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Stroke is a common central nervous system disease in clinical practice. Stroke patients often have infectious complications, such as pneumonia and infections of the urinary tract and gastrointestinal tract. Although it has been shown that translocation of the host gut microbiota to the lungs and immune dysfunction plays a vital role in the development of infection after ischemic stroke, the occurrence and mechanism of pulmonary infection at different time points after hemorrhagic cerebral remain unclear. In this study, the changes in the immune system and intestinal barrier function in mice during disease development were investigated at 1 day (M 1 d), 3 days (M 3 d) and 7 days (M 7 d) following hemorrhagic stroke to clarify the mechanism of secondary pulmonary infection. The experimental results revealed that after hemorrhagic stroke, model mice showed increased brain damage from day 1 to 3, followed by a trend of brain recovery from day 3 to 7 . After hemorrhagic stroke, the immune system was disturbed in model mice. Significant immunosuppression of the peripheral immune system was observed in the M 3 d group but improved in the M 7 d group. Staining of lung tissues with hematoxylin and eosin (H&E) and for inflammatory factors revealed considerable disease and immune disorders in the M 7 d group. Stroke seriously impaired intestinal barrier function in mice and significantly changed the small intestine structure. From 1 to 7 d after stroke, intestinal permeability was increased, whereas the levels of markers for intestinal tight junctions, mucus and immunoglobulin A were decreased. Analysis based on 16S rRNA suggested that the microflora in the lung and ileum was significantly altered after stroke. The composition of microflora in lung and ileum tissue was similar in the M 7d group, suggesting that intestinal bacteria had migrated to lung tissue and caused lung infection at this time point after hemorrhagic stroke. In stroke mice, the aggravation of intestinal barrier dysfunction and immune disorders after intracerebral hemorrhage, promoted the migration of enteric bacteria, and increased the risk of pneumonia poststroke. Our findings reveal the dynamic process of infection after hemorrhagic stroke and provide clues for the optimal timing of intervention for secondary pulmonary infection in stroke patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Huang, Li, Han, Hu, Wang, Zhang, Zhuang and Zhang.)

Osteosarcoma (OSA), the malignant bone tumor, predominantly affecting children and adolescents, threatens the life and life quality of the patients. An increasing number of studies have indicated the role of long non-coding RNA (lncRNA) dysregulation in cancer biology. Herein, the study was aimed to explore the role of FGD5 antisense RNA 1 (FGD5-AS1), a lncRNA, in OSA. Expression levels of FGD5-AS1, miR-506-3p and RAB3D mRNA were quantified utilizing qRT-PCR. The expression of RAB3D protein was examined employing Western blot. A series of functional experiments including CCK-8 assay, BrdU assay, wound healing assay, Transwell assay were performed for studying the effects of FGD5-AS1 on the malignancy of OSA cell lines 143B and HOS. The binding site between miR-506-3p and FGD5-AS1 was identified and validated by luciferase reporter assay and RNA immunoprecipitation assay. It was demonstrated that the expression of FGD5-AS1 was up-regulated in OSA tissues and cell lines, and its high expression is associated with higher Enneking stage and poorer histological differentiation. Gain-of-function and loss-of-function studies suggested that FGD5-AS1 facilitated OSA cells proliferation and migration. The promoting effects of FGD5-AS1 overexpression on OSA cell proliferation and migration could be counteracted by miR-506-3p. Moreover, FGD5-AS1 competitively adsorbed miR-506-3p to repress its expression so as to up-regulate the expression of RAB3D. These results indicate that FGD5-AS1 is capable of expediting OSA cell proliferation and migration via sponging miR-506-3p to up-regulate RAB3D.

Pulsatillae radix, a traditional Chinese medicine (TCM), is often used in combination with florfenicol for treatment of intestinal infection in Chinese veterinary clinics. Anemoside B4 (AB4) is the major effective saponin in Pulsatillae radix. This study aimed to investigate whether the pharmacokinetics of florfenicol in broilers was affected by the combination of AB4. In this study, broilers were given AB4 (50 mg/kg BW), or 0.9% sodium chloride solution by oral administration for 7 days. They were then fed florfenicol orally (30 mg/kg BW) on the eighth day. The results showed that the AUC (0-∞) , MRT (0-∞) , t 1/2z and C max of florfenicol were significantly decreased, and the Vz/F and CLz/F were significantly increased by AB4; the mRNA expression levels of CXR, CYP3A37 and MDR1 (except CXR and CYP3A37 in the liver) were up-regulated by AB4. In conclusion, AB4 altered the pharmacokinetics of florfenicol, resulting in lower plasma concentrations of florfenicol, this was probably related to the mRNA expression of CXR, CYP3A37 and MDR1 in the jejunum and liver (except CXR and CYP3A37) increased by AB4. The implications of these findings on the effect of traditional Chinese medicine containing AB4 on the effectiveness of florfenicol in veterinary practice deserve study.

Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.

The spleen is a unique lymphoid organ that plays a key role in immune regulation. Interferon-tau induces upregulation of interferon-stimulated gene 15-kDa protein (ISG15) in the uterus during early pregnancy in sheep. Prostaglandins (PGs) have important effects in both the activation and the inhibition of immune response through an autocrine and paracrine manner. In this study, splenic samples were obtained at Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy from ewes, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, PGE synthase (PTGES), and PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1), was detected through quantitative real-time PCR, western blot, and immunohistochemistry analysis. Our results showed that there was upregulation of COX-2 mRNA and protein at Day 25 of pregnancy, and ISG15 mRNA and conjugated proteins, and AKR1B1 mRNA and dimer at Days 16 and 25 of pregnancy. COX-2 and AKR1B1 proteins were limited to the capsule, trabeculae, and splenic cords. However, the expression of COX-1 and PTGES was not affected by early pregnancy. In conclusion, ISG15-conjugated proteins, COX-2 and AKR1B1 upregulated in maternal spleen during early pregnancy, which may be beneficial for the placentation in sheep., (© 2018 Japanese Society of Animal Science.)

Background: Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Extracellular expression of pullulanase from recombinant B. subtilis is still limited due to the issues on promoters of B. subtilis expression system. This study was undertaken to develop a new, high-level expression system in B. subtilis ATCC6051., Results: To further optimize B. subtilis ATCC6051 as a expression host, eight extracellular proteases (aprE, nprE, nprB, epr, mpr, bpr, vpr and wprA), the sigma factor F (spoIIAC) and a surfactin (srfAC) were deleted, yielding the mutant B. subtilis ATCC6051∆10. ATCC6051∆10 showed rapid growth and produced much more extracellular protein compared to the widetype strain ATCC6051, due to the inactivation of multiple proteases. Using this mutant as the host, eleven plasmids equipped with single promoters were constructed for recombinant expression of pullulanase (PUL) from Bacillus naganoensis. The plasmid containing the P spovG promoter produced the highest extracellular PUL activity, which achieved 412.9 U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter P amyL -P spovG produced the maximum extracellular PUL activity (625.5 U/mL) and showed the highest expression level (the dry cell weight of 18.7 g/L)., Conclusions: Taken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter P amyL -P spovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051.

Aims: The study aimed to investigate the renoprotective effect of glucagon-like peptide-1 (GLP-1) against renal tubular injury in C57BL/6 mice induced by a high-fat diet (HFD)., Methods: Twenty C57BL/6 mice were fed HFD for 12 weeks. Ten of these mice were treated with GLP-1 at 200 µg/kg subcutaneously twice daily for 4 weeks (HFDG group), and the other ten mice received vehicle only (HFD group). Ten mice fed standard rodent chow served as controls (Con group). Body weight, kidney weight, food intake, and systolic blood pressure were measured. The expression of endoplasmic reticulum stress (ERS) markers (BIP, p-eIF2α, ATF4, and CHOP) and apoptosis in the kidney were examined utilizing western blotting, immunohistochemistry and TUNEL, respectively. Angiotensin II and angiotensin II type 1 receptor (AT1R) were examined by ELISA. Human proximal tubule epithelial cells (HK2) were treated with GLP-1(150 nM) followed by treatment with palmitic acid (500 nM [PA]) for 24 h. HK2 cells treated with BSA were used as controls. The protein levels of ERS markers, apoptosis-associated protein, and AT1R were measured by western blotting., Results: Increase of body weight, food intake, and systolic blood pressure was less pronounced in GLP-1 treated HFDG mice compared to HFD mice. The levels of ERS markers (BIP, p-eIF2α, ATF4, and CHOP) and apoptosis decreased following GLP-1 treatment in vivo and in vitro (p<0.05). Increased AT1R induced by HFD and PA were blocked with GLP-1 treatment. In contrast, the level of angiotensin II after GLP-1 treatment was not significantly different between the HFD and HFDG mice., Conclusion: The study indicated that saturated fatty acids induced ERS and apoptosis in the kidney and increased AT1R expression. GLP-1 treatment exerted renoprotective effects against saturated fatty acid-induced kidney tubular cell ERS and apoptosis together with inhibition of AT1R expression in vivo and in vitro., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

A correlation method was recently adopted to identify selection-favored 'optimal' codons from 675 bacterial genomes. Surprisingly, the identities of these optimal codons were found to track the bacterial GC content, leading to a conclusion that selection would generally shape the codon usages to the same direction as the overall mutation does. Raising several concerns, here we report a thorough comparative study on 203 well-selected bacterial species, which strongly suggest that the previous conclusion is likely an illusion. Firstly, the previous study did not preclude species that are suffering weak or no selection pressures on their codon usages. For these species, as showed in this study, the optimal codon identities are prone to be incorrect and follow GC content. Secondly, the previous study only adopted the correlation method, without considering another method to test the reliability of inferred optimal codons. Actually by definition, optimal codons can also be identified by simply comparing codon usages between high- and low-expression genes. After using both methods to identify optimal codons for the selected species, we obtained highly conflicting results, suggesting at least one method is misleading. Further we found a critical problem of correlation method at the step of calculating gene bias level. Due to a failure of accurately defining the background mutation, the problem would result in wrong optimal codon identities. In other words, partial mutational effects on codon choices were mistakenly regarded as selective influences, leading to incorrect and biased optimal codon identities. Finally, considering the translational dynamics, optimal codons identified by comparison method can be well-explained by tRNA compositions, whereas optimal codons identified by correlation method can not be. For all above reasons, we conclude that real optimal codons actually do not track the genomic GC content, and correlation method is misleading in identifying optimal codons and better be avoided.

Nerve growth factor (NGF) is mainly secreted by the neuroglia cells, which can exert biological effect through its receptors on the specific target cell surface. NGF is closely related to neurocyte growth, differentiation and apoptosis. As a neurotropic virus, HSV-1 an easily lead to neurocyte, neuroglia cells death or apoptosis. In this study, the U251 human glioma cells were chosen as target cells to study the change of NGF and its receptors in the apoptosis process of HSV-1 infection. Our results showed that U251 cells were permissive to HSV-1 replication. In the apoptosis process of HSV-1 infected U251 cells, the expression of both NGF and P75NTR increased and then decreased, while the expression of TrkA decreased gradually. These result indicated that HSV-1 was able to induce the abnormal expression of NGF and its receptors in U251 cells.

Study Design: Comparison of melatonin receptor mRNA expression in bilateral paravertebral muscles in adolescent idiopathic scoliosis (AIS). OBJECTIVES.: To investigate the change of melatonin receptor mRNA expression in bilateral paravertebral muscles in AIS, congenital scoliosis (CS), and control in order to analyze its association to the pathogenesis of AIS., Summary of Background Data: Muscle imbalance and asymmetry of stretch receptors in the paravertebral muscles of patients with AIS were supposed to have a large role to play in the development and production of the deformity. Melatonin is a focus of studies of the mechanism underlying the development of scoliosis, and there is no research on the expression of melatonin receptors in the paravertebral muscles of patients with AIS., Methods: Twenty cases with average age of 15.1 +/- 2.2 years and average Cobb angle of 56.2 degrees +/- 16.1 degrees, including 10 cases with Cobb angle >50 degrees and 10 cases with Cobb angle < or =50 degrees, were included in AIS group. The apical vertebrae were from T6 to T11. Twelve cases with an average age of 11.6 +/- 3.2 years and average Cobb angle of 59.2 degrees +/- 33.3 degrees were included in CS group. The apical vertebrae were from T7 to T12. Ten cases without scoliosis were in the control group. The mRNA expression of melatonin receptor subtype MT1 and MT2 was detected by the RT-PCR method., Results: The MT2 mRNA expression on the concave side of the paravertebral muscle was higher than that on the convex side in AIS and CS groups (P < 0.05), but the MT1 mRNA expression showed no significant difference (P > 0.05). In the AIS group, the ratio of MT2 mRNA expression on the concave side compared with the convex side in cases with Cobb angle >50 degrees and cases with Cobb angle < or =50 degrees showed no significant difference (P > 0.05). The MT1 and MT2 mRNA expression showed no significant difference in control group (P > 0.05)., Conclusion: The melatonin receptor expression in bilateral paravertebral muscles in AIS is asymmetric, which may be a secondary change. The bilateral asymmetry in force exerted on the scoliotic spine may be the cause.

The purpose of this study is to investigate the properties of recombinant Cecropin-XJ isolated from Xinjing silkworm and expressed in Pichia yeast. According to Agarose Diffusion Assay, this recombinant Cecropin-XJ has exhibited an extreme heat-stable characteristic and the ability to kill ampicillin-resistant S. aureus and S. enterica. Moreover, we have observed that the Cecropin-XJ well tolerant to extreme acidic, basic, and high salt environments as well as resistant to 24 hours digestion by artificial gastric juice. The inhibition capability to S. aureus with 1mg Cecropin-XJ is equal to 1200U ampicillin. With a broad spectrum of antibacterial activities, the Cecropin-XJ is able to inhibit the Gram-positive bacteria and Gram-negative bacteria. These findings could lead it to a broad applications for agriculture, medical, domestic animal and food industry. The further investigation of the antibacterial mechanism of cecropin-XJ is needed.

This study aimed to explore the role of the trehalose-6-phosphate synthase (TPS) gene family in the adaptation of peas to environmental stress. A comprehensive analysis of the PsTPS gene family identified 20 genes with conserved domains and specific chromosomal locations. Phylogenetic analysis delineated evolutionary relationships, while gene structure analysis revealed compositional insights, and motif analysis provided functional insights. Cis-regulatory element identification predicted gene regulation patterns. Tissue-specific and stress-induced expression profiling highlighted eight genes with ubiquitous expression, with PsTPS15 and PsTPS18 displaying elevated expression levels in roots, nodules, and young stems, and PsTPS13 and PsTPS19 expression downregulated in seeds. Transcriptome analysis identified a differential expression of 20 PsTPS genes, highlighting the significance of 14 genes in response to drought and salinity stress. Notably, under drought conditions, the expression of PsTPS4 and PsTPS6 was initially upregulated and then downregulated, whereas that of PsTPS15 and PsTPS19 was downregulated. Salinity stress notably altered the expression of PsTPS4, PsTPS6, and PsTPS19. Taken together, these findings elucidate the regulatory mechanisms of the PsTPS gene family and their potential as genetic targets for enhancing crop stress tolerance. [ABSTRACT FROM AUTHOR]

The functional role and molecular mechanisms of small‐nucleolar RNA host gene 14 (SNHG14) in triple‐negative breast cancer (TNBC) progression remain unclear. The expression levels of SNHG14 in breast cancer samples and cell lines were determined using real‐time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion abilities were detected using MTS and transwell assays. By RNA sequencing, differentially expressed genes were identified between the SNHG14 siRNA and the negative control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to predict the targets and pathways regulated by SNHG14. pRAF, pMEK, and pERK expression were measured by western blot. The xenograft model was constructed to access the biological function of SNHG14 in vivo. A minimal patient‐derived xenograft model was established to evaluate the sensitivity to chemotherapy drugs. Our data indicated that SNHG14 expression was increased in TNBC tissues and cell lines. SNHG14 knockdown attenuated the proliferation, migration, and invasion abilities of TNBC cells both in vivo and in vitro. High SNHG14 expression was associated with lymph node metastasis and a high Ki67 index. The targets of SNHG14 were mainly enriched in the MAPK signaling pathway. pRAF, pMEK, and pERK expression were downregulated after being transfected with SNHG14 siRNA. Compared with the negative control group, the expression of CACNA1I, DUSP8, FGF17, FGFR4, FOS, PDGFRB, and DDIT3 was increased, and the expression of MKNK1 was decreased in the SNHG14 siRNA group. Minimal patient‐derived xenograft model demonstrated that knockdown of SNHG14 enhanced the sensitivity to Docetaxel in vivo. Compared with the DMSO group, the proliferation of Docetaxel‐resistant MDA‐MB‐231 cells was decreased in Dabrafenib, PD184352, and FR180204 treatment groups. SNHG14 knockdown inhibits TNBC progression by regulating the ERK/MAPK signaling pathway, which provides evidence for SNHG14 as a potential target for TNBC therapy. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) infection can exacerbate liver disease progression through multiple mechanisms, eventually leading to hepatocellular carcinoma (HCC). HBV-encoded oncogene X protein (HBx), a key regulatory protein of HBV infection, serves as a positive regulator of hepatocarcinogenesis. The indispensability of the M2 subunit of ribonucleotide-diphosphate reductase (RRM2) lies in its role in facilitating DNA replication and repair processes. In our previous investigation, it was postulated that the gene RRM2 exhibits elevated expression levels in several categories of malignant tumors, particularly in HBV-related HCC. Additionally, it was observed that RRM2 is present within protein complexes that are centered on HBx. In the present investigation, the objective of this work was to investigate the potential relationship between the elevated expression of RRM2 in HBV-related HCC and the influence of HBx on this expression. The study attempted to determine the specific mechanism by which RRM2 is implicated in the promotion of hepatocarcinogenesis by HBx. There have been multiple scholarly proposals suggesting that the induction of autophagy by HBx is a significant intermediary factor in the development of HCC. However, the precise carcinogenic function of HBx-induced autophagy remains a subject of debate. Results: This work initially investigated the impact of suppressing cellular autophagy on the malignant biological behaviors of HBx-promoted cells using an in vitro cellular model. The findings revealed that the suppression of cellular autophagy partially disrupted the oncogenic effects of HBx. In light of this, we proceeded to conduct more investigations into the regulatory association between RRM2 and HBx-induced autophagy in the upstream-downstream context. Our data indicate that HBx proteins increase the expression of RRM2. Suppression of RRM2 expression not only hinders HBx-induced autophagy, but also worsens the cellular G1/S blockage and reduces the HBx-induced malignant growth of hepatocellular carcinoma tumors, while stimulating apoptosis. Conclusions: Therefore, we hypothesised that RRM2 is a potential downstream target of HBx-induced hepatocarcinogenesis, and mining the oncogenic mechanism of RRM2 is significant in exploring the preventive treatment of HBV-related HCC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Excessive body weight and obesity elevate the risk of chronic non‐communicable diseases. The judicious application of the gut microbiome, encompassing both microorganisms and their derived compounds, holds considerable promise in the treatment of obesity. RESULTS: In this study, we showed that a cocktail of gut microbiota‐derived metabolites, comprising indole 3‐propionic acid (IPA), sodium butyrate (SB) and valeric acid (VA), alleviated various symptoms of obesity in both male and female mice subjected to a high‐fat diet (HFD). The 16S ribosomal RNA (rRNA) sequencing revealed that administering the cocktail via oral gavage retained the gut microbiota composition in obese mice. Fecal microbiota transplantation using cocktail‐treated mice as donors mitigated the obesity phenotype of HFD‐fed mice. Transcriptomic sequencing analysis showed that the cocktail preserved the gene expression profile of hepatic tissues in obese mice, especially up‐regulated the expression level of leptin receptor. Gene delivery via in vivo fluid dynamics further validated that the anti‐obesity efficacy of the cocktail was dependent on leptin signaling at least partly. The cocktail also inhibited the expression of appetite stimulators in hypothalamus. Together, the metabolite cocktail combated adiposity by retaining the gut microbiota configuration and activating the hepatic leptin signaling pathway. CONCLUSIONS: Our findings provide a sophisticated regulatory network between the gut microbiome and host, and highlight a cocktail of gut microbiota‐derived metabolites, including IPA, SB, and VA, might be a prospective intervention for anti‐obesity in a preclinical setting. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Spinibarbus sinensis, also known as Qingbo, is an important economic fish in China. However, the detailed mechanisms underlying its growth are still unknown. To excavate the genes and signaling pathways related to its growth, we compared the transcriptome profiles of the hepatopancreas tissues of S. sinensis, with two groups of growth rate for evaluation. An average of 66,304,909 and 68,739,585 clean reads were obtained in the fast growth (FG) and slow growth (SG) group, respectively. The differential gene expression analysis results showed that 272 differentially expressed genes (DEGs) were screened between the FG and SG groups, including 101 up-regulated genes and 171 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results showed that GO terms related to metabolic process, organic substance metabolic process, and catalytic activity were enriched, pathway signals related to steroid biosynthesis and protein digestion and absorption were also detected. Meanwhile, the potential key regulatory genes sst2, fndc4, and cckra related to the growth of S. sinensis were screened. Reverse transcript fluorescence quantitative PCR (RT-qPCR) validation of 18 DEGs associated with growth differences showed that the RT-qPCR results were consistent with RNA-seq analysis, and nine genes, stk31, gpr149, angptl1, fstl1, sik1, ror2, nlrc3, pdlim2, and nav2 were significantly expressed in the FG group. bmp1, stc1, gpatch8, sstrt2, s100a1, ktf6, cckar6, sync1, bhlha15, a total of nine genes were significantly expressed in the SG group. This study provides basic information for improving the growth characteristics of S. sinensis and the functional research of candidate genes. [ABSTRACT FROM AUTHOR]

Background: The present study aims to identify the differential miRNA expression profile in middle ear cholesteatoma and explore their potential roles in its pathogenesis. Methods: Cholesteatoma and matched normal retroauricular skin tissue samples were collected from patients diagnosed with acquired middle ear cholesteatoma. The miRNA expression profiling was performed using small RNA sequencing, which further validated by quantitative real-time PCR (qRT-PCR). Target genes of differentially expressed miRNAs in cholesteatoma were predicted. The interaction network of 5 most significantly differentially expressed miRNAs was visualized using Cytoscape. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses were processed to investigate the biological functions of miRNAs in cholesteatoma. Results: The miRNA expression profile revealed 121 significantly differentially expressed miRNAs in cholesteatoma compared to normal skin tissues, with 56 upregulated and 65 downregulated. GO and KEGG pathway enrichment analyses suggested their significant roles in the pathogenesis of cholesteatoma. The interaction network of the the 2 most upregulated (hsa-miR-21-5p and hsa-miR-142-5p) and 3 most downregulated (hsa-miR-508-3p, hsa-miR-509-3p and hsa-miR-211-5p) miRNAs identified TGFBR2, MBNL1, and NFAT5 as potential key target genes in middle ear cholesteatoma. Conclusions: This study provides a comprehensive miRNA expression profile in middle ear cholesteatoma, which may aid in identifying therapeutic targets for its management. [ABSTRACT FROM AUTHOR]

Background: Vegetable soybean is an important vegetable crop in world. Seed size and soluble sugar content are considered crucial indicators of quality in vegetable soybean, and there is a lack of clarity on the molecular basis of grain quality in vegetable soybean. Results: In this context, we performed a comprehensive comparative transcriptome analysis of seeds between a high-sucrose content and large-grain variety (Zhenong 6, ZN6) and a low-sucrose content and small-grain variety (Williams 82, W82) at three developmental stages, i.e. stage R5 (Beginning Seed), stage R6 (Full Seed), and stage R7 (Beginning Maturity). The transcriptome analysis showed that 17,107 and 13,571 differentially expressed genes (DEGs) were identified in ZN6 at R6 (vs. R5) and R7 (vs. R6), respectively, whereas 16,203 and 16,032 were detected in W82. Gene expression pattern and DEGs functional enrichment proposed genotype-specific biological processes during seed development. The genes participating in soluble sugar biosynthesis such as FKGP were overexpressed in ZN6, whereas those responsible for lipid and protein metabolism such as ALDH3 were more enhanced in W82, exhibiting different dry material accumulation between two genotypes. Furthermore, hormone-associated transcriptional factors involved in seed size regulation such as BEH4 were overrepresented in ZN6, exhibiting different seed size regulation processes between two genotypes. Conclusions: Herein, we not only discovered the differential expression of genes encoding metabolic enzymes involved in seed composition, but also identified a type of hormone-associated transcriptional factors overexpressed in ZN6, which may regulate seed size and soluble content. This study provides new insights into the underlying causes of differences in the soybean metabolites and appearance, and suggests that genetic data can be used to improve its appearance and textural quality. [ABSTRACT FROM AUTHOR]

Background and purpose: Silent information regulator proteins (sirtuins, SIRT) are a class III histone deacetylases with nicotinamide adenine dinucleotide (NAD+) as coenzyme. YME1 like 1 ATPase (YME1L1) is essential for the maintenance of mitochondrial morphology, function and plasticity. Optic atrophy 1 (OPA1) mainly mediates mitochondrial fusion. The aim of this study was to explore the expression of SIRT3 in the endocrine resistance of breast cancer, the relationship between SIRT3 and YME1L1 and OPA1, and the mechanism of SIRT3 in the endocrine resistance of breast cancer. Methods: 4-hydroxytamoxifen was used to induce tamoxifen-resistant MCF-7/TAM cells. cell counting kit-8 (CCK-8) was used to detect cell proliferation and verify drug resistance. The mitochondrial morphology was observed by transmission electron microscopy (TEM) and immunofluorescence staining. The expressions of SIRT3 and OPA1 were detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. JC-1 staining was used to detect mitochondrial membrane potential, and dihydroethidium (DHE) staining was used to detect reactive oxygen species (ROS) to verify mitochondrial function. SIRT3 was knocked down in drug-resistant cells by RNA interference, and SIRT3 and YME1L1 wild type (WT), simulated acetylation state mutant (MUT K237Q), and simulated deacetylation state mutant (MUT K237R) were overexpressed in parental cells by overexpression plasmid. Immunoprecipitation assay (IP) and immunofluorescence (IF) were used to verify the interaction between SIRT3 and YME1L1. Results: RTFQ-PCR and Western blot results showed that SIRT3 gene expression and protein level was significantly higher in drug-resistant cells than in parental cells. Overexpression of SIRT3 in parental cells decreased the sensitivity of breast cancer cells to tamoxifen. Knockdown of SIRT3 in drug-resistant cells enhanced the sensitivity of drug-resistant cells to tamoxifen. DHE staining showed that the ROS level was lower in tamoxifen resistant cells than in parental cells at the same concentration. Transmission electron microscopy and fluorescence staining showed that the mitochondria of the drug-resistant cells were elongated compared with the parental cells. Western blot results showed that the expression level of L-OPA1 protein was higher in drug-resistant cells than in parental cells. Overexpression of SIRT3 in the parental cells resulted in enhanced mitochondrial function and longer mitochondrial morphology compared with the control cells. Western blot showed that the expression of L-OPA1 was upregulated. When SIRT3 was knocked down in drug-resistant cells, the opposite result was obtained. We further verified how SIRT3 regulated OPA1 protein, affected the morphology and function of mitochondria, and promoted drug resistance of breast cancer. Overexpression of YME1L1 (wild-type and mutant plasmids) in parental cells showed that overexpression of YME1L1 in the simulated deacetylation state resulted in similar results as overexpression of SIRT3, and overexpression of YME1L1 in the acetylated state resulted in similar results as knockdown of SIRT3. IP assay confirmed the interaction between SIRT3 and YME1L1 in breast cancer cells. The acetylation level of YME1L1 was different at different SIRT3 expression levels. IF assay showed that YME1L1 was co-localized with SIRT3 in MCF-7 cells. Conclusion: SIRT3 is highly expressed in tamoxifen-resistant breast cancer cells. SIRT3 upregulates L-OPA1 expression by deacetylating YME1L1, thereby promoting mitochondrial fusion and enhancing mitochondrial function, and promotes tamoxifen resistance in breast cancer. [ABSTRACT FROM AUTHOR]

'Ruixue' apples were used as the test material to study the effect of 10 μM methyl jasmonate (MeJA) on the quality and cell wall metabolism of apples after 18 d of storage. The results showed that MeJA significantly decreased the respiratory rate, reduced the titratable acid content and maintained a high soluble solids content. MeJA has been shown to suppress the activities and gene expressions of WSP, CSP, ISP, and cellulose in contrast to the control group, thereby maintaining a lower cell permeability and higher exocarp firmness. MeJA significantly decreased the expression of MdACS, MdACO, MdPL, Mdgal, and MdPG genes in the apple exocarp when compared to the control group. In addition, the overexpression of MdPL18 increased the content of cell wall polysaccharides such as WSP and CSP, enhanced cell wall-degrading enzyme activities, and accelerated fruit ripening and softening, whereas silencing MdPL18 did the opposite. Together, these results demonstrate that exogenous MeJA maintains the Ruixue apple fruit quality by regulating the metabolism of cell wall substances. [ABSTRACT FROM AUTHOR]

Objective: Ubiquitin-specific peptidase 39 (USP39) plays a carcinogenic role in many cancers, but little research has been conducted examining whether it is involved in head and neck squamous cell carcinoma (HNSCC). Therefore, this study explored the functional role of USP39 in HNSCC. Method: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins (DEPs) between the HNSCC tumor and adjacent healthy tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to assess the functional enrichment of DEPs. Immunohistochemistry was used to detect protein expression. The viability and migration of two HNSCC cell lines, namely CAL27 and SCC25, were detected using the cell counting kit-8 assay and a wound healing assay, respectively. Quantitative real-time PCR was used to detect the expression level of signal transducer and activator of transcription 1 (STAT1) mRNA. Results: LC-MS/MS results identified 590 DEPs between HNSCC and adjacent tissues collected from 4 patients. Through GO and KEGG pathway analyses, 34 different proteins were found to be enriched in the spliceosome pathway. The expression levels of USP39 and STAT1 were significantly higher in HNSCC tumor tissue than in adjacent healthy tissue as assessed by LC-MS/MS analysis, and the increased expression of USP39 and STAT1 protein was confirmed by immunohistochemistry in clinical samples collected from 7 additional patients with HNSCC. Knockdown of USP39 or STAT1 inhibited the viability and migration of CAL27 and SCC25 cells. In addition, USP39 knockdown inhibited the expression of STAT1 mRNA in these cells. Conclusion: Our findings indicated that USP39 knockdown may inhibit HNSCC viability and migration by suppressing STAT1 expression. The results of this study suggest that USP39 may be a potential new target for HNSCC clinical therapy or a new biomarker for HNSCC. [ABSTRACT FROM AUTHOR]

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3′-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism. NEW & NOTEWORTHY: A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations. [ABSTRACT FROM AUTHOR]

Motilin is a gastrointestinal hormone that is mainly produced in the duodenum of mammals, and it is responsible for regulating appetite. However, the role and expression of motilin are poorly understood during starvation and the weaning stage, which is of great importance in the seeding cultivation of fish. In this study, the sequences of Yangtze sturgeon (Acipenser dabryanus Motilin (AdMotilin)) motilin receptor (AdMotilinR) were cloned and characterized. The results of tissue expression showed that by contrast with mammals, AdMotilin mRNA was richly expressed in the brain, whereas AdMotilinR was highly expressed in the stomach, duodenum, and brain. Weaning from a natural diet of T. Limnodrilus to commercial feed significantly promoted the expression of AdMotilin in the brain during the period from day 1 to day 10, and after re-feeding with T. Limnodrilus the change in expression of AdMotilin was partially reversed. Similarly, it was revealed that fasting increased the expression of AdMotilin in the brain (3 h, 6 h) and duodenum (3 h), and the expression of AdMotilinR in the brain (1 h) in a time-dependent manner. Furthermore, it was observed that peripheral injection of motilin-NH2 increased food intake and the filling index of the digestive tract in the Yangtze sturgeon, which was accompanied by the changes of AdMotilinR and appetite factors expression in the brain (POMC, CART, AGRP, NPY and CCK) and stomach (CCK). These results indicate that motilin acts as an indicator of nutritional status, and also serves as a novel orexigenic factor that stimulates food intake in Acipenser dabryanus. This study lays a strong foundation for the application of motilin as a biomarker in the estimation of hunger in juvenile Acipenser dabryanu during the weaning phase, and enhances the understanding of the role of motilin as a novel regulator of feeding in fish. [ABSTRACT FROM AUTHOR]

Objective of this study is to identify a set of internal control genes for the analysis of gene expression. Methods: This study employs reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) to assess gene expression in bone tissue. We selected fourteen housekeeping genes and assessed their stability in the cortical bone of mouse models for obesity and diabetes using four well-established algorithms (GeNorm, BestKeeper, NormFinder, and the comparative Delta Ct method). Results and Conclusion: We identified Rpl13a as the mostly stably expressed reference gene in cortical bone tissue from mouse models of obesity and diabetes (db/db), while Gapdh was found to be the most stable reference gene in another diabetes model, KKAy mice. Additionally, Ef1a, Ppia, Rplp0, and Rpl22 were identified as alternative genes suitable for normalizing gene expression in cortical bone from obesity and diabetes mouse models. These findings enhance RT-qPCR accuracy and reliability, offering a strategic guide to select reference gene for studying bone tissue gene expression in metabolic disorders. [ABSTRACT FROM AUTHOR]

Ankylosing spondylitis (AS) is an immune chronic inflammatory disease, resulting in back pain, stiffness, and thoracolumbar kyphotic deformity. Based on the reported anti‐inflammatory and antioxidant capacities of cynarin (Cyn), this study explored its protective role and molecular mechanisms in mesenchymal stem cells (MSCs) from AS. The target pathways and genes were verified using Western blotting, quantitative real‐time polymerase chain reaction, and immunofluorescent staining, while molecular docking analysis was conducted. In AS‐MSCs, we found that the expression levels of p‐NF‐κB, IL‐6, IL‐1β, and TNF‐α were higher and IκB‐α, Nrf‐2, and HO‐1 were lower compared with healthy control (HC)‐MSCs. With molecular docking analysis, the biding affinities between Cyn and Keap1‐Nrf‐2 and p65‐IκB‐α were predicted. The mRNA and protein expression of p‐NF‐κB, IL‐6, IL‐1β, and TNF‐α and the reactive oxygen species (ROS) generation were downregulated following Cyn administration. Meanwhile, the expression level of IκB‐α, Nrf‐2, and HO‐1 were significantly increased after Cyn pretreatment. The results suggested that the protective mechanisms of Cyn in AS‐MSCs were based on enhancing the antioxidation and suppression of excessive inflammatory responses via Nrf‐2/ROS/NF‐κB axis. Our findings demonstrate that Cyn is a potential candidate for alleviating inflammation in AS. [ABSTRACT FROM AUTHOR]

Chemotherapeutic resistance is one of the most common reasons for poor prognosis of patients with nasopharyngeal carcinoma (NPC). We found that CENPN can promote the growth, proliferation and apoptosis resistance of NPC cells, but its relationship with chemotherapeutic resistance in NPC is unclear. Here we verified that the CENPN expression level in NPC patients was positively correlated with the degree of paclitaxel (PTX) resistance and a poor prognosis through analysis of clinical cases. VAMP8 expression was significantly increased after knockdown of CENPN by transcriptome sequencing. We found in cell experiments that CENPN inhibited macroautophagy/autophagy and VAMP8 expression and significantly increased PTX resistance. Overexpression of CENPN reduced the inhibitory effects of PTX on survival, cell proliferation, cell cycle progression and apoptosis resistance in NPC cells by inhibiting autophagy. In turn, knockdown of CENPN can affect the phenotype of NPC cells by increasing autophagy to achieve PTX sensitization. Sequential knockdown of CENPN and VAMP8 reversed the PTX-sensitizing effect of CENPN knockdown alone. Experiments in nude mice confirmed that knockdown of CENPN can increase VAMP8 expression, enhance autophagy and increase the sensitivity of NPC cells to PTX. Mechanistic studies showed that CENPN inhibited the translocation of p-CREB into the nucleus of NPC cells, resulting in the decreased binding of p-CREB to the VAMP8 promoter, thereby inhibiting the transcription of VAMP8. These results demonstrate that CENPN may be a marker for predicting chemotherapeutic efficacy and a potential target for inducing chemosensitization to agents such as PTX. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; CENPN: centromere protein N; CQ: chloroquine; CREB: cAMP responsive element binding protein; ChIP: chromatin immunoprecipitation assay; IC50: half-maximal inhibitory concentration; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC: nasopharyngeal carcinoma; NPG: nasopharyngitis; oeCENPN: overexpressed CENPN; PTX: paclitaxel; RAPA: rapamycin; RNA-seq: transcriptome sequencing; shCENPN: small hairpin RNA expression vector targeting the human CENPN gene; shCENPN-shVAMP8: sequential knockdown targeting the human CENPN gene and VAMP8 gene; shVAMP8: small hairpin RNA expression vector targeting the human VAMP8 gene; TEM: transmission electron microscopy; TIR: tumor inhibitory rate; VAMP8: vesicle associated membrane protein 8. [ABSTRACT FROM AUTHOR]

Ischemic ulcers pose a multifaceted clinical dilemma for patients with atherosclerosis, frequently compounded by suboptimal wound healing mechanisms. The dual function of Transforming Growth Factor Beta 3 (TGF‐β3) in ischemic ulcer healing is not fully comprehended, despite its involvement in modulating inflammatory responses and tissue regeneration. The main aim of this investigation was to clarify the functions and mechanisms by which TGF‐β3 regulates inflammatory responses and promotes wound healing in patients with ischemic ulcers who have atherosclerosis. Between August 2022 and November 2023, this cross‐sectional investigation was conducted on 428 patients diagnosed with atherosclerotic ischemic ulcers in Haikou, China. The expression and function of TGF‐β3 were examined throughout the different stages of wound healing, including inflammation, proliferation and remodelling. In addition to documenting patient demographics and ulcer characteristics, an analysis was conducted on biopsy samples to determine the expression of TGF‐β3, pro‐inflammatory and anti‐inflammatory markers. A subset of patients were administered topical TGF‐β3 in order to evaluate its therapeutic effects. The expression pattern of TGF‐β3 was found to be stage‐dependent and significant, exhibiting increased levels during the phase of inflammation and reduced activity in subsequent phases. TGF‐β3 levels were found to be greater in ulcers that were larger and deeper, especially in inflammatory phase. TGF‐β3 applied topically induced discernible enhancement in ulcer healing parameters, such as reduction in ulcer depth and size. The therapeutic significance of TGF‐β3 was emphasised due to its twofold function of regulating the inflammatory environment and facilitating the regeneration of damaged tissues. Ischemic ulcer lesion healing is significantly influenced by TGF‐β3, which functions as an anti‐inflammatory and pro‐inflammatory mediator. Its correlation with ulcer characteristics and stages of healing suggests that it may have utility as a targeted therapeutic agent. [ABSTRACT FROM AUTHOR]

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. Key points: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases globally, leading to chronic disability and poor prognosis. One of the approaches for optimizing OA treatment is to find early effective diagnostic biomarkers. The contribution of microRNAs (miRNAs) in OA progression is now being increasingly recognized. This review provides a comprehensive summary on studies reporting the expression profiling of miRNAs in OA and associated signaling pathways. We performed a systematic search of the Embase, Web of Science, PubMed, and Cochrane library databases. This systematic review is reported according to the PRISMA checklist. Studies which identified miRNAs with aberrant expression compared to controls during OA progression were included, and a meta-analysis was performed. Results from the random effects model were provided as log10 odds ratios (logORs) and 95% confidence intervals. Sensitivity analysis was conducted to confirm the accuracy of the results. Subgroup analysis was conducted based on tissue source. The target genes of miRNAs identified in this study were extracted from the MiRWalk database, and these target genes were enriched in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. A total of 191 studies reporting 162 miRNAs were included in our meta-analysis. Among them, 36 miRNAs distributed across 96 studies were expressed in the same direction in at least two studies (13 up-regulated and 23 down-regulated). Subgroup analysis of tissue source revealed that the highest number of studies was conducted using articular cartilage, where the most up-regulated miRNAs were miR-146a-5p (logOR 7.355; P < 0.001) and miR-34a-5p (logOR 6.955; P < 0.001), and the most down-regulated miRNAs were miR-127-5p (logOR 6.586; P < 0.001) and miR-140-5p (logOR 6.373; P < 0.001). Enrichment analysis of 752 downstream target genes of all identified miRNAs was performed, and the regulatory relationships among them were displayed. Mesenchymal stem cells and transforming growth factor-β were found to be the most important downstream effectors regulated by miRNA in OA. This study highlighted the importance of miRNA signaling in OA progression and identified a number of prominent miRNAs including miR-146a-5p, miR-34a-5p, miR-127-5p, and miR-140-5p which might be considered as potential biomarkers for OA. [ABSTRACT FROM AUTHOR]

This study is to observe the upregulation effect of astragaloside IV on ghrelin in diabetic cognitive impairment (DCI) rats and to investigate the pathway in prevention and treatment by reducing oxidative stress. The DCI model was induced with streptozotocin (STZ) in conjunction with a high-fat and high-sugar diet and divided into three groups: model, low-dose (40 mg/kg), and high-dose (80 mg/kg) astragaloside IV. After 30 days of gavage, the learning and memory abilities of rats, as well as their body weight and blood glucose levels, were tested using the Morris water maze and then detection of insulin resistance, SOD activity, and serum MDA levels. The whole brain of rats was sampled for hematoxylin–eosin and Nissl staining to observe pathological changes in the hippocampal CA1 region. Immunohistochemistry was used to detect ghrelin expression in the hippocampal CA1 region. A Western blot was used to determine changes in GHS-R1α/AMPK/PGC-1α/UCP2. RT-qPCR was used to determine the levels of ghrelin mRNA. Astragaloside IV reduced nerve damage, increased superoxide dismutase (SOD) activity, decreased MDA levels, and improved insulin resistance. Ghrelin levels and expression increased in serum and hippocampal tissues, and ghrelin mRNA levels increased in rat stomach tissues. According to Western blot, it increased the expression of the ghrelin receptor GHS-R1α and upregulated the mitochondrial function associated-protein AMPK-PGC-1α-UCP2. Astragaloside IV increases ghrelin expression in the brain to reduce oxidative stress and delay diabetes-induced cognitive impairment. It may be related to the promotion of ghrelin mRNA levels. [ABSTRACT FROM AUTHOR]

SUMMARY: Chromosomal rearrangements (CRs) may occur in newly formed polyploids due to compromised meiotic fidelity. Moreover, CRs can be more readily tolerated in polyploids allowing their longer‐term retention and hence potential spreading/fixation within a lineage. The direct functional consequences of CRs in plant polyploids remain unexplored. Here, we identified a heterozygous individual from a synthetic allohexaploid wheat in which the terminal parts of the long‐arms of chromosomes 2D (approximately 193 Mb) and 4A (approximately 167 Mb) were reciprocally translocated. Five homogeneous translocation lines including both unbalanced and balanced types were developed by selfing fertilization of the founder mutant (RT [2DL; 4AL]‐ter/1, reciprocal translocation). We investigated impacts of these translocations on phenotype, genome‐wide gene expression and metabolome. We find that, compared with sibling wild‐type, CRs in the form of both unbalanced and balanced translocations induced substantial changes of gene expression primarily via trans‐regulation in the nascent allopolyploid wheat. The CRs also manifested clear phenotypic and metabolic consequences. In particular, the genetically balanced, stable reciprocal translocations lines showed immediate enhanced reproductive fitness relative to wild type. Our results underscore the profound impact of CRs on gene expression in nascent allopolyploids with wide‐ranging phenotypic and metabolic consequences, suggesting CRs are an important source of genetic variation that can be exploited for crop breeding. Significance Statement: Our results have revealed the profound impact of chromosomal rearrangements on gene expression in nascent allopolyploids with phenotypic and metabolic consequences likely relevant to polyploid crop breeding. [ABSTRACT FROM AUTHOR]

Allopolyploidy through the combination of divergent genomes into a common nucleus at doubled dosage is known as a potent genetic and evolutionary force. As a macromutation, a striking feature of allopolyploidy in comparison with other mutational processes is that 'genome shock' can be evoked, thereby generating rapid and saltational biological consequences. A major manifestation of genome shock is genome-wide gene expression rewiring, which previously remained to be fully elucidated. Here, using a large set of RNAseq-based transcriptomic data of a synthetic allotetraploid wheat (genome AADD) and its parental species, we performed in-depth analyses of changes in the genome-wide gene expression under diverse environmental conditions at the subgenome (homoeolog) level and investigated the additional effects of homoeologous chromosomal segment exchanges (abbreviated HEs). We show that allopolyploidy caused large-scale changes in gene expression that were variable across the conditions and exacerbated by both stresses and HEs. Moreover, although both subgenomes (A and D) showed clear commonality in the changes, they responded differentially under variable conditions. The subgenome- and condition-dependent differentially expressed genes were enriched for different gene ontology terms implicating different biological functions. Our results provide new insights into the direct impacts of allopolyploidy on condition-dependent changes in subgenome expression and the additional effects of HEs in nascent allopolyploidy. [ABSTRACT FROM AUTHOR]

Background: N6-methyladenosine (m6A) is an important epigenetic modification involved in RNA stability and translation regulation. Manipulating the expression of RNA m6A methyltransferases or demethylases makes it difficult to study the effect of specific RNA methylation. Results: In this study, we report the development of Plant m6A Editors (PMEs) using dLwaCas13a (from L. wadei) and human m6A demethylase ALKBH5 catalytic domain. PMEs specifically demethylates m6A of targeted mRNAs (WUS, STM, FT, SPL3 and SPL9) to increase mRNAs stability. In addition, we discovered that a double ribozyme system can significantly improve the efficiency of RNA editing. Conclusion: PMEs specifically demethylates m6A of targeted mRNAs to increase mRNAs stability, suggesting that this engineered tool is instrumental for biotechnological applications. [ABSTRACT FROM AUTHOR]

Background: The function of mesenchymal stem cells (MSCs) from patients with osteoporosis (OP) is impaired and worsens in patients with type 2 diabetes mellitus (T2DM). Icariin (ICA) is the major active flavonoid glucoside isolated from traditional Chinese herbal Epimedium pubescens, and confirmed able to improve bone mass of OP patients. Objective: To investigate the effect of ICA on the proliferation and osteogenic differentiation of bone-derived MSCs (BMSCs) from patients with OP and T2DM and uncover the potential mechanism. Methods: BMSCs were treated with ICA, and proliferation and osteogenic potency were evaluated using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and detection of osteogenic markers (ALP, RUNX2, SPP1, COL1A1, and mineralized nodules) was performed. RNA sequencing and bioinformatic analysis were performed to identify differentially expressed genes (DEGs) after ICA treatment and screen proliferation- and osteogenic differentiation-related processes. Gene gain and loss were performed to confirm the role of the key candidate gene. Results: ICA significantly promoted the proliferation and osteogenic differentiation of BMSCs. A total of 173 DEGs were identified after ICA treatment. Six DEGs (GLI-1, IGF2, BMP6, WNT5A, PTHLH, and MAPK14) enriched in both proliferation- and osteogenic differentiation-related processes were screened; GLI-1 had the highest validated |log2FC| value. Overexpression of GLI-1 enhanced the proliferation and osteogenic differentiation of BMSCs, and knockdown of GLI-1 weakened the positive effect of ICA on BMSCs. Conclusion: ICA promoted the proliferation and osteogenic differentiation of impaired BMSCs by upregulating GLI-1. [ABSTRACT FROM AUTHOR]

Aquaponics is a new type of composite farming system, which combines aquaculture and hydroponics through ecological design to achieve scientific synergism. However, the effects of aquaponics on the welfare status and stress parameters of fish are unclear. In this study, 150 crucian carp with an average initial body weight of 7.06 ± 0.32 g were selected. Nine fish were randomly selected as the control group (NC group and hypoxia group) for stress, antioxidant and gene expression parameters after acclimation and hypoxia stress, respectively. The remaining crucian carps were randomly divided into three experimental groups with 20 crucian carps in each group (T0, T1 and T2, respectively), with three replicates. The fresh weight of the lettuce root in the T2 group had no significant effect (p > 0.05). Compared with the control group (T0 and T1), there were significant differences in the specific growth rate, the weight gain rate, the fresh leaf weight, the chlorophyll content and the water quality parameters in group T2 (p < 0.05). Regarding the biochemical parameters, superoxide dismutase and catalase showed significant differences between the T2 and T1 groups (p < 0.05). At the same time, the HSP70 and Prdx3 genes were upregulated in the liver of the T2 group compared to the NC group and the hypoxia group. The research suggests that aquaponics may reduce the hypoxia stress of crucian carp without affecting the growth of crucian carp and lettuce. [ABSTRACT FROM AUTHOR]

Problem: Postmenopausal osteoporosis (PMO) is a common osteoporosis. Hyperoside (Hyp), a natural flavonoid compound, has anti‐osteoporotic effects, but the underlying mechanisms remain poorly understood. Inflammatory cytokine IL‐17A is upregulated in PMO and plays vital roles in bone loss, but the upstream regulatory factors and mechanisms are still unknown. Method of study: Twenty PMO patients and 20 healthy control subjects were included to analyze IL‐17A expression changes and screen dys‐regulated miRNAs in the peripheral blood of PMO patients. miR‐19a‐5p mimics and inhibitor were transfected into RAW264.7 osteoclasts, and injected into bilateral ovariectomized (OVX) mice to explore the regulatory effect of miR‐19a‐5p on IL‐17A. OVX mice were randomly grouped and treated with different doses of Hyp to uncover the effective targets for the medicine in PMO disease. Results: MiR‐19a‐5p was downregulated in PMO patients and the expression level was negatively correlated with that of IL‐17A. miR‐19a‐5p could directly bind to the 3′UTR of IL‐17A and regulate its expression. Both in vitro and in vivo studies demonstrated that miR‐19a‐5p mimics decreased the expression of IL‐17A, RANK and Cathepsin K, while miR‐19a‐5p inhibitor significantly increased the expression of IL‐17A, RANK, and Cathepsin K. Importantly, the Hyp could improve bone structure of OVX mice by enhancing miR‐19a‐5p‐mediated IL‐17A downregulation. Conclusion: Overall, these data demonstrated that miR‐19a‐5p/IL‐17A axis might serve as novel therapeutic candidate for PMO. Hyp could relieve bone resorption by targeting the miR‐19a‐5p/IL‐17A axis in OVX mice and exhibited prospective for the treatment of PMO. [ABSTRACT FROM AUTHOR]

Maize is a valuable raw material for feed and food production. Healthy seed germination is important for improving the yield and quality of maize. However, the molecular mechanisms that regulate maize seed germination remain unclear. In this study, multi-omics was used to reveal the molecular mechanism of seed germination induced by gibberellin (GA) in maize. The results indicated that 25,603 genes were differentially expressed (DEGs) and annotated in the GO database, of which 2515 genes were annotated in the KEGG database. In addition, 791 mature miRNAs with different expression levels were identified, of which 437 were known in the miRbase database and 354 were novel miRNAs. Integrative analysis of DEGs and miRNAs suggested that carbohydrate, lipid, amino acid, and energy metabolisms are the primary metabolic pathways in maize seed germination. Interestingly, a lipid metabolism-related gene named ZmSLP was found to negatively regulate maize germination. We transformed this gene into Arabidopsis thaliana to verify its function. The results showed that the germination rate of transgenic Arabidopsis seeds was obviously decreased, and the growth of seedlings was weaker and slower than that of WT plants, suggesting that this gene plays an important role in promoting seed germination. These findings provide a valuable reference for further research on the mechanisms of maize seed germination. [ABSTRACT FROM AUTHOR]

Background: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. Results: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. Conclusions: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis. [ABSTRACT FROM AUTHOR]

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone h2a.z, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm. A screen for clock mutants uncovered a conserved novel auxiliary NuA4 histone acetylase complex, containing orthologs of BRD-8 and BYE-1, needed for maintaining the timely Negative Element expression required for sustaining a normal circadian rhythm. [ABSTRACT FROM AUTHOR]

The tumor-promoting function of IGF2BP3 has been reported in several cancers. The present study aimed to explore the function and molecular mechanisms of IGF2BP3 are elusive in lung adenocarcinoma (LUAD). IGF2BP3 expression in LUAD and its prognostic value were estimated by bioinformatics. RT-qPCR was employed to detect the expression of IGF2BP3 and confirm the transfection efficiency following the knockdown or overexpression of IGF2BP3. Functional assays, including CCK-8, TUNEL, and Transwell assays, were used to determine the role of IGF2BP3 in tumor cell viability, apoptosis, migration and invasion. Gene Set Enrichment Analysis (GSEA) was used to identify signaling pathways related to IGF2BP3 expression. The effects of IGF2BP3 on the PI3K/AKT pathway were detected by western blotting. In this study, we found that IGF2BP3 was overexpressed in LUAD, and patients with high IGF2BP3 levels had a lower probability of overall survival. Moreover, ectopic expression of IGF2BP3 enhanced cell viability and metastasis, and reduced apoptosis. Conversely, IGF2BP3 silencing reduced the viability, migration, and invasion while enhancing the apoptosis of LUAD cells. In addition, it was disclosed that overexpression of IGF2BP3 could activate PI3K/AKT signaling in LAUD, while silencing of IGF2BP3 deactivated this pathway. Moreover, 740Y-P (PI3K agonist) reversed the inhibitory effects on cell viability and metastasis, and the promotion effect on metastasis caused by IGF2BP3 silencing. Our findings demonstrated that IGF2BP3 contributed to the tumorigenesis of LUAD by activating the PI3K/AKT signaling. [ABSTRACT FROM AUTHOR]

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis. [ABSTRACT FROM AUTHOR]

The impact of depression on spermatogenic function and protein expression was investigated by reference to a bioinformatics database for the prediction of key targets and pathways. Experimental validation was performed using a rat model established using the chronic restraint stress method. Organ coefficients and semen parameters were measured. Hematoxylin and eosin (HE) staining was performed to compare testicular tissue between model and control rats. Western blotting and RT–qPCR were conducted to evaluate protein and mRNA expression. As a result, depression and male infertility (MI) were found to share 81 common targets, and the PI3K/Akt/FOXO1 signaling pathway was involved in both conditions. Animal experiments showed testis and epididymis coefficients to be lower in the model group. HE staining showed damage to the seminiferous tubules of model rats. PI3K and p-Akt mRNA and protein were present at lower levels and FOXO1 at higher levels in the model group. Depression led to reduced spermatogenic function in rats and might be associated with differential expression of the PI3K/Akt/FOXO1 signaling pathway. [ABSTRACT FROM AUTHOR]

SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) are engines for almost all of the membrane fusion and exocytosis events in organism cells. In this study, we identified 84 SNARE genes from banana (Musa acuminata). Gene expression analysis revealed that the expression of MaSNAREs varied a lot in different banana organs. By analyzing their expression patterns under low temperature (4 °C), high temperature (45 °C), mutualistic fungus (Serendipita indica, Si) and fungal pathogen (Fusarium oxysporum f. sp. Cubense Tropical Race 4, FocTR4) treatments, many MaSNAREs were found to be stress responsive. For example, MaBET1d was up-regulate by both low and high temperature stresses; MaNPSN11a was up-regulated by low temperature but down-regulated by high temperature; and FocTR4 treatment up-regulated the expression of MaSYP121 but down-regulated MaVAMP72a and MaSNAP33a. Notably, the upregulation or downregulation effects of FocTR4 on the expression of some MaSNAREs could be alleviated by priorly colonized Si, suggesting that they play roles in the Si-enhanced banana wilt resistance. Foc resistance assays were performed in tobacco leaves transiently overexpressing MaSYP121, MaVAMP72a and MaSNAP33a. Results showed that transient overexpression of MaSYP121 and MaSNPA33a suppressed the penetration and spread of both Foc1 (Foc Race 1) and FocTR4 in tobacco leaves, suggesting that they play positive roles in resisting Foc infection. However, the transient overexpression of MaVAMP72a facilitated Foc infection. Our study can provide a basis for understanding the roles of MaSNAREs in the banana responses to temperature stress and mutualistic and pathogenic fungal colonization. [ABSTRACT FROM AUTHOR]

Asthma is a common chronic respiratory disease characterized by wheezing and shortness of breath. Its risk factors include genetic and acquired factors. The acquired factors are closely related to the environment, especially cold conditions. Autophagy plays a regulatory role in asthma. Therefore, we hypothesized that asthma can be controlled by drug intervention at the autophagy level under cold conditions. The Xiaoqinglong decoction (XQLT) was freeze-dried. The compounds in the freeze-dried powder were identified and quantified using reference standards via the high-performance liquid chromatography method. Ovalbumin (OVA)-sensitized rats were subjected to cold stimulation. The effect of cold stimulation on autophagy levels was determined, and it was confirmed that cold stimulation affected autophagy. The effects and mechanisms of XQLT in an asthmatic rat model (OVA-sensitized rats stimulated with cold) were explored. The concentrations of paeoniflorin, liquiritin, trans-cinnamic acid, glycyrrhizic acid, 6-gingerol, schisandrol A, and asarinin in XQLT freeze-dried powder were 14.45, 3.85, 1.03, 3.93, 0.59, 0.24, and 0.091 mg/g, respectively. Cold stimulation is an important cause of asthma. The inflammatory factors in bronchoalveolar lavage fluid and serum were increased in the model group, accompanied by a decline in autophagy level. The treatment with XQLT increased the expression of autophagy genes and decreased the expression of inflammatory factors. Histological studies showed that XQLT improved inflammatory infiltration and collagen fiber deposition in the lungs of rats. XQLT intervention increased autophagy in asthmatic rats. Autophagy plays a role in phagocytosis and reduces the accumulation of abnormal metabolites in the body to reduce airway inflammation and promote asthma recovery. [ABSTRACT FROM AUTHOR]