2-bromoacetamide exposure impairs oocyte maturation in mice and humans primarily via disrupting the cytoskeleton.

2-bromoacetamide (BAcAm) is an emerging class of unregulated disinfection by-products (DBPs), with potent cytogenetic and developmental toxicity in animals. However, whether BAcAm exerts toxic effects on mammalian oocyte quality remains to be elucidate. In this research, we investigated the effect of BAcAm on mouse and human oocyte maturation with an in vitro culture system. Our results revealed that BAcAm exposure hindered the extrusion of the first polar body, disrupted the spindle organization and reduced the competence of embryo development after fertilization in the mouse oocytes. Results of single-cell RNA sequencing (scRNA-seq) showed that 605 differentially expressed genes (DEGs) were identified in the BAcAm exposed mouse oocytes, among which 366 were up-regulated and 239 were down-regulated. Gene Ontology (GO) analysis further revealed that DEGs were mainly enriched in mitochondrial functions, oxidative stress, cytoskeleton, endoplasmic reticulum (ER), Golgi and protein synthesis, DNA damage and apoptosis. We then conducted further tests in these aspects and discovered that BAcAm exposure principally perturbed the function of microtubule and actin cytoskeleton. This finding was confirmed in human oocytes. Overall, our data suggest that BAcAm exposure disturbs the cytoskeleton function, thus impairing oocyte maturation. These data, for the first time, provide a comprehensive view for the toxic effects of BAcAm on oocyte maturation.

Transcriptome Identification and Analysis of Fatty Acid Desaturase Gene Expression at Different Temperatures in Tausonia pullulans 6A7.

Tausonia pullulans 6A7 is a low-temperature yeast strain that can produce lipases. Yeast, which is made up of chassis cells, is an important part of synthetic biology, and the use of the lipase-producing properties of T. pullulans 6A7 for the production of fatty acids provides a new pathway for targeted synthesis in yeast cell factories. In this study, we performed RNA-seq on lipase-producing T. pullulans 6A7 at different temperatures (15 °C, 20 °C, 20 °C without corn oil, and 25 °C). Therefore, a total of 8455 differentially expressed genes were screened, and 16 of them were FAD candidate genes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of group A (15 °C) vs. group D (25 °C) showed that the pathways of fatty acid biosynthesis (map00061) and the biosynthesis of unsaturated fatty acids (map01040) were significantly enriched. In the proposed temporal analysis of differentially expressed genes among the four temperature modulations, we found differentially expressed genes in nine clusters that had the same expression trends; these genes may be jointly involved in multiple biological processes in T. pullulans 6A7. In addition, we found 16 FAD candidate genes involved in fatty acid biosynthesis, and the expression of these genes had similar expression in the transcriptome trends with the different temperature treatments. These findings will help in future in-depth studies of the function and molecular mechanisms of these important FAD genes involved in fatty acid metabolism in yeast, and they could also be conducive to the establishment of a cellular factory for targeted fatty acid production by using yeast.

Post-ovulatory aging affects mitochondria, spindle and protein metabolism in mouse oocytes.

In brief: Post-ovulatory aging (POA) results in a decline in oocyte quality and embryonic developmental capacity although the underlying mechanisms remain elusive. This study provides comprehensive mRNA expression profiles of fresh and aging oocytes in mice for the first time. Abstract: POA impairs the quality of mammalian oocytes with harmful effects on the developmental potential of the embryo. This is a major problem for humans since it is associated with low rate of natural fertility, with high rate of spontaneous abortion and low efficiency of in vitro fertilization. However, the molecular mechanisms underlying this process remain unclear and new methods are demanded to control POA. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis on fresh and aging MII mouse oocytes and compared their global RNA transcription patterns. Nine hundred and twenty-one differentially expressed genes (DEGs) were identified. Five hundred and sixty-nine genes were downregulated, while 356 were upregulated in the group of aging oocytes. Gene ontology (GO) enrichment analysis demonstrated that a series of DEGs were significantly enriched involving mitochondrial functions, spindle functions and protein metabolism. The results of qPCR and a series of functional tests further confirmed that the disorder of mitochondrial functions, spindle functions and impairment of protein metabolism were actually involved in the progression of POA. In this study, panoramic mRNA expression profiles of fresh and aging oocytes were depicted and fully validated. Our data will provide a useful resource for further research on the regulation of gene expression of POA and suggest potential strategies to delay and reverse POA.

Characterization of Two Na+(K+, Li+)/H+ Antiporters from Natronorubrum daqingense.

The Na+/H+ antiporter NhaC family protein is a kind of Na+/H+ exchanger from the ion transporter (IT) superfamily, which has mainly been identified in the halophilic bacteria of Bacillus. However, little is known about the Na+/H+ antiporter NhaC family of proteins in the extremely halophilic archaea. In this study, two Na+/H+ antiporter genes, nhaC1 and nhaC2, were screened from the genome of Natronorubrum daqingense based on the gene library and complementation of salt-sensitive Escherichia coli KNabc. A clone vector pUC18 containing nhaC1 or nhaC2 could make KNabc tolerate 0.6 M/0.7 M NaCl or 30 mM/40 mM LiCl and a pH of up to 8.5/9.5, respectively. Functional analysis shows that the Na+(K+, Li+)/H+ antiport activities of NhaC1 and NhaC2 are both pH-dependent in the range of pH 7.0-10.0, and the optimal pH is 9.5. Phylogenetic analysis shows that both NhaC1 and NhaC2 belong to the Na+/H+ antiporter NhaC family of proteins and are significantly distant from the identified NhaC proteins from Bacillus. In summary, we have identified two Na+(K+, Li+)/H+ antiporters from N. daqingense.

Engineering 3-Hydroxypropionic Acid Production from Glucose in Yarrowia lipolytica through Malonyl-CoA Pathway.

3-Hydroxypropionic acid (3-HP) is an important intermediate compound in the chemical industry. Green and environmentally friendly microbial synthesis methods are becoming increasingly popular in a range of industries. Compared to other chassis cells, Yarrowia lipolytica possesses advantages, such as high tolerance to organic acid and a sufficient precursor required to synthesize 3-HP. In this study, gene manipulations, including the overexpression of genes MCR-NCa, MCR-CCa, GAPNSm, ACC1 and ACSSeL641P and knocking out bypass genes MLS1 and CIT2, leading to the glyoxylate cycle, were performed to construct a recombinant strain. Based on this, the degradation pathway of 3-HP in Y. lipolytica was discovered, and relevant genes MMSDH and HPDH were knocked out. To our knowledge, this study is the first to produce 3-HP in Y. lipolytica. The yield of 3-HP in recombinant strain Po1f-NC-14 in shake flask fermentation reached 1.128 g·L-1, and the yield in fed-batch fermentation reached 16.23 g·L-1. These results are highly competitive compared to other yeast chassis cells. This study creates the foundation for the production of 3-HP in Y. lipolytica and also provides a reference for further research in the future.

Developmental toxicity of 2-bromoacetamide on peri- and early post-implantation mouse embryos in vitro.

2-bromoacetamide (BAcAm), a new class of disinfection by-products (DBPs), is widely detected in drinking water across the world. Reports of the high cytogenetic toxicity of BAcAm have aroused public attention concerning its toxic effects on early embryonic development. In this study, we optimized an in vitro culture (IVC) system for peri- and early post-implantation mouse embryos and used this system to determine the developmental toxicity of BAcAm. We found that exposure to BAcAm caused a reduction in egg cylinder formation rate and abnormal lineage differentiation in a dose-dependent manner. Transcriptomic analysis further revealed that BAcAm exposure at early developmental stages altered the abundance of transcripts related to a variety of biological processes including gene expression, metabolism, cell proliferation, cell death and embryonic development, thus indicating its toxic effects on embryonic development. Thus, we developed a robust tool for studying the toxicology of chemicals at the early stages of embryonic development and demonstrated the developmental toxicity of BAcAm in the early embryonic development of mammals.

Identification of a Novel Pleiotropic Transcriptional Regulator Involved in Sporulation and Secondary Metabolism Production in Chaetomium globosum.

Chaetoglobosin A (CheA), a well-known macrocyclic alkaloid with prominently highly antimycotic, antiparasitic, and antitumor properties, is mainly produced by Chaetomium globosum. However, a limited understanding of the transcriptional regulation of CheA biosynthesis has hampered its application and commercialization in agriculture and biomedicine. Here, a comprehensive study of the CgXpp1 gene, which encodes a basic helix-loop-helix family regulator with a putative role in the regulation of fungal growth and CheA biosynthesis, was performed by employing CgXpp1-disruption and CgXpp1-complementation strategies in the biocontrol species C. globosum. The results suggest that the CgXpp1 gene could be an indirect negative regulator in CheA production. Interestingly, knockout of CgXpp1 considerably increased the transcription levels of key genes and related regulatory factors associated with the CheA biosynthetic. Disruption of CgXpp1 led to a significant reduction in spore production and attenuation of cell development, which was consistent with metabolome analysis results. Taken together, an in-depth analysis of pleiotropic regulation influenced by transcription factors could provide insights into the unexplored metabolic mechanisms associated with primary and secondary metabolite production.

Proteomic analysis implicates that postovulatory aging leads to aberrant gene expression, biosynthesis, RNA metabolism and cell cycle in mouse oocytes.

BACKGROUND: In mammals, oocytes display compromised quality after experiencing a process of postovulatory aging. However, the mechanisms underlying are not yet fully understood. Here, we portrayed a protein expression profile of fresh and aging metaphase II (MII) mouse oocytes by means of four-dimensional label-free quantification mass spectrometry (4D-LFQ). RESULTS: The analysis of 4D-LFQ data illustrated that there were seventy-six differentially expressed proteins (DEPs) between two groups of MII stage oocytes. Fifty-three DEPs were up-regulated while twenty-three DEPs were down-regulated in the MII oocytes of the aging group, and Gene Ontology (GO) analysis revealed that these DEPs were mainly enriched in regulation of gene expression, biosynthesis, RNA metabolism and cell cycle. Our detailed analysis revealed that the expression of proteins that related to gene expression processes such as transcription, translation, post-translational modifications and epigenome was changed; the relative protein expression of RNA metabolic processes, such as RNA alternative splicing, RNA export from nucleus and negative regulation of transcription from RNA polymerase II promoter was also altered. CONCLUSION: In conclusion, we identified considerable DEPs and discussed how they agreed with previous researches illustrating altered protein expression associated with the quality of oocytes. Our research provided a new perspective on the mechanisms of postovulatory aging and established a theoretical support for practical methods to control and reverse postovulatory aging.

Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation.

Translational regulation plays a critical role during the oocyte-to-embryo transition (OET) and zygotic genome activation (ZGA). Here, we integrated ultra-low-input ribosome profiling (Ribo-lite) with messenger RNA sequencing to co-profile the translatome and transcriptome in human oocytes and early embryos. Comparison with mouse counterparts identified widespread differentially translated gene functioning in epigenetic reprogramming, transposon defense, and small RNA biogenesis, in part driven by species-specific regulatory elements in 3' untranslated regions. Moreover, PRD-like homeobox transcription factors, including TPRXL, TPRX1, and TPRX2, are highly translated around ZGA. TPRX1/2/L knockdown leads to defective ZGA and preimplantation development. Ectopically expressed TPRXs bind and activate key ZGA genes in human embryonic stem cells. These data reveal the conservation and divergence of translation landscapes during OET and identify critical regulators of human ZGA.

Functional substitution of zona pellucida with modified sodium hyaluronate gel in human embryos.

PURPOSE: Zona pellucida-free (ZP-free) embryos often fail to achieve good developmental outcomes and are routinely discarded in assisted reproductive laboratories. Existing attempts to rescue ZP-free embryos are not widely used due to operational complexity and high technical requirements. To handle cases with missing ZP, we applied modified sodium hyaluronate gel (MSHG) to embryo culture to determine if it can function as a substitute for human zona pellucida. METHODS: The developmental process and the blastocyst formation rate of embryos were analyzed in both mouse and human. The first clinical application of MSHG was reported, and the pregnancy outcome was continuously followed up. RESULTS: Human and mouse ZP-free embryos cultured with MSHG showed a blastocyst formation rate similar to ZP-intact embryos. MSHG improves blastocysts formation rate by maintaining blastomere spatial arrangement at early stages. Compared to ZP-free embryos, the proportion of tetrahedrally arranged blastomeres at the 4-cell stage increased significantly in embryos cultured with MSHG in humans. A ZP-free blastocyst cultured in MSHG with the highest score was successfully implanted after day 5 transplantation and developed normally. CONCLUSION: These data demonstrate that MSHG can substitute the function of zona pellucida and rescue human ZP-free embryos during assisted reproductive technology.

Screening and identification of a strain of Aureobasidium pullulans and its application in potato starch industrial waste.

Pullulan and melanin have become important secondary metabolites that are now widely studied. In this study, a strain of Aureobasidium pullulans HIT-LCY3T was used to ferment potato starch industrial waste to produce pullulan and melanin. After optimization, the culture conditions for the fermentation medium were obtained: inoculum age of 48 h, initial pH of 6.0, inoculation quantity of 1.5%, temperature of 26 °C, fermentation time of 5 d and speed of 160 rpm. Under these conditions, the yield of pullulan was 23.47 g/L with a molecular weight (MW) of 1.21 × 106 Da and the yield of melanin was 18.98 g/L. In addition, the adaptive evolution could significantly increase the yield of pullulan and melanin and the air-floating fermenters was more conductive to product accumulation. Through the 5 L small-scale test and 1000 L pilot test, the yield of pullulan reached 16.52 g/L with molecular weight of 0.92 × 106 Da and the yield of melanin was 12.08 g/L at the trial production of 30,000 L. This work could provide strong support for industrial production and new guidance for waste utilization and environmental protection.

Requirement of LaeA for sporulation, pigmentation and secondary metabolism in Chaetomium globosum.

The global regulator LaeA has been confirmed to govern the production of secondary metabolites in fungi. Herein, we examined the role of LaeA in Chaetomium globosum. Similarly as observed in other filamentous, CgLaeA had a significant effect on the secondary metabolism. The ΔCglaeA mutant strain did not exhibit chaetoglobosin A, whereas its production was restored in the CglaeAC strain. In addition, CglaeA overexpression led to an increase in chaetoglobosin A production. Transcriptional examination of the mutants indicated that CgLaeA positively regulated the expression of pathway-specific transcription factor CgcheR, while another global regulator CgvelB was negatively regulated by CgLaeA. Furthermore, CgLaeA also affected the morphological phenotypes of fungi. The ΔCglaeA mutant strains exhibited decreased sporulation and pigmentation compared with the wild-type strain, whereas the phenotypes were restored in the CglaeAC strain. Moreover, OE::CglaeA exhibited increased levels of sporulation and pigmentation. Moreover, inhibition activity against phytopathogenic fungi affected by decreased mycotoxin production of the ΔCglaeA mutant strain.

Functional analysis of a chaetoglobosin A biosynthetic regulator in Chaetomium globosum.

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including chaetoglobosin A production. Chaetoglobosin A is produced by Chaetomium globosum and has potential antifungal activity. Bioinformatics analysis of the chaetoglobosin A gene cluster (che) showed it that consists of nine open reading frames, including those encoding polyketide synthases (PKSs), PKS extender units, post-PKS modifications, and proposed regulators. Here, the role of the CgcheR regulator was investigated using gene disruption experiments. The CgcheR disruptant (ΔCgcheR) completely abolished the production of chaetoglobosin A, which was restored by the introduction of a copy of the wild-type CgcheR gene, suggesting that CgcheR is involved in chaetoglobosin A biosynthesis. A transcriptional analysis of the CgcheR disruptant indicated that CgCheR activates the transcription of chaetoglobosin biosynthetic genes in a pathway-specific manner. Furthermore, constitutive overexpression of CgcheR significantly improved the production of chaetoglobosin A from 52 to 260 mg/L. Surprisingly, CgcheR also played a critical role in sporulation; the CgcheR disruptant lost the ability to produce spores, suggesting that the regulator modulates cellular development. Our results not only shed light on the regulation of chaetoglobosin A biosynthesis, but also indicate a relationship between secondary metabolism and fungal morphogenesis.

Visible-Light-Promoted C2 Selective Arylation of Quinoline and Pyridine N-Oxides with Diaryliodonium Tetrafluoroborate.

A protocol of visible-light-promoted C2 selective arylation of quinoline and pyridine N-oxides, with diaryliodonium tetrafluoroborate as an arylation reagent, using eosin Y as a photocatalyst for the construction of N-heterobiaryls was presented. This methodology provided an efficient way for the synthesis of 2-aryl-substituted quinoline and pyridine N-oxides. This strategy has the following advantages: specific regioselectivity, simple operation, good functional group tolerance, and high to moderate yields under mild conditions.

A novel expression vector for Corynebacterium glutamicum with an auxotrophy complementation system.

Corynebacterium glutamicum is an important industrial strain used for the production of amino acids and vitamins. Most tools developed for overexpression of genes in C. glutamicum are based on the inducible promoter regulated by the lacIq gene or contain an antibiotic resistance gene as a selection marker. These vectors are essential for rapid identification of recombinant strains and detailed study of gene functions, but, as a considerable disadvantage, these vectors are not suitable for large-scale industrial production due to the need for the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) and antibiotics. In this study, the novel Escherichia coli-C. glutamicum shuttle expression vector pLY-4, derived from the expression vector pXMJ19, was constructed. The constitutive vector pLY-4 contains a large multiple cloning site, the strong promoter tacM and two selective markers: the original chloramphenicol resistance gene cat is used for molecular cloning operations, and the auxotrophy complementation marker alr, which can be stably replicated in the auxotrophic host strain without antibiotic selection pressure, is used for industrial fermentation. Heterologous expression of the gapC gene using the vector pLY-4 in C. glutamicum for L-methionine production indicated the potential application of pLY-4 in the development of C. glutamicum strain engineering and industrial fermentation.

ATP-Binding Cassette Transporter Regulates N,N'-diacetylchitobiose Transportation and Chitinase Production in Trichoderma asperellum T4.

ATP-binding cassette (ABC) transporters are a superfamily of proteins that transport nutrient substances and secondary metabolites through cell membranes. They also act as an uptake system for N,N'-diacetylchitobiose (GlcNAc)2 in Streptomyces coelicolor. (GlcNAc)2 is an important inducer of chitinase. However, whether the ABC transporter in Trichoderma spp. is also responsible for (GlcNAc)2 uptake and chitinase induction has not yet been confirmed. In this study, we applied RNA interference and overexpression technologies to alter the expression level of the ABC-B transporter in order to detect changes in its transportation ability and the expression level of inducible endo-chitinase ECH42-an important biocontrol enzyme in Trichoderma asperellum. The results revealed that, after interference with the expression of the ABC-B transporter, T. asperellum T4 was only able to grow normally when glucose was the only carbon source. Compared with the wild-type, the efficiency of (GlcNAc)2 by the overexpression strain evidently increased, along with the activity level of ECH42. In conclusion, one of the functions of the ABC-B transporter in T. asperellum is the uptake and transport of (GlcNAc)2 into cells, and chitobiose is a strong inducer of ECH42 in T. asperellum T4.

New production process of the antifungal chaetoglobosin A using cornstalks

Abstract Chaetoglobosin A is an antibacterial compound produced by Chaetomium globosum, with potential application as a biopesticide and cancer treatment drug. The aim of this study was to evaluate the feasibility of utilizing cornstalks to produce chaetoglobosin A by C. globosum W7 in solid-batch fermentation and to determine an optimal method for purification of the products. The output of chaetoglobosin A from the cornstalks was 0.34 mg/g, and its content in the crude extract was 4.80%. Purification conditions were optimized to increase the content of chaetoglobosin A in the crude extract, including the extract solvent, temperature, and pH value. The optimum process conditions were found to be acetone as the extractant, under room temperature, and at a pH value of 13. Under these conditions, a production process of the antifungal chaetoglobosin A was established, and the content reached 19.17%. Through further verification, cornstalks could replace crops for the production of chaetoglobosin A using this new production process. Moreover, the purified products showed great inhibition against Rhizoctonia solani, with chaetoglobosin A confirmed as the main effective constituent (IC50 = 3.88 µg/mL). Collectively, these results demonstrate the feasibility of using cornstalks to synthesize chaetoglobosin A and that the production process established in this study was effective.

Identification and characterization of the major antifungal substance against Fusarium Sporotrichioides from Chaetomium globosum.

Fusarium sporotrichioides, is a common soil-borne plant pathogen causing dry rot of potato in Northeast China. The objective of this study was to identify the main antifungal substances from Chaetomium globosum W7 against F. sporotrichioides. Strain W7 can significantly inhibit F. sporotrichioides without direct contact, suggesting that its antifungal substance was extracellular, and the solubility of this antifungal substance in ethyl acetate was superior to that in water. Acetone was selected as the optimum solvent for the extraction of the metabolites of C. globosum. Metabolites were then separated with thin-layer chromatography. Following antifungal tests on bands, a dark brown band with Rf value of 0.20 was determined as the antifungal substance, and identified as chaetoglobosin A. The antifungal activity test showed that the minimum inhibitory concentration of chaetoglobosin A to F. sporotrichioides was 9.45-10.50 µg/mL, IC50 being 4.344 µg/mL. Chaetoglobosin A also proved to have an excellent preventive effect on potato dry rot caused by F. sporotrichioides. To summarize, chaetoglobosin A was identified as the main active substance of C. globosum to inhibit F. sporotrichioides for the first time, and demonstrated a potential application value in agriculture.

New production process of the antifungal chaetoglobosin A using cornstalks.

Chaetoglobosin A is an antibacterial compound produced by Chaetomium globosum, with potential application as a biopesticide and cancer treatment drug. The aim of this study was to evaluate the feasibility of utilizing cornstalks to produce chaetoglobosin A by C. globosum W7 in solid-batch fermentation and to determine an optimal method for purification of the products. The output of chaetoglobosin A from the cornstalks was 0.34mg/g, and its content in the crude extract was 4.80%. Purification conditions were optimized to increase the content of chaetoglobosin A in the crude extract, including the extract solvent, temperature, and pH value. The optimum process conditions were found to be acetone as the extractant, under room temperature, and at a pH value of 13. Under these conditions, a production process of the antifungal chaetoglobosin A was established, and the content reached 19.17%. Through further verification, cornstalks could replace crops for the production of chaetoglobosin A using this new production process. Moreover, the purified products showed great inhibition against Rhizoctonia solani, with chaetoglobosin A confirmed as the main effective constituent (IC50=3.88µg/mL). Collectively, these results demonstrate the feasibility of using cornstalks to synthesize chaetoglobosin A and that the production process established in this study was effective.

Expression and Characterization of a Novel Antifungal Exo-ß-1,3-glucanase from Chaetomium cupreum.

A novel ß-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 ß-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein's molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 °C. Heavy metal ions Fe2+, Mn2+, Cu2+, Co2+, Ag+, and Hg2+ had inhibitory effects on Ccglu17A, but Ba2+ promoted the enzyme's activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing ß-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 µmol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-ß-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.