Benchmarking of long-read sequencing, assemblers and polishers for yeast genome.

BACKGROUND: The long reads of the third-generation sequencing significantly benefit the quality of the de novo genome assembly. However, its relatively high single-base error rate has been criticized. Currently, sequencing accuracy and throughput continue to improve, and many advanced tools are constantly emerging. PacBio HiFi sequencing and Oxford Nanopore Technologies (ONT) PromethION are two up-to-date platforms with low error rates and ultralong high-throughput reads. Therefore, it is urgently needed to select the appropriate sequencing platforms, depths and genome assembly tools for high-quality genomes in the era of explosive data production. METHODS: We performed 455 (7 assemblers with 4 polishing pipelines or without polishing on 13 subsets with different depths) and 88 (4 assemblers with or without polishing on 11 subsets with different depths) de novo assemblies of Yeast S288C on high-coverage ONT and HiFi datasets, respectively. The assembly quality was evaluated by Quality Assessment Tool (QUAST), Benchmarking Universal Single-Copy Orthologs (BUSCO) and the newly proposed Comprehensive_score (C_score). In addition, we applied four preferable pipelines to assemble the genome of nonreference yeast strains. RESULTS: The assembler plays an essential role in genome construction, especially for low-depth datasets. For ONT datasets, Flye is superior to other tools through C_score evaluation. Polishing by Pilon and Medaka improve accuracy and continuity of the preassemblies, respectively, and their combination pipeline worked well in most quality metrics. For HiFi datasets, Flye and NextDenovo performed better than other tools, and polishing is also necessary. Enough data depth is required for high-quality genome construction by ONT (>80X) and HiFi (>20X) datasets.

Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum.

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Protective effects of bioactive components targeting ß2-adrenergic receptors and muscarinic-3 acetylcholine receptor in Zhisou San on ovalbumin-induced allergic asthma.

One promising approach to overcome drug resistance in asthma treatments involves dual-target therapy, specifically targeting the ß2 adrenergic receptor (ß2-AR) and muscarinic-3 acetylcholine receptor (M3R). This study investigated the anti-asthma effects and dual-target mechanisms of glycyrrhizic acid, hesperidin, and platycodin D (GHP) from Zhisou San. GHP administration effectively attenuated OVA-induced inflammatory infiltration and overproduction of mucus in asthmatic mice. Additionally, GHP treatment significantly suppressed M3R and promoted ß2-AR activation, resulting in the relaxation of tracheal smooth muscle. These findings concluded that GHP mitigated asthma by targeting ß2-AR and M3R to ameliorate airway inflammation and modulate airway smooth muscle relaxation.

Evolution of cyclic di-GMP signalling on a short and long term time scale.

Diversifying radiation of domain families within specific lineages of life indicates the importance of their functionality for the organisms. The foundation for the diversifying radiation of the cyclic di-GMP signalling network that occurred within the bacterial kingdom is most likely based in the outmost adaptability, flexibility and plasticity of the system. Integrative sensing of multiple diverse extra- and intracellular signals is made possible by the N-terminal sensory domains of the modular cyclic di-GMP turnover proteins, mutations in the protein scaffolds and subsequent signal reception by diverse receptors, which eventually rewires opposite host-associated as well as environmental life styles including parallel regulated target outputs. Natural, laboratory and microcosm derived microbial variants often with an altered multicellular biofilm behaviour as reading output demonstrated single amino acid substitutions to substantially alter catalytic activity including substrate specificity. Truncations and domain swapping of cyclic di-GMP signalling genes and horizontal gene transfer suggest rewiring of the network. Presence of cyclic di-GMP signalling genes on horizontally transferable elements in particular observed in extreme acidophilic bacteria indicates that cyclic di-GMP signalling and biofilm components are under selective pressure in these types of environments. On a short and long term evolutionary scale, within a species and in families within bacterial orders, respectively, the cyclic di-GMP signalling network can also rapidly disappear. To investigate variability of the cyclic di-GMP signalling system on various levels will give clues about evolutionary forces and discover novel physiological and metabolic pathways affected by this intriguing second messenger signalling system.

Intracellular accumulation of c-di-GMP and its regulation on self-flocculation of the bacterial cells of Zymomonas mobilis.

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Methyltransferase-like 3 promotes the progression of lung cancer via activating PI3K/AKT/mTOR pathway.

Methyltransferase-like 3 (METTL3) catalyses N6-methyladenosine (m6 A) modification on messenger RNA (mRNA) and participates in a wide range of biological functions via epigenetically regulating gene expression. Recent studies suggested that dysregulation of METTL3 is associated with multiple human cancers; however, the role of METTL3 in lung cancer remains unclear. In the present study, through transcriptome analysis of lung cancer patients, we found that METTL3 is overexpressed in lung cancer patients and is associated with poor patient survival. More importantly, combining both in vitro and in vivo models, we revealed that in lung cancer cells, METTL3 overexpression activates PI3K/AKT/mTOR pathway and mTOR-mediated protein synthesis. Mechanistically, METTL3 promotes PI3K expression by introducing m6 A modification in PI3K 3' untranslated region (3' UTR). Elevated PI3K level then activates downstream AKT and mTOR signalling pathway and results in rapid cancer cell proliferation and metastasis. Taken together, our study reveals that METTL3-mediated m6 A methylation promotes lung cancer progression via activating PI3K/AKT/mTOR pathway.

Analysis of Prevention and Treatment of Anastomotic Leakage after Sphincter-Preserving Surgery for Middle- and Low-Grade Rectal Cancer under Laparoscopy.

Background: Anastomotic leakage is one of the most serious complications that can occur after laparoscopic-assistedsphincter-preserving surgery for middle- and low-grade rectal cancer. Objectives: To explore the cause, prevention, and treatment of anastomotic leakage after sphincter-preserving surgery for middle- and low-grade rectal cancer under laparoscopy. Methods: The clinical data from patients with mid- and low-grade rectal cancer who underwent laparoscopic-assistedanus-preserving surgery in the anorectal surgery department of our hospital have been analyzed. Patients with a definite diagnosis, indications for laparoscopic surgery, and sphincter-preserving surgery were included in the analysis, and patients with a protective loop ileostomy and laparotomy were excluded. Results: Among the 126 patients with middle- and low-grade rectal cancer undergoing sphincter-preserving surgery under laparoscopy. There were 75 male patients and 51 female patients, ranging in age from 37 to 89 years old, with an average age of 60.2 ± 6.7. The distance from the lower edge of the rectal tumor to the anal edge was ≤10 cm. 6 developed anastomotic leakage after the operation (leakage rate of 4.7%). Moreover, turbid purulent fluid was drained from the abdominal drainage tube in three patients on the third and fourth days after the operation, and the abdominal drainage tube drained serous drainage in three more patients on the fifth and sixth days, with signs of peritonitis appearing locally. All patients received continuous flushing and negative pressure drainage with a self-made double cannula and symptomatic treatment, and all were cured and discharged. Conclusion: Many factors can cause anastomotic leakage after this operation, and adequate perioperative preparation, meticulous operation during surgery, and careful postoperative management are key factors in preventing it.

Occurrence of polycyclic aromatic hydrocarbons (PAHs) in the seafood from an important fishing area in the East China Sea and a comparison between seafood from different origins.

Polycyclic aromatic hydrocarbons (PAHs) were analyzed in a variety of coastal marine seafood from the Wentai fishing area, which is in proximity to highly urbanized areas and provides a significant portion of the marine aquatic products in Eastern China. Specifically, total PAHs ranged from 320 to 2500 ng/g d.w., whereas carcinogenic PAHs ranged from 21 to 92 ng/g d.w. According to the source diagnosis, the PAHs in the seafood were generally from sources highly related to fossil fuel. Thus, it is necessary to control the unintentional use of fossil fuels and optimize the energy structure in the terrestrial area nearby. Moreover, the food chain structure and the environmental behavior of PAHs through the food chain, which are relevant to dietary risk, were assessed. The δ15N values fell in a narrow range, which indicates a relatively short food chain, which is due to overfishing in this area. Furthermore, in this shortened food chain, the trophic magnification factors (TMFs) ranged from 0.23 to 6.6, which were generally higher than those in more complex food chains. The cancer risk (ILCR) ranged from 2.2 × 10-8 to 2.2 × 10-6. It is noted that the carcinogenic risk of consuming yellow croaker, which is one of the most popular seafood among people on the east coast of China, was the highest. In conclusion, trophic magnification implied a possible elevated risk through this marine food chain, and overfishing may have increased the uncertainty associated with TMF estimations.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbon metabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.

[Effects of silencing HMGB1 combined with docetaxel chemotherapy on the proliferation and apoptosis of prostate cancer cells and its action mechanism].

Objective: To investigate the effect of silencing the high-mobility group box-1 protein (HMGB1) combined with docetaxel (DTX) on the proliferation and apoptosis of PCa cells and its possible action mechanism. METHODS: The expression of HMGB1 mRNA in different PCa cell lines and normal prostatic epithelial cells was detected by RT-qPCR. The PC-3 cells were transfected with different HMGB1 small interfering RNAs (si-HMGB1, si-HMGB1-2 and si-HMGB1-3), and the silencing effect was detected. The effects of different concentrations of DTX on the proliferation of the PC-3 cells was determined by MTT. Then the PC-3 cells were randomly divided into five groups: control (conventional culture), si-HMGB1-NC (si-HMGB1-NC transfection), si-HMGB1 (si-HMGB1-3 transfection), DTX (20 nmol/L DTX), and si-HMGB1+DTX (si-HMGB1-3+20 nmol/L DTX transfection), followed by measurement of the survival rate of the cells by MTT, their apoptosis rate by flow cytometry, and the expressions of HMGB1, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins in different groups by Western blot. RESULTS: The expression of HMGB1 mRNA in the PC-3 cells was the highest and the lowest after transfection with si-HMGB1-3. DTX inhibited the proliferation of the PC-3 cells at various concentrations. Compared with the control group, the si-HMGB1 and DTX groups showed significantly decreased A values, cell survival rates and HMGB1 and Bcl-2 expressions, but increased cell apoptosis rates and Bax expressions (P < 0.05). In comparison with the si-HMGB1 and DTX groups, the si-HMGB1+DTX group exhibited a remarkably decreased A value, cell survival rate and Bcl-2 expression, but increased cell apoptosis and Bax expression. The expression of the HMGB1 protein was markedly lower in the si-HMGB1+DTX than in the DTX group (P < 0.05). CONCLUSIONS: Silencing HMGB1 combined with DTX chemotherapy can inhibit the proliferation and promote the apoptosis of PCa cells, which may be attributed to its regulatory effect on the expressions of the Bcl-2 family-related proteins.、.

Genome sequencing, assembly, and annotation of the self-flocculating microalga Scenedesmus obliquus AS-6-11.

BACKGROUND: Scenedesmus obliquus belongs to green microalgae and is widely used in aquaculture as feed, which is also explored for lipid production and bioremediation. However, genomic studies of this microalga have been very limited. Cell self-flocculation of microalgal cells can be used as a simple and economic method for harvesting biomass, and it is of great importance to perform genome-scale studies for the self-flocculating S. obliquus strains to promote their biotechnological applications. RESULTS: We employed the Pacific Biosciences sequencing platform for sequencing the genome of the self-flocculating microalga S. obliquus AS-6-11, and used the MECAT software for de novo genome assembly. The estimated genome size of S. obliquus AS-6-11 is 172.3 Mbp with an N50 of 94,410 bp, and 31,964 protein-coding genes were identified. Gene Ontology (GO) and KEGG pathway analyses revealed 65 GO terms and 428 biosynthetic pathways. Comparing to the genome sequences of the well-studied green microalgae Chlamydomonas reinhardtii, Chlorella variabilis, Volvox carteri and Micractinium conductrix, the genome of S. obliquus AS-6-11 encodes more unique proteins, including one gene that encodes D-mannose binding lectin. Genes encoding the glycosylphosphatidylinositol (GPI)-anchored cell wall proteins, and proteins with fasciclin domains that are commonly found in cell wall proteins might be responsible for the self-flocculating phenotype, and were analyzed in detail. Four genes encoding both GPI-anchored cell wall proteins and fasciclin domain proteins are the most interesting targets for further studies. CONCLUSIONS: The genome sequence of the self-flocculating microalgal S. obliquus AS-6-11 was annotated and analyzed. To our best knowledge, this is the first report on the in-depth annotation of the S. obliquus genome, and the results will facilitate functional genomic studies and metabolic engineering of this important microalga. The comparative genomic analysis here also provides new insights into the evolution of green microalgae. Furthermore, identification of the potential genes encoding self-flocculating proteins will benefit studies on the molecular mechanism underlying this phenotype for its better control and biotechnological applications as well.

Identification of a novel repressor encoded by the putative gene ctf1 for cellulase biosynthesis in Trichoderma reesei through artificial zinc finger engineering.

Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-ß-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Cytotoxic Trichothecene Macrolides Produced by the Endophytic Myrothecium roridum.

Six new macrolides named myrothecines D-G (1-4), 16-hydroxymytoxin B (5), and 14'-dehydrovertisporin (6), including four 10,13-cyclotrichothecane derivatives, in addition to 12 known compounds (7-18), were isolated from three endophytic Myrothecium roridum, IFB-E008, IFB-E009, and IFB-E012. The isolated compounds were characterized by MS, NMR, CD, and single-crystal X-ray crystallography. The isolated macrolides exhibited an antiproliferation effect against chronic myeloid leukemia K562 and colorectal carcinoma SW1116 cell lines. Compounds 1-6 were cytotoxic, with IC50 values ranging between 56 nM and 16 µM. Since slight structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional quantitative structure-activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic, and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure-activity relationships of these metabolites.

Sesquiterpenoid Compounds from Curcuma kwangsiensis.

Two previously undescribed guaiane-type sesquiterpenes (1 and 2), a pair of new salvialane-type sesquiterpenes (3a and 3b), together with 11 known compounds were isolated and purified from the rhizomes of Curcuma kwangsiensis. Their structures were elucidated by the extensive spectroscopic data (1D- and 2D-NMR) analysis. All the isolated compounds were assessed for their anti-neuroinflammatory activity by inhibiting the nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells in vitro assay, and the isolates 3 and 11 showed anti-neuroinflammatory activity with IC50 values of 1.85 and 20.05 µm, respectively.

Nesfatin-1 functions as a switch for phenotype transformation and proliferation of VSMCs in hypertensive vascular remodeling.

The phenotypic transformation from differentiated to dedifferentiated vascular smooth muscle cells (VSMCs) plays a crucial role in VSMC proliferation and vascular remodeling in many cardiovascular diseases including hypertension. Nesfatin-1, a multifunctional adipocytokine, is critically involved in the regulation of blood pressure. However, it is still largely unexplored whether nesfatin-1 is a potential candidate in VSMC phenotypic switch and proliferation in hypertension. Experiments were carried out in Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), human VSMCs and primary rat aortic VSMCs. We showed that the expression of nesfatin-1 was upregulated in media layer of the aorta in SHR and SHR-derived VSMCs. Nesfatin-1 promoted VSMC phenotypic transformation, accelerated cell cycle progression and proliferation. Knockdown of nesfatin-1 inhibited the VSMC phenotype switch from a contractile to a synthetic state, attenuated cell cycle progression and retarded VSMC proliferation in SHR-derived VSMCs. Moreover, nesfatin-1-activated PI3K/Akt/mTOR signaling was abolished by JAK/STAT inhibitor WP1066, and the increased phosphorylation levels of JAK2/STAT3 in response to nesfatin-1 were suppressed by inhibition of PI3K/Akt/mTOR in VSMCs. Pharmacological blockade of the forming feedback loop between PI3K/Akt/mTOR and JAK2/STAT3 prevented the proliferation of nesfatin-1-incubated VSMCs and primary VSMCs from SHR. Chronic intraperitoneal injection of nesfatin-1 caused severe hypertension and cardiovascular remodeling in normal rats. In contrast, silencing of nesfatin-1 gene ameliorated hypertension, phenotype switching, and vascular remodeling in the aorta of SHR. Therefore, our data identified nesfatin-1 as a key modulator in hypertension and vascular remodeling by facilitating VSMC phenotypic switching and proliferation.

Contribution of cellulose synthesis, formation of fibrils and their entanglement to the self-flocculation of Zymomonas mobilis.

Due to the unique Entner-Doudoroff pathway, Zymomonas mobilis has been acknowledged as a potential host to be engineered for biorefinery to produce biofuels and biobased chemicals. The self-flocculation of Z. mobilis can make the bacterial cells self-immobilized within bioreactors for high density to improve product productivities, and in the meantime enhance their tolerance to stresses, particularly product inhibition and the toxicity of byproducts released during the pretreatment of lignocellulosic biomass. In this work, we explored mechanism underlying such a phenotype with the self-flocculating strain ZM401 developed from the regular non-flocculating strain ZM4. Cellulase de-flocculation and the restoration of the self-flocculating phenotype for the de-flocculated bacterial cells subjected to culture confirmed the essential role of cellulose biosynthesis in the self-flocculation of ZM401. Furthermore, the deactivation of both Type I and Type IV restriction-modification systems was performed for ZM4 and ZM401 to improve their transformation efficiencies. Comparative genome analysis detected the deletion of a thymine from ZMO1082 in ZM401, leading to a frame-shift mutation for the putative gene to be integrated into the neighboring downstream gene ZMO1083 encoding the catalytic subunit A of cellulose synthase, and consequently created a new gene to encode a larger transmembrane protein BcsA_401 for more efficient synthesis of cellulose as well as the development of cellulose fibrils and their entanglement for the self-flocculation of the mutant. These speculations were confirmed by the morphological observation of the bacterial cells under scanning electron microscopy, the impact of the gene deletion on the self-flocculation of ZM401, and the restoration of the self-flocculating phenotype of ZM401 ΔbcsA by the gene complementation. The progress will lay a foundation not only for fundamental research in deciphering molecular mechanisms underlying the self-flocculation of Z. mobilis and stress tolerance associated with the morphological change but also for technological innovations in engineering non-flocculating Z. mobilis and other bacterial species with the self-flocculating phenotype.