) as the proportion of sweet sorghum increased. Linear (p = .003) and quadratic (p = .046) increased effects were observed in milk fat as corn silage was replaced with sorghum silage. Compared with the CON diet group, the CS2 and CS3 diet groups had lower dry matter (DM) (linear, p < .001), ether extract (EE) (linear, p < .001), and gross energy (GE) (linear, p = .001) digestibility of the dairy cows. The ruminal fluid aspartate (Asp) level decreased (linear, p = .003) as the proportion of sweet sorghum increased. Linear (p < .05) and quadratic (p < .05) increased effects were observed for the contents of threonine (Thr), glycine (Gly), valine (Val), leucine (Leu), tyrosine (Tyr), and histidine (His) in rumen fluid with the replacement of corn silage with sorghum silage. Cows fed the CS3 diet had greater Faecalibacterium, Bacteroides, and Prevotella ruminicola content/copy number than those fed the CON diet (p < .05). In conclusion, feeding sorghum silage as a replacement for corn silage could increase the milk yield and fat, promote the growth of rumen microbes, and provide more rumen fluid amino acids for the body and microbial utilization. We believe that sorghum silage is feasible for dairy cows, and it is reasonable to replace corn silage with 75% sorghum silage.

Flocculation of oleaginous green algae with Mortierella alpina fungi.

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80-85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3-10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

Tuber rugosum, a new species from northeastern North America: Slug mycophagy aides in electron microscopy of ascospores.

Species in the genus Tuber are ascomycetous fungi that produce hypogeous fruiting bodies commonly called truffles. These fungi are ecologically relevant owing to the ectomycorrhizal symbiosis they establish with plants. One of the most speciose lineages within Tuber is the Rufum clade, which is widely distributed throughout Asia, Europe, and North America and is estimated to include more than 43 species. Most species in this clade have spiny spores, and many still have not been formally described. Here, we describe T. rugosum based on multigene phylogenetic analysis and its unique morphological characters. Tuber rugosum (previously designated in literature as Tuber sp. 69) has been collected throughout the Midwest, USA, and Quebec, Canada, and is an ectomycorrhizal symbiont of Quercus trees, as confirmed through morphological and molecular analyses of root tips presented here. We also present a novel method for preparing Tuber ascospores for scanning electron microscope imaging that includes feeding, digestion, and spore excretion by the slug Arion subfuscus. Following this method, spores become free from ascus and other mycelial debris that could obscure morphological traits during their passage through the snail gut while maintaining ornamentation. Finally, we report the fatty acid analysis, a fungicolous species association, and we provide an updated taxonomic key of the Rufum clade.

In vivo cadmium-assisted dilute acid pretreatment of the phytoremediation sweet sorghum for enzymatic hydrolysis and cadmium enrichment.

Phytoremediation with energy crops is considered an integrated technology that provides both environment and energy benefits. Herein, the sweet sorghum cultivated on Cd-contaminated farmland (1.21 mg/kg of Cd in the soil) showed promising phytoremediation potential, and the approach for utilizing sorghum stalks was explored. Sweet sorghum bagasse with Cd contamination was pretreated with dilute acid in order to improve enzymatic saccharification and achieve Cd recovery, resulting in harmless and value-added utilization. After pretreatment, hemicelluloses were dramatically degraded, and the lignocellulosic structures were partially deconstructed with xylan removal up to 98.1%. Under the optimal condition (0.75% H2SO4), the highest total sugar yield was 0.48 g/g of raw bagasse; and nearly 98% of Cd was enriched in the liquid phase. Compared with normal biomass, Cd reduced the biomass recalcitrance and further facilitated the deconstruction of biomass under super dilute acid conditions. This work provided an example for the subsequent valorization of Cd-containing biomass and Cd recovery, which will greatly facilitate the development of phytoremediation of heavy metal contaminated soil.

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds.

Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene-an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g-1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl- d-erythritol 4-phosphate pathway, Coleus forskohlii 1-deoxy- d-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g-1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Structure-Based Discovery of a Series of NSD2-PWWP1 Inhibitors.

Overexpression, point mutations, or translocations of protein lysine methyltransferase NSD2 occur in many types of cancer cells. Therefore, it was recognized as onco-protein and considered as a promising anticancer drug target. NSD2 consists of multiple domains including a SET catalytic domain and two PWWP domains binding to methylated histone proteins. Here, we reported our efforts to develop a series of NSD2-PWWP1 inhibitors, and further structure-based optimization resulted in a potent inhibitor 38, which has high selectivity toward the NSD2-PWWP1 domain. The detailed biological evaluation revealed that compound 38 can bind to NSD2-PWWP1 and then affect the expression of genes regulated by NSD2. The current discovery will provide a useful chemical probe to the future research in understanding the specific regulation mode of NSD2 by PWWP1 recognition and pave the way to develop potential drugs targeting NSD2 protein.

Structure-Based Discovery of Potent CARM1 Inhibitors for Solid Tumor and Cancer Immunology Therapy.

CARM1 is a protein arginine methyltransferase and acts as a transcriptional coactivator regulating multiple biological processes. Aberrant expression of CARM1 has been related to the progression of multiple types of cancers, and therefore CARM1 was considered as a promising drug target. In the present work, we report the structure-based discovery of a series of N1-(3-(pyrimidin-2-yl)benzyl)ethane-1,2-diamines as potent CARM1 inhibitors, in which compound 43 displays high potency and selectivity. With the advantage of excellent tissue distribution, compound 43 demonstrated good in vivo efficacy for solid tumors. Furthermore, from the detailed immuno-oncology study with MC38 C57BL/6J xenograft model, we confirmed that this chemical probe 43 has profound effects in tumor immunity, which paves the way for future studies on the modulation of arginine post-translational modification that could be utilized in solid tumor treatment and cancer immunotherapy.

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5.

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein-protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein.

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world's most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; KD 0.11 µM) and stearoyl-CoA (18:0-CoA; KD 0.13 µM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; KD 6.4 µM) and linoleoyl-CoA (18:2-CoA; KD 21.4 µM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast alters its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.