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The urgency of ensuring adequate moral courage in clinical nursing practice is evident. However, currently, there are few formal intervention plans targeted at enhancing the moral courage of nurses.To develop a training program for improving the moral courage of nurses using the modified Delphi method.A modified Delphi study.From November to December 2022, a literature review and expert group discussion were conducted to develop a preliminary training plan framework. From January to March 2023, a two-round Delphi survey was performed, and a consensus was reached regarding the plan through online questionnaires. Descriptive statistics were used to analyze the data.This study was approved by the institutional ethics committee (No. 138, 30 August 2021). All participants provided written informed consent.Consensus was reached on eight themes and 33 items to strengthen the moral courage training program for nurses.Guided by a unified goal of moral education, a multi-level and acceptable intervention plan was designed to enhance the moral courage of nurses. [ABSTRACT FROM AUTHOR]

• A combined approach of QSAR modeling, enzymatic kinetics and molecular docking were used. • The QSAR models of Gly-Pro-type peptides were successfully constructed by 5z-scale descriptor. • The complex formed by DPP-IV and Gly-Pro-type penta- and hexa- peptides had lower acylation. • Gly-Pro-type penta- and hexa- peptides interact more with S2 extensive and S2 sites of DPP-IV. Our previous study has demonstrated that both the amino acid at N3 position and peptide length affected the DPP-IV inhibitory activity of Gly-Pro-type peptides. To further elucidate their molecular mechanism, a combined approach of QSAR modeling, enzymatic kinetics and molecular docking was used. Results showed that the QSAR models of Gly-Pro-type tripeptides and Gly-Pro-type peptides containing 3–12 residues were successfully constructed by 5z-scale descriptor with R2 of 0.830 and 0.797, respectively. The lower values of electrophilicity, polarity, and side-chain bulk of amino acid at N3 position caused higher DPP-IV inhibitory activity of Gly-Pro-type peptides. Moreover, an appropriate increase in the length of Gly-Pro-type peptides did not change their competitive inhibition mode, but decreased their inhibition constants (K i values) and increased interactions with DPP-IV. More importantly, the interactions between the residues at C-terminal of Gly-Pro-type peptides containing 5 ∼ 6 residues with S2 extensive subsites (Ser209, Phe357, Arg358) of DPP-IV increased the interactions of Gly residue at N1 position with the S2 subsites (Glu205, Glu206, Asn710, Arg125, Tyr662) and decreased the acylation level of DPP-IV-peptide complex, and thereby increasing peptides' DPP-IV inhibitory activity. [ABSTRACT FROM AUTHOR]

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements. [ABSTRACT FROM AUTHOR]

Background: Sequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing). Results: By using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases. Conclusions: Pime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes. [ABSTRACT FROM AUTHOR]

Previously, we prepared a chondroitin sulfate-soluble undenatured type II collagen complex (CS-SC II) with low salt content. This paper further explored the differences between CS-SC II and SC II in terms of gastrointestinal digestive characteristics and osteoarthritis (OA) improvement. In vitro and in vivo experiments showed that the gastric digestive stability of CS-SC II was high under both pH 2.0 and pH 3.0, the α1 chain and triple helix structure of type II collagen retained >60 %. However, SC II had high gastric digestive stability only under pH 3.0. Furthermore, intestinal digestion had little effect on α1 chains of CS-SC II and SC II, and distribution experiments showed that they might exert their biological activities in the intestine. CS-SC II had obvious improvement in OA rats at 1.0 mg/kg/d, that is, the joint swelling was significantly reduced and the weight-bearing ratio of the right hind limb was increased to 49 %, which was close to that of 4.0 mg/kg/d SC II. The wear of articular cartilage, Mankin and OARSI scores of rats in CS-SC II group were significantly reduced. The effects of low-dose CS-SC II on the proportion of regulatory T cells (Treg), mRNA expression of OA key biomarkers (Il6, Ccl7, MMP-3 and MMP13) and signaling pathway genes (NF-κB, AKT or AMPKα) were comparable to those of high-dose SC II. These results showed that CS-SC II might have greater potential to improve OA at a lower dose than SC II due to its high gastrointestinal digestive stability at a wide range of pH conditions. [Display omitted] • Study on chondroitin sulfate-soluble undenatured type II collagen complex (CS-SC II) • The gastric digestive stability of CS-SC II was high under both pH 2.0 and pH 3.0. • SC II had high gastric digestive stability only under pH 3.0. • The improvement of 1.0 mg/kg/d CS-SC II in OA rats was close to 4.0 mg/kg/d SC II. • CS-SC II regulated the expression of key OA biomarkers and signaling pathway genes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Temperature regulation improved the aroma quality of HLFSS produced in winter. • 11 odor-active markers primarily determined temperature-mediated aroma difference. • Dominant and low abundance genus together drove volatile profiles variations. • Synthesis network of representative furanones HDMF and HEMF was elucidated. The flavor regulation of soy sauce fermented in winter is imminent challenge for the industry, while fermentation temperature is considered as an effective method to fortify soy sauce flavor. Thus, industrial-level fermentation systems with controlled temperature at 30°C (SSCT) and regular temperature (SSRT) in winter were designed to elucidate molecular basis and microbial regulatory mechanism of temperature-controlled flavor enhancement of soy sauce. Sensory evaluation suggested 30°C fermentation enhanced caramel-like, floral, fruity, roasted nut and smoky aroma. A total of 160 volatiles were identified, of which 39 components were evaluated for odor activity value (OAV). Eleven volatiles were determined as the odor markers distinguishing the aroma profiles of SSRT and SSCT, among which 2,5-dimethyl-4-hydroxy-3(2H)-furanone (HDMF, caramel-like), β-damascenone (floral), ethyl 2-methylpropanoate (fruity), ethyl acetate (fruity) and 2/3-methyl-1-butanol (malty, alcoholic) were largely responsible for the flavor enhancement. Moreover, high-throughput sequencing results demonstrated the temperature intervention induced more differential bacterial structure (R = 0.324, P = 0.001) than fungal structure (R = 0.069, P = 0.058). Correlation analysis revealed dominant and low-abundance genus together drove the formation and variation of volatile profile, particularly Weissella , Tetragenococcus , Starmerella and Pediococcus. Representatively, the formation pathways of key aroma substances HDMF and 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (HEMF) were elaborated. Both temperature-mediated abiotic reactions and gene functions of microbiota were proposed to favor the yields of HDMF and C 5 precursor of HEMF, whereas the small populations of Zygosaccharomyces and insufficient acetaldehyde limited the elevation of the HEMF level through the biosynthesis pathway. This study provided the practical and theoretical basis for the industrial applications of temperature control in soy sauce fermentation. [ABSTRACT FROM AUTHOR]

Background: Chromosomal microarray analysis (CMA) has been widely applied to explore the genetic etiology in recurrent pregnancy loss (RPL). However, the reproductive prognosis in RPL couples with different types of chromosomally abnormal miscarriage remains unclear. Objectives: The main purpose of this study was to evaluate the reproductive prognosis among RPL couples after genetic testing in products of conception (POCs) by CMA. Study design: In this retrospective study, 1101 RPL couples referred for genetic testing in POCs by CMA. A total of 830 couples who met the inclusion criteria were followed up for at least 24 months after the index miscarriage. The rates of live birth and adverse pregnancy events in subsequent pregnancy and cumulative pregnancies were examined. Results: For couples with three or more miscarriage, compared with those with chromosomally normal miscarriage, a significantly higher subsequent live birth rate was found in couples with chromosomally abnormal miscarriage (66.9% vs 71.6%, P =.040). However, differences in cumulative live birth rate among couples with chromosomally abnormal miscarriage and normal miscarriage were nonsignificant (82.7% vs 80.2%, P =.131). Women with advanced maternal age showed a significant decrease in the live birth rate (P < 0.01) and an increase in the miscarriage rate (P < 0.01) than those aged < 35 years old, regardless of whether the miscarriage was chromosomally normal or abnormal. RPL couples with chromosomally normal miscarriage showed a significant decrease in live birth rates in subsequent pregnancy and cumulative pregnancies, when they had experienced a large number of previous miscarriages; however, no significant difference was observed in those with chromosomally abnormal miscarriage. Conclusion: For women with three or more previous miscarriages, RPL couples with chromosomally normal miscarriage manifested a poorer reproductive prognosis than those with chromosomally abnormal miscarriage in subsequent pregnancy, while the cumulative live birth rate was similar. Advanced maternal age was a predictor of adverse pregnancy events, regardless of embryonic chromosomal results. Furthermore, among RPL women with large numbers of previous miscarriages, the supportive care and counselling regarding individual risk is necessary for those with chromosomally normal miscarriage. [ABSTRACT FROM AUTHOR]

Recombinant proteins produced by cell factories are now widely used in various fields. Many efforts have been made to improve the secretion capacity of cell factories to meet the increasing demand for recombinant proteins. Recombinant protein production usually causes cell stress in the endoplasmic reticulum (ER). The overexpression of key genes possibly removes limitations in protein secretion. However, inappropriate gene expression may have negative effects. There is a need for dynamic control of genes adapted to cellular status. In this study, we constructed and characterized synthetic promoters that were inducible under ER stress conditions in Saccharomyces cerevisiae. The unfolded protein response element UPRE2, responding to stress with a wide dynamic range, was assembled with various promoter core regions, resulting in UPR-responsive promoters. Synthetic responsive promoters regulated gene expression by responding to stress level, which reflected the cellular status. The engineered strain using synthetic responsive promoters P4UPRE2 − TDH3 and P4UPRE2 − TEF1 for co-expression of ERO1 and SLY1 had 95% higher α-amylase production compared with the strain using the native promoters PTDH3 and PTEF1. This work showed that UPR-responsive promoters were useful in the metabolic engineering of yeast strains for tuning genes to support efficient protein production. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae is a widely used cell factory for protein production. Increasing the protein production capacity of a yeast strain may be beneficial for obtaining recombinant proteins as a product or exerting its competence in consolidated bioprocessing. However, heterologous protein expression usually imposes stress on cells. Improving the cell's ability to cope with stress enhances protein yield. HAC1 is a key transcription factor in the unfolded protein response (UPR). In this study, several genes related to the UPR signal pathway, including unfolded protein sensing, HAC1 mRNA splicing, mRNA ligation, mRNA decay, translation, and Hac1p degradation, were selected as targets to engineer yeast strains. The final engineered strain produced α-amylase 3.3-fold, and human serum albumin 15.3-fold, greater than that of the control strain. Key regulation and metabolic network changes in the engineered strains were identified by transcriptome analysis and physiological characterizations. This study demonstrated that cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. The verified genetic modifications of this study provide useful targets in the construction of yeast cell factories for efficient protein production. • Cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. • The final engineered strain had a 3.3-fold higher α-amylase yield and a 15.3-fold higher HSA yield. • Engineered strains showed a better stress tolerance. • Transcriptome analysis revealed changes in gene expression patterns. [ABSTRACT FROM AUTHOR]

Cordyceps militaris , widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks, expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to further research on C. militaris across various fields, including computational biology, existing DNA elements, and genome editing approaches. This review aims to describe specific and promising opportunities for the in-depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic biology development are illustrated. • C. militaris is a medicinal and edible mushroom. • C. militaris has the potential and possibility to be built as a cell factory. • Computational and advanced CRISPR technique were applied in C. militaris. • Metabolic engineering instructs in producing high-value compounds. • Synthetic biology facilitates the development of cell factory construction. [ABSTRACT FROM AUTHOR]

Previously, we presented a green and efficient separation method for soluble undenatured type II collagen (SC II) based on the complex coacervation of SC II and anionic polysaccharides, and demonstrated the significant impact of molecular weight (M w). In this study, we combined differential scanning calorimetry (DSC), microscale thermophoresis (MST) and molecular dynamics simulation (MDS) to further explore the affinity and interaction types of SC II with different anionic polysaccharides. Results showed that the denaturation temperature of SC II increased (about 3 °C) after complex coacervating with chondroitin sulfate (CS, a small anionic polysaccharide), which might be related to their strong affinity (dissociation constant of K d and binding energy were about 3.04 × 10−6 mg/mL and −4054.97 kJ/mol, respectively), and the molecular weight of anionic polysaccharides had a greater impact than chain conformation. The non-covalent interactions between SC II and anionic polysaccharides were mainly hydrogen bonds, followed by hydrophobic interactions and ionic bonds. The hydroxyl and sulfate groups in polysaccharides could form extensive hydrogen bonds and small amount of ionic bonds with a large number of amino acids in SC II, and the hydrophobic groups such as methyl also formed hydrophobic interactions with hydrophobic parts in SC II (such as Pro82, Pro85, Val88). Importantly, the smaller the M w of anionic polysaccharides, the faster they formed stable interactions with SC II. Therefore, anionic polysaccharides with smaller M w had higher affinity and faster non-covalent interactions formation speed with SC II, resulting in more compact structure and higher thermal stability of the complexes. [Display omitted] • Anionic polysaccharides with smaller M w had stronger affinity with SC Ⅱ. • Thermal stability of SC II increased after combining with chondroitin sulfate and pectin. • Hydrogen bonds, hydrophobic interactions were the main non-covalent interactions. • Small M w of anionic polysaccharides was beneficial to the interactions. [ABSTRACT FROM AUTHOR]

• Rate of chromosomal abnormality in the non-isolated was higher than the isolated. • Theoretical residual risks in all NIPS models were higher than the control cohort. • The isolated and normal CMA showed higher normal infant rate than the non-isolated. • Theoretical residual risk should be considered in genetic counseling. It remains controversial whether prenatal screening or diagnostic testing should be offered to fetuses with nasal bone (NB) absence or hypoplasia, and there are no studies comparing the yield of chromosomal microarray analysis (CMA) to non-invasive prenatal screening (NIPS). The aim of this study was to evaluate the residual risk of clinically significant copy number variations (CNVs) in fetuses with NB absence or hypoplasia after excluding theoretically NIPS-detectable abnormalities, and to assess their clinical outcomes. This prospective study encompassed 400 fetuses with NB absence or hypoplasia undergoing CMA testing between 2015 and 2022. Clinically significant CMA findings were categorized into three subgroups, including three-NIPS-detectable (trisomies 21, 18 and 13), five-NIPS-detectable (trisomies 21, 18 and 13 and sex chromosome aneuploidies) and genome-wide NIPS-detectable (variants over 7 Mb). We calculated the theoretical residual risk and compared it with the results of a control cohort of low-risk pregnancies. We further evaluated their clinical outcomes. The overall diagnostic yield in our cohort was 7.8% (31/400). The detection rate of clinically significant CMA findings in fetuses with non-isolated NB absence or hypoplasia was significantly higher than that in fetuses with isolated NB absence or hypoplasia (20.0% vs. 6.6%, P =.005). The theoretical residual risks in all NIPS models were significantly higher when compared with the control cohort. The normal infant rate in fetuses with normal CMA results was 97.9% (323/330), and a significant higher incidence was observed in fetuses with isolated NB absence or hypoplasia compared with non-isolated NB absence or hypoplasia (98.4% vs. 91.7%, P =.028). The residual risk of clinically significant CNVs in fetuses with NB absence or hypoplasia following the exclusion of theoretically NIPS-detectable findings was higher than that in low-risk pregnancies. This risk should be considered in genetic counseling to make a more comprehensive and precise choice regarding prenatal genetic testing. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microbiota correlation with soy sauce flavor was explored by different source samples. • Flavor-producing ability for 5 of 15 potential flavor-producing bacteria was verified. • Not only yeast, but also bacteria were closely linked to the volatiles in soy sauce. • L. fermentum high yielding succinic acid, verifying their positive correlation. Flavor, the most important quality index of soy sauce, is mostly influenced by the microbiota in fermented food ecosystem, however, the association between microorganisms and soy sauce flavor is still poorly understood. Therefore, the bacterial and fungal profiles, physicochemical parameters, and flavor compounds (9 organic acids, 17 free amino acids and 97 volatile flavor compounds) of 5 different source soy sauce were investigated using high-throughput sequencing, HPLC, amino acid analyzer and SPME/LLE-GC–MS, and their correlations were explored. A total of 3 fungal genera and 12 bacterial genera were identified as potential flavor-producing microorganisms by multivariate data and correlation analysis. Notably, Lactobacillus and Tetragenococcus were strongly positively correlated with succinic acid and lactic acid, respectively. Moreover, not only fungi, but also bacteria were found to be closely correlated with volatiles. Finally, 5 screened potential flavor-producing microorganisms were validated using a rapid fermentation model, with multiple strains showing the potential to improve the soy sauce flavor, with Lactobacillus fermentum being the most significant. Our research will provide a theoretical basis for the regulation and enhancement of soy sauce flavor. [ABSTRACT FROM AUTHOR]

In order to avoid the problem of high salt content caused by salt-out method when separating soluble undenatured type II collagen (SC II), the complex coacervation of SC II and anionic polysaccharides, including carrageenan (CG), chondroitin sulfate (CS), xanthan gum (XG), sodium alginate (SA) and pectin (PE) was investigated to separate SC II. Furthermore, their effects on the recovery rate of SC II and the possible mechanisms were discussed. Results showed that CS-SC II (pH 3.0 and 4.0), SA-SC II (pH 4.0) and PE-SC II (pH 4.0 and 5.0) had lower moisture and higher SC II recovery rate, while CG-SC II, XG-SC II and SA-SC II (pH 3.0) showed opposite results. Based on the analysis of microstructure, molecular weight (Mw) and chain length, the smaller Mw and shorter chain of CS and PE might lead to the compact structure of their complexes with SC II. Fourier transform infrared (FTIR) analysis showed that, the amide II band of SC II blue shifted with the increase of pH value, and the α-helix structure of SC II decreased from 60.47% (pH 3.0) to 40.51% (pH 4.0). Moreover, the amide II band and α-helix structure of SC Ⅱ disappeared when it combined with CS, PE or SA (pH 4.0). Therefore, the molecular conformation of anionic polysaccharide and environmental pH, which might change the molecular charge and the secondary structure of SC II, had great impact on the complex coacervation of SC II with anionic polysaccharide. Small, short and flexible anionic polysaccharides may be suitable for SC II precipitation under charge neutralization and weakly acidic conditions. [Display omitted] • Complexation of soluble undenatured type II collagen (SC II) with anionic polysaccharides. • Complex coacervation of polysaccharides and SC II can be a new SC II separating method. • PH alteration and complex coacervation could change secondary structure of SC II. • Small, soft and short anionic polysaccharides were suitable for separating SC II. [ABSTRACT FROM AUTHOR]

The authors have reanalyzed the data using the correct conversion calculation. B Correction: Biotechnology for Biofuels and Bioproducts (2022) 15:89 b https://doi.org/10.1186/s13068-022-02186-y Following publication of the original article [[1]], the author noticed an error in Additional file 1: Fig. Correction: Secretion of collagenases by Saccharomyces cerevisiae for collagen degradation. [Extracted from the article]

Myclobutanil (MYC) is a common triazole fungicide widely applied in agriculture. MYC extensively exists in the natural environment and can be detected in organisms. However, little is known about MYC-induced embryonic developmental damage. This study aimed to unravel the cardiotoxicity of MYC and the underlying mechanisms, as well as the cardioprotective effect of curcumin (CUR, an antioxidant polyphenol) using the zebrafish model. Here, zebrafish embryos were exposed to MYC at concentrations of 0, 0.5, 1 and 2 mg/L from 4 to 96 h post fertilization (hpf) and cardiac development was assessed. As results, MYC reduced the survival and hatching rate, body length and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal cardiac morphology and function in myl7:egfp transgenic zebrafish, and downregulated cardiac developmental genes. MYC promoted oxidative stress through excessive reactive oxygen species (ROS) accumulation and suppressed the activities of antioxidant enzymes, triggering cardiomyocytic apoptosis via upregulated expression of apoptosis-related genes. These adverse toxicities could be significantly ameliorated by the antioxidant properties of CUR, indicating that CUR rescued MYC-induced cardiotoxicity by inhibiting oxidative stress and apoptosis. Overall, our study revealed the potential mechanisms of oxidative stress and apoptosis in MYC-induced cardiotoxicity in zebrafish and identified the cardioprotection of CUR in this pathological process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myclobutanil (MYC), a typical broad-spectrum triazole fungicide, is often detected in surface water. This study aimed to explore the neurotoxicity of MYC and the underlying mechanisms in zebrafish and in PC12 cells. In this study, zebrafish embryos were exposed to 0, 0.5 and 1 mg/L of MYC from 4 to 96 h post fertilization (hpf) and neurobehavior was evaluated. Our data showed that MYC decreased the survival rate, hatching rate and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal neurobehaviors characterized by decreased swimming distance and movement time. MYC impaired cerebral histopathological morphology and inhibited neurogenesis in HuC:egfp transgenic zebrafish. MYC also reduced the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and downregulated neurodevelopment related genes (gfap, syn2a, gap43 and mbp) in zebrafish and PC12 cells. Besides, MYC activated autophagy through enhanced expression of the LC3-II protein and suppressed expression of the p62 protein and autophagosome formation, subsequently triggering apoptosis by upregulating apoptotic genes (p53, bax, bcl-2 and caspase 3) and the cleaved caspase-3 protein in zebrafish and PC12 cells. These processes were restored by the autophagy inhibitor 3-methyladenine (3-MA) both in vivo and in vitro, indicating that MYC induces neurotoxicity by activating autophagy and apoptosis. Overall, this study revealed the potential autophagy and apoptosis mechanisms of MYC-induced neurotoxicity and provided novel strategies to counteract its toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow., Methods: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES)., Results: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES., Conclusions: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD., (© 2024. The Author(s).)

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories., Competing Interests: Competing interests statement:C.X., X.L., Y.P., and M.H. applied a patent for protecting UPRE2m development and its application. The remaining authors declare no competing interest.

Saccharomyces cerevisiae has become a key microbial cell factory for producing biofuels, recombinant proteins, and natural products. The development of efficient cell factories relies on the precise control and fine-tuning of gene expression, underscoring the pivotal role of promoters in pathway engineering. However, natural promoters often have limited transcriptional capacity and thus fall short of the metabolic engineering requirements. This chapter provides protocols and guidelines for constructing and evaluating synthetic promoters in S. cerevisiae. Moreover, these protocols are applicable for creating and testing various synthetic promoters in other host systems., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fenvalerate (FEN), a typical type II pyrethroid pesticide, is widely used in agriculture. FEN has been detected in the environment and human body. However, the neurotoxicity of FEN has not been well elucidated. This study aimed to explore the mechanisms underlying FEN-induced neurotoxicity using the zebrafish (Danio rerio) model. We also investigated whether curcumin (CUR), a polyphenol antioxidant that exhibits neuroprotective properties, can prevent FEN-induced neurotoxicity. Here, zebrafish embryos were exposed to 0, 3.5, 7 and 14 μg/L of FEN from 4 to 96 h post fertilization (hpf) and neurotoxicity was assessed. Our results showed that FEN decreased the survival rate, heart rate, body length and spontaneous movement, and increased malformation rate. FEN caused neurobehavioral alterations, including decreased swimming distance and velocity, movement time and clockwise rotation times. FEN also suppressed neurogenesis in transgenic HuC:egfp zebrafish, reduced cholinesterase activity and downregulated the expression of neurodevelopment related genes (elavl3, gfap, gap43 and mbp). In addition, FEN enhanced oxidative stress via excessive reactive oxygen species and antioxidant enzyme inhibition, then triggered apoptosis by upregulation of apoptotic genes (p53, bcl-2, bax and caspase 3). These adverse outcomes were alleviated by CUR, indicating that CUR mitigated FEN-induced neurotoxicity by inhibiting oxidative stress. Overall, this study revealed that CUR ameliorated FEN-induced neurotoxicity via its antioxidant, indicating a promising protection of CUR against environmental pollutant-induced developmental anomalies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design-Build-Test-Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products , such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion . This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.