Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

Discovery and mechanism explanation of a novel green biocatalyst esterase Bur01 from Burkholderia ambifaria for ester synthesis under aqueous phase.

Biocatalyst catalyzing the synthesis of esters under aqueous phase is an alternative with green and sustainable characteristics. Here, a biocatalyst esterase Bur01 was identified through genome sequencing and gene library construction from a Burkholderia ambifaria BJQ0010 with efficient ester synthesis property under aqueous phase for the first time. Bur01 was soluble expressed and the purified enzyme showed the highest activity at pH 4.0 and 40 °C. It had a broad substrate spectrum, especially for ethyl esters. The structure of Bur01 was categorized as a member of α/ß fold hydrolase superfamily. The easier opening of lid under aqueous phase and the hydrophobicity of substrate channel contribute to easier access to the active center for substrate. Molecular docking and site-directed mutation demonstrated that the oxyanion hole Ala22, Met112 and π-bond stacking between His24 and Phe217 played essential roles in catalytic function. The mutants V149A, V149I, L159I and F137I enhanced enzyme activity to 1.42, 1.14, 1.32 and 2.19 folds due to reduced spatial resistance and increased hydrophobicity of channel and ethyl octanoate with the highest conversion ratio of 68.28 % was obtained for F137I. These results provided new ideas for developing green catalysts and catalytic basis of mechanistic studies for ester synthetase under aqueous phase.

Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

AIMS: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. METHODS AND RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. CONCLUSION: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

Evolution of fermented grain yeast communities in strong-flavored baijiu and functional validation of yeasts that produce superior-flavored substances.

BACKGROUND: Baijiu is a well-known alcoholic beverage in China and the quality is determined by various microorganisms during the fermentation process. Yeast is one of the most important microorganisms in the fermentation of baijiu. It has a strong esterification capacity and also affects the aroma. RESULTS: High-throughput sequencing results showed that the fermented grains (jiupei) during baijiu production were mainly composed of eight highly abundant yeast species. The species and abundance of yeasts changed significantly with the fermentation process. The flavor of 30 yeast strains in the jiupei was determined by a sniffing test and gas chromatography-mass spectrometry (GC-MS). The strain with the highest flavor substance content (2.34 mg L-1 ), named YX3205, was identified as Clavispora lusitaniae. Tolerance results showed that C. lusitaniae YX3205 can tolerate up to 15% (v v-1 ) ethanol. In a solid-state simulated fermentation experiment, the content of 24 flavor substances was significantly increased in the fortified group, and the total ester content reached 4240.73 µg kg-1 , which was 2.8 times higher than that of the control group. CONCLUSION: The present study demonstrated the potential of C. lusitaniae YX3205 to enhance the flavor of baijiu, thereby serving as a valuable strain for the improvement of the flavor quality of baijiu. © 2024 Society of Chemical Industry.

Study on Cavitation, Warpage Deformation, and Moisture Diffusion of Sop-8 Devices during Molding Process.

Plastic packaging has shown its advantages over ceramic packaging and metal packaging in lightweight, thin, and high-density electronic devices. In this paper, the reliability and moisture diffusion of Sop-8 (Small Out-Line Package-8) plastic packaging devices are studied, and we put forward a set of complete optimization methods. Firstly, we propose to improve the reliability of plastic packaging devices by reducing the amount of cavitation and warpage deformation. Structural and process factors were investigated in the injection molding process. An orthogonal experiment design was used to create 25 groups of simulation experiments, and Moldflow software was used to simulate the flow mode analysis. Then, the simulation results are subjected to range analysis and comprehensive weighted score analysis. Finally, different optimization methods are proposed according to different production conditions, and each optimization method can reduce cavitation or warpage by more than 9%. The moisture diffusion of the Sop-8 plastic packing devices was also investigated at the same time. It was determined that the contact surface between the lead frame and the plastic packaging material was more likely to exhibit delamination under the condition of MSL2 moisture diffusion because the humidity gradient was easily produced at the crucial points of different materials. The diffusion of moisture is related to the type of plastic packaging material and the diffusion path.

Comparison of fungal communities and flavour substances in surface and inner layers of fermented grains during stacking fermentation of sauce-flavour baijiu.

The stacking fermentation process plays a vital role in the production of sauce-flavour baijiu. The aim of this paper is to elucidate the effects of environmental variables on the fungal communities of different layers of fermented grains (zaopei) during the sixth round of stacking and the changes in volatile flavour substances during this process. The composition of the fungal communities in different layers during the stacking fermentation process was analysed. Principal coordinate analysis (pCoA) showed that the dominant fungal communities in different layers differed significantly with the stacking fermentation process. The dominant fungal genera were Thermoascus, Thermomyces and Issatchenkia. The total content of flavouring substances in the surface layer of zaopei were higher, but the types of flavouring substances were less than in the inner layer. The relationship between temperature, moisture content, acidity, starch content and reducing sugar content and microbial community was analysed by Redundancy analysis. The results showed that the correlation between microbial communities and physicochemical indexes in different layers of zaopei varied. The core fungal genera in the surface layer were mainly influenced by acidity, and the microorganisms in the inner layer were more strongly correlated with temperature. Spearman correlation coefficient revealed the correlation between fungal community and volatile flavour substances, and the results showed that microorganisms in different layers of zaopei have different correlations with flavour substances. This study contributes to the understanding of the evolution of different layers fungal communities during the stacking of sauce-flavour baijiu and their relationship with volatile aroma substances.

Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation.

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

Using Chiplet Encapsulation Technology to Achieve Processing-in-Memory Functions.

With the rapid development of 5G, artificial intelligence (AI), and high-performance computing (HPC), there is a huge increase in the data exchanged between the processor and memory. However, the "storage wall" caused by the von Neumann architecture severely limits the computational performance of the system. To efficiently process such large amounts of data and break up the "storage wall", it is necessary to develop processing-in-memory (PIM) technology. Chiplet combines processor cores and memory chips with advanced packaging technologies, such as 2.5D, 3 dimensions (3D), and fan-out packaging. This improves the quality and bandwidth of signal transmission and alleviates the "storage wall" problem. This paper reviews the Chiplet packaging technology that has achieved the function of PIM in recent years and analyzes some of its application results. First, the research status and development direction of PIM are presented and summarized. Second, the Chiplet packaging technologies that can realize the function of PIM are introduced, which are divided into 2.5D, 3D packaging, and fan-out packaging according to their physical form. Further, the form and characteristics of their implementation of PIM are summarized. Finally, this paper is concluded, and the future development of Chiplet in the field of PIM is discussed.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

Flavor mystery of Chinese traditional fermented baijiu: The great contribution of ester compounds.

Chinese traditional fermented baijiu is a famous alcoholic beverage with unique flavor. Despite its consumption for millennia, the flavor mystery behind baijiu is still unclear. Studies indicate that esters are the most important flavor substances, and bring health benefits. However, the aroma contribution and formation mechanism of esters still need to be clarified to reveal the flavor profile of baijiu. This review systematically summarizes all the 510 esters and finds 9 ethyl esters contribute greatly to the flavor of baijiu. The 508 different microbial species that have been identified affect the synthesis of esters through fatty acid and amino acid metabolism. The determination of minimum functional microbial groups and the analysis of their metabolic characteristics are crucial to reveal the mechanism of formation of baijiu flavor, and ensure the reproducible formation of flavor substances.

Molecular mechanism of LIP05 derived from Monascus purpureus YJX-8 for synthesizing fatty acid ethyl esters under aqueous phase.

Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu. Monascus purpureus YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to α/ß fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The π-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu.

5'-UTR SNP of FGF13 causes translational defect and intellectual disability.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

An efficient phthalate ester-degrading Bacillus subtilis: Degradation kinetics, metabolic pathway, and catalytic mechanism of the key enzyme.

Phthalate ester pollution in the environment and food chain is frequently reported. Microbial treatment is a green and efficient method for solving this problem. The isolation and systematic investigation of microorganisms generally recognized as safe (GRAS) will provide useful resources. A GRAS Bacillus subtilis strain, BJQ0005, was isolated from Baijiu fermentation starter and efficiently degraded phthalate esters (PAEs). The half-lives for di-isobutyl phthalate, di-butyl phthalate and di-(2-ethylhexyl) phthalate were 3.93, 4.28, and 25.49 h, respectively, from the initial amount of 10 mg per 10 mL reaction mixture, which are records using wild-type strains. Genome sequencing and metabolic intermediate analysis generated the whole metabolic pathway. Eighteen enzymes from the α/ß hydrolase family were expressed. Enzymes GTW28_09400 and GTW28_13725 were capable of single ester bond hydrolysis of PAEs, while GTW28_17760 hydrolyzed di-ester bonds of PAEs. Using molecular docking, a possible mechanism affecting enzymatic ester bond hydrolysis of mono-butyl phthalate was proposed of GTW28_17760. The carboxyl group generated by the first hydrolysis step interacted with histidine in the catalytic active center, which negatively affected enzymatic hydrolysis. Isolation and systematic investigation of the PAE degradation characteristics of B. subtilis will promote the green and safe treatment of PAEs in the environment and food industry.

MicroRNA­539 inhibits cell proliferation, colony formation and invasion in pancreatic ductal adenocarcinoma by directly targeting IGF­1R.

MicroRNAs (miRNAs) possess oncogenic and tumour­suppressive roles in the carcinogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) by regulating the expression of numerous cancer­related genes. Thus, the investigation on the expression and roles of miRNAs in PDAC may facilitate the identification of novel and effective targets for the clinical diagnosis and treatment of patients with PDAC. miRNA­539 (miR­539) has been studied in multiple types of human cancer. However, its expression and potential biological function in PDAC remain unclear. In the current study, the expression level, clinical significance, roles and underlying molecular mechanism of miR­539 in PDAC. The present results demonstrated that miR­539 expression was downregulated in PDAC tissues and cell lines. A low miR­539 level was associated with TNM stage and lymph node metastasis of patients with PDAC. miR­539 overexpression induced a significant reduction in the proliferation, colony formation and invasion of PDAC cells. Insulin­like growth factor 1 receptor (IGF­1R) was confirmed as a direct target gene of miR­539 in PDAC. Further analysis indicated that IGF­1R was overexpressed in PDAC tissues. Notably, the mRNA expression of IGF­1R was negatively correlated with miR­539 levels in PDAC tissues. In addition, the recovered IGF­1R expression also partially counteracted the suppressive roles of miR­539 overexpression in PDAC cells. Overall, miR­539 may inhibit the aggressive behaviour of PDAC by directly targeting IGF­1R and may serve as a novel therapeutic target for patients with this disease.

Screening of potential biomarkers in uterine leiomyomas disease via gene expression profiling analysis.

The present study aimed to screen potential biomarkers for uterine leiomyomas disease, particularly target genes associated with the mediator of RNA polymerase II transcription subunit 12 (MED12) mutation. The microarray data of GSE30673, including 10 MED12 wild-type myometrium, 8 MED12 mutation leiomyoma and 2 MED12 wild-type leiomyoma samples, were downloaded from the Gene Expression Omnibus database. Compared with myometrium samples, differently-expressed genes (DEGs) in the MED12 mutation and wild-type leiomyoma samples were identified using the Limma package. The two sets of DEGs obtained were intersected to screen common DEGs. The DEGs in the MED12 mutation and wild-type leiomyoma samples, and common DEGs were defined as group A, B and C. Gene Ontology (GO) and pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery online tool. Based on the Kyoto Encyclopedia of Genes and Genomes database, pathway relation networks were constructed. DEGs in GO terms and pathways were intersected to screen important DEGs. Subsequently, a gene co­expression network was constructed and visualized using Cytoscape software. Reverse transcription­quantitative polymerase chain reaction was used to detect the expression levels of important DEGs. A total of 1,258 DEGs in group A were screened, and enriched for extracellular matrix (ECM) organization and ECM­receptor interaction. In addition, a total of 1,571 DEGs in group B were enriched for cell adhesion. Furthermore, 391 DEGs were involved in extracellular matrix organization. Pathway relation networks of group A, B and C were constructed with nodes of 48, 39, and 28, respectively. Finally, 135 important DEGs were obtained, including Acyl­CoA synthetase medium­chain family member 3, protein S (α) (PROS1) and F11 receptor. A gene co­expression network with 68 nodes was constructed. The expression of caspase 1 (CASP1) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was significant higher in SK­UT­1 compared with that in PHM1­31 cells, while the expression of PROS1 was significant lower in SK­UT­1 cells. These results that CASP1, ALDH1A1 and PROS1 may be potential biomarkers for uterine leiomyomas. Furthermore, hematopoietic prostaglandin D synthase and carbonyl reductase 3 (CBR3) may be particular genes associated with the MED12 mutation in this disease.

Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity.

Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.