Preeclampsia and STOX1 (storkhead-box protein 1): Molecular evaluation of STOX1 in preeclampsia.

Preeclampsia (PE) is clinically defined as a part of pregnancy characterized by hypertension and multiple organ failure. PE is broadly categorized into two types: "placental" and "maternal". Placental PE is associated with fetal growth restriction and adverse maternal and neonatal outcomes. STOX1 (Storkhead box 1), a transcription factor, discovered through a complete transcript analysis of the PE susceptibility locus of 70,000 bp on chromosome 10q22.1. So far, studies investigating the relationship between STOX1 and PE have focused on STOX1 overexpression, STOX1 isoform imbalance, and STOX1 variations that could have clinical consequence. Initially, the Y153H variation of STOX was associated with the placental form of PE. Additionally, studies focusing on the maternal and fetal interface have shown that NODAL and STOX1 variations play a role together in the unsuccessful remodeling of the spiral arteries. Research specifically addressing the overexpression of STOX1 has shown that its disruption of cellular hemoastasis, leading to impaired hypoxia response, disruption of the cellular antioxidant system, and nitroso/redox imbalance. Furthermore, functional studies have been conducted showing that the imbalance between STOX1 isoforms contributes to the pathogenesis of placental PE. Research indicates that STOX1B competes with STOX1A and that the overexpression of STOX1B reverses cellular changes that STOX1A induces to the pathogenesis of PE. In this review, we aimed at elucidating the relationship between STOX1 and PE as well as function of STOX1. In conclusion, based on a comprehensive literature review, numerous studies support the role of STOX1 in the pathogenesis of PE.

LbCOPT1 is a copper transporter induced in Lycium barbarum mycorrhizal roots, which allows tobacco with improved growth and nutrient uptake.

KEY MESSAGE: Overexpressing the copper transporter LbCOPT1 leads to a notable increase in the abundance of mycorrhizal arbuscules that suggests the potential application of LbCOPT1 in breeding programs aimed at enhancing symbiotic nutrient uptake in Lycium barbarum L.

Enhanced resistance of Lactiplantibacillus plantarum by expression of albumin.

BACKGROUND: Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing-strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113-pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild-type strain AR113-pIB184. RESULTS: The results demonstrated that the survival rate of AR113-pLLY01 was also significantly better than that of AR113-pIB184 after lyophilization. In addition, AR113-pLLY01 exhibited a significantly better protective effect than AR113-pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113-pLLY01 had a significant protective effect. CONCLUSION: This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry.

Stem cell-like circulating tumor cells identified by Pep@MNP and their clinical significance in pancreatic cancer metastasis.

Objective: The circulating tumor cells (CTCs) could be captured by the peptide functionalized magnetic nanoparticles (Pep@MNP) detection system in pancreatic ductal adenocarcinoma (PDAC). CTCs and the CXCR4 expression were detected to explore their clinical significance. The CXCR4+ CTCs, this is highly metastatic-prone stem cell-like subsets of CTCs (HM-CTCs), were found to be associated with the early recurrence and metastasis of PDAC. Methods: CTCs were captured by Pep@MNP. CTCs were identified via immunofluorescence with CD45, cytokeratin antibodies, and the CXCR4 positive CTCs were assigned to be HM-CTCs. Results: The over-expression of CXCR4 could promote the migration of pancreatic cancer cell in vitro and in vivo. In peripheral blood (PB), CTCs were detected positive in 79.0% of all patients (49/62, 9 (0-71)/2mL), among which 63.3% patients (31/49, 3 (0-23)/2mL) were HM-CTCs positive. In portal vein blood (PVB), CTCs were positive in 77.5% of patients (31/40, 10 (0-40)/2mL), and 67.7% of which (21/31, 4 (0-15)/2mL) were HM-CTCs positive CTCs enumeration could be used as diagnostic biomarker of pancreatic cancer (AUC = 0.862), and the combination of CTCs positive and CA19-9 increase shows improved diagnostic accuracy (AUC = 0.963). in addition, PVB HM-CTCs were more accurate to predict the early recurrence and liver metastasis than PB HM-CTCs (AUC 0.825 vs. 0.787 and 0.827 vs. 0.809, respectively). Conclusions: The CTCs identified by Pep@MNP detection system could be used as diagnostic and prognostic biomarkers of PDAC patients. We identified and defined the CXCR4 over-expressed CTC subpopulation as highly metastatic-prone CTCs, which was proved to identify patients who were prone to suffering from early recurrence and metastasis.

Mitofusin 2 inhibits high glucose-induced apoptosis of human lens epithelial cells via modulating mitochondrial function and autophagy.

INTRODUCTION: Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear. MATERIALS AND METHODS: DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined. RESULTS: STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects. CONCLUSIONS: Our findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.

Analyzing the impact of T7L variants overexpression on the metabolic profile of Escherichia coli.

INTRODUCTION: Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for clinical/therapeutic use. AIM AND OBJECTIVE: This study aims to assess the impact of overexpressing T7L variants on the metabolic profiles of E. coli. The two variants considered include T7L-H37A, which has enhanced lytic activity compared to its wild-type protein, and T7L-H48K, a dead mutant with no significant activity. METHODS: 1H NMR-based metabolomics was employed to compare the metabolic profiles of E. coli cells overexpressing T7L wild-type protein and its variants. RESULTS: Overexpression of the T7L wild-type (T7L-WT) protein and its variants (T7L-H48K and T7L-H37A) was compared to RNAP overexpression in E. coli cells using 1H NMR-based metabolomics, analyzing a total of 75 annotated metabolites, including organic acids, amino acids, sugars, and nucleic acids. The results showed distinct clustering patterns for the two T7L variant groups compared with the WT, in which the dead mutant (H48K) group showed clustering close to that of RNAP. Pathway impact analysis revealed different effects of T7L variants on E. coli metabolic profiles, with T7L-H48K showing minimal alterations in energy and amino acid pathways linked to osmotic stress compared to noticeable alterations in these pathways for both T7L-H37A and T7L-WT. CONCLUSIONS: This study uncovered distinct metabolic fingerprints when comparing the overexpression of active and inactive mutants of T7L lytic enzymes in E. coli cells. These findings could contribute to the optimization and enhancement of suitable endolysins as potential alternatives to antibiotics.

Engineering strategies for enhanced 1', 4'-trans-ABA diol production by Botrytis cinerea.

BACKGROUND: Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application. RESULTS: In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain. CONCLUSIONS: We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.

A Sweet Potato MYB Transcription Factor IbMYB330 Enhances Tolerance to Drought and Salt Stress in Transgenic Tobacco.

MYB transcription factors (TFs) play vital roles in plant growth, development, and response to adversity. Although the MYB gene family has been studied in many plant species, there is still little known about the function of R2R3 MYB TFs in sweet potato in response to abiotic stresses. In this study, an R2R3 MYB gene, IbMYB330 was isolated from sweet potato (Ipomoea batatas). IbMYB330 was ectopically expressed in tobacco and the functional characterization was performed by overexpression in transgenic plants. The IbMYB330 protein has a 268 amino acid sequence and contains two highly conserved MYB domains. The molecular weight and isoelectric point of IbMYB330 are 29.24 kD and 9.12, respectively. The expression of IbMYB330 in sweet potato is tissue-specific, and levels in the root were significantly higher than that in the leaf and stem. It showed that the expression of IbMYB330 was strongly induced by PEG-6000, NaCl, and H2O2. Ectopic expression of IbMYB330 led to increased transcript levels of stress-related genes such as SOD, POD, APX, and P5CS. Moreover, compared to the wild-type (WT), transgenic tobacco overexpression of IbMYB330 enhanced the tolerance to drought and salt stress treatment as CAT activity, POD activity, proline content, and protein content in transgenic tobacco had increased, while MDA content had decreased. Taken together, our study demonstrated that IbMYB330 plays a role in enhancing the resistance of sweet potato to stresses. These findings lay the groundwork for future research on the R2R3-MYB genes of sweet potato and indicates that IbMYB330 may be a candidate gene for improving abiotic stress tolerance in crops.

The Role of Z Chromosome Localization Gene psmd9 in Spermatogenesis of Cynoglossus semilaevis.

Proteasome 26S Subunit, Non-ATPase 9 (psmd9) plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this study, we cloned the psmd9 of Cynoglossus semilaevis and analyzed its expression pattern. psmd9 was identified on the Z chromosome of C. semilaevis, which is considered an interesting candidate gene for spermatogenesis. qRT-PCR and FISH experiments showed that the psmd9 gene was significantly highly expressed in the testes. It is worth noting that the expression level of psmd9 in male fish testes is significantly higher than that in pseudomales. In order to further explore the role of psmd9 in spermatogenesis, a male testicular cell line was used as the experimental material. The results of the psmd9-RNAi and overexpression experiments showed that psmd9 had a synergistic effect with spermatogenesis-related genes dnd1, cfap69, dnah3 and dnajb13, but had an antagonistic effect with ccne2. Our findings offer a scientific foundation for comprehending the role of psmd9 in the spermatogenesis regulatory network of C. semilaevis.

NSP4 promotes replication of porcine reproductive and respiratory syndrome virus-2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3 C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24 h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.

Reverse Genetics Applied to Immunobiology of Tumor Necrosis Factor, a Multifunctional Cytokine.

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

A Comprehensive Pan-Cancer Analysis Reveals Cyclin-Dependent Kinase Inhibitor 2A Gene as a Potential Diagnostic and Prognostic Biomarker in Colon Adenocarcinoma.

Introduction Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a suppressor carcinogenic gene that is upregulated across various types of cancer including breast, liver, thyroid, and bile duct cancer due to its crucial role in cell cycle regulation and cell division. Nevertheless, it is mostly investigated at the genetic level, but it is still poorly studied on pan-cancer analysis as a biomarker and this study shows its significant potential diagnostic and prognostic characteristics. However, this study aims to investigate the role of CDKN2A as a diagnostic and prognostic biomarker across various types of cancer focusing primarily on colon adenocarcinoma (COAD). Methods We investigated CDKN2A gene expression in a pan-cancer analysis across different types of cancer to show its diagnostic potential characteristics by using various bioinformatic tools, including Tumor Immune Estimation Resource (TIMER) 2.0, Gene Expression Profiling Interactive Analysis (GEPIA), and University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) database. TIMER was used to profile gene expression across 32 types of cancer composed of 10,000 RNA-seq samples obtained from the Cancer Genome Atlas (TCGA) and to analyze the tumor-infiltrating immune cells. In addition, GEPIA and UALCAN were further used to analyze gene expression, in terms of gene regulation, pathological stages, and clinical parameters, including gender, age, and race. Therefore, we used GEPIA, UALCAN, and Kaplan-Meier plotter particularly across adenocarcinoma to investigate CDKN2A prognosis by studying its high expression association with the patient's overall survival rate to show the tumor progression. Then, we looked into the genetic alteration of CDKN2A by using the cBio Cancer Genomics Portal (cBioPortal), including 10 pan-cancer studies. We concluded the analysis with gene validation by using a public cohort in Gene Expression Omnibus (GEO). Results CDKN2A showed a trend of upregulation in most cancers and it was significantly upregulated in five cancers, which were commonly identifiable in three databases, including breast invasive carcinoma (p < 0.001), kidney chromophobe (p < 0.001), kidney renal clear cell carcinoma (p < 0.001), kidney renal papillary cell carcinoma (p < 0.001), and COAD (p < 0.001). The upregulation was significantly different in association with pathogenic stages II and III (pr(>F) = 0.00234) which was identifiable significantly in COAD more than in other cancers. The gene showed a high upregulation in association with poor prognosis of patient survival in three cancers, including COAD (log-rank p = 0.011), mesothelioma (log-rank p = 5.9e-07), and liver hepatocellular carcinoma (log-rank p = 0.0045). Therefore, COAD was the only comprehensively analyzed tumor to show a diagnostic and prognostic potential characteristic during high upregulation of CDKN2A. Furthermore, CDKN2A displayed a rare mutation in the form of deep deletion (9%) and revealed an upregulation associated with CD4+ T cells (p = 0.0108), macrophage (p = 0.0073), and neutrophils (p = 0.0272) as immune cells infiltrating COAD.  Conclusion Our study demonstrates the pan-cancer relevance of CDKN2A and revealed a novelty in showing CDKN2A underscores its potential as a diagnostic prognostic biomarker in COAD since CDKN2A is mostly studied at a genetic level across COAD.

Contrasting Impacts of Ubiquitin Overexpression on Arabidopsis Growth and Development.

In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply.

Jasmonate ZIM Domain Protein (JAZ) Gene SLJAZ15 Increases Resistance to Orobanche aegyptiaca in Tomato.

Orobanche aegyptiaca Pers. is a holoparasitic plant that severely reduces tomato (Solanum lycopersicum L.) production in China. However, there is a lack of effective control methods and few known sources of genetic resistance. In this study, we focused on key genes in the JAZ family, comparing the JAZ family in Arabidopsis thaliana (L. Heynh.) to the tomato genome. After identifying the JAZ family members in S. lycopersicum, we performed chromosomal localization and linear analysis with phylogenetic relationship analysis of the JAZ family. We also analyzed the gene structure of the JAZ gene family members in tomato and the homology of the JAZ genes among the different species to study their relatedness. The key genes for O. aegyptiaca resistance were identified using VIGS (virus-induced gene silencing), and the parasitization rate of silenced tomato plants against O. aegyptiaca increased by 47.23-91.13%. The genes were localized in the nucleus by subcellular localization. Heterologous overexpression in A. thaliana showed that the key gene had a strong effect on the parasitization process of O. aegyptiaca, and the overexpression of the key gene reduced the parasitization rate of O. aegyptiaca 1.69-fold. Finally, it was found that the SLJAZ15 gene can positively regulate the hormone content in tomato plants and affect plant growth and development, further elucidating the function of this gene.

Identifying differentially expressed genes in goat mammary epithelial cells induced by overexpression of SOCS3 gene using RNA sequencing.

The suppressor of cytokine signaling 3 (SOCS3) is a key signaling molecule that regulates milk synthesis in dairy livestock. However, the molecular mechanism by which SOCS3 regulates lipid synthesis in goat milk remains unclear. This study aimed to screen for key downstream genes associated with lipid synthesis regulated by SOCS3 in goat mammary epithelial cells (GMECs) using RNA sequencing (RNA-seq). Goat SOCS3 overexpression vector (PC-SOCS3) and negative control (PCDNA3.1) were transfected into GMECs. Total RNA from cells after SOCS3 overexpression was used for RNA-seq, followed by differentially expressed gene (DEG) analysis, functional enrichment analysis, and network prediction. SOCS3 overexpression significantly inhibited the synthesis of triacylglycerol, total cholesterol, non-esterified fatty acids, and accumulated lipid droplets. In total, 430 DEGs were identified, including 226 downregulated and 204 upregulated genes, following SOCS3 overexpression. Functional annotation revealed that the DEGs were mainly associated with lipid metabolism, cell proliferation, and apoptosis. We found that the lipid synthesis-related genes, STAT2 and FOXO6, were downregulated. In addition, the proliferation-related genes BCL2, MMP11, and MMP13 were upregulated, and the apoptosis-related gene CD40 was downregulated. In conclusion, six DEGs were identified as key regulators of milk lipid synthesis following SOCS3 overexpression in GMECs. Our results provide new candidate genes and insights into the molecular mechanisms involved in milk lipid synthesis regulated by SOCS3 in goats.

Expression of circRNA-0036474 in esophageal cancer and its clinical significance.

Circular RNAs are considered to play important roles in the progression of different cancers such as esophageal squamous cell carcinoma. However, the functions of circular RNAs in esophageal squamous cell carcinoma are still not clear. This study aimed to investigate the role and mechanism of circRNA-0036474 in the progression of esophageal squamous cell carcinoma. The hsa_circ_0036474 expression levels were found to be elevated in both EC109 cells and esophageal squamous cell carcinoma tissue samples. Moreover, knockdown of circRNA-0036474 expression in the EC109 cells induced migration and invasion, characterized by the down-regulation of E-cadherin, and up-regulation of N-cadherin and vimentin. In addition, the over-expressed hsa_circ_0036474 significantly decreased the activity of EC109 cells, elevated E-cadherin expression but declined N-cadherin and vimentin expression. Moreover, over-expressed mir-223-3p levels and interfered RERG expression verified the role of hsa_circ_0036474 in inhibiting the invasion and migration of EC109 cells, reducing the expression of N-cadherin and vimentin, and promoting the expression of E-cadherin. In conclusion, circRNA-0036474 mitigated the progression of esophageal squamous cell carcinoma through regulating mir-223-3p/RERG axis, presenting a potential therapeutic target for the treatment.

Tannic acid and silicate-functionalized polyvinyl alcohol-hyaluronic acid hydrogel for infected diabetic wound healing.

Healing of chronic diabetic wounds is challenging due to complications of severe inflammatory microenvironment, bacterial infection and poor vascular formation. Herein, a novel injectable polyvinyl alcohol-hyaluronic acid-based composite hydrogel was developed, with tannic acid (TA) and silicate functionalization to fabricate an 'all-in-one' hydrogel PTKH. On one hand, after being locally injected into the wound site, the hydrogel underwent a gradual sol-gel transition in situ, forming an adhesive and protective dressing for the wound. Manipulations of rheological characteristics, mechanical properties and swelling ability of PTKH could be performed via regulating TA and silicate content in hydrogel. On the other hand, PTKH was capable of eliminating reactive oxygen species overexpression, combating infection and generating a cell-favored microenvironment for wound healing acceleration in vitro. Subsequent animal studies demonstrated that PTKH could greatly stimulate angiogenesis and epithelization, accompanied with inflammation and infection risk reduction. Therefore, in consideration of its impressive in vitro and in vivo outcomes, this 'all-in-one' multifunctional hydrogel may hold promise for chronic diabetic wound treatment.

Incautious design of shRNAs for stable overexpression of miRNAs could result in generation of undesired isomiRs.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

Integrating GC-MS and comparative transcriptome analysis reveals that TsERF66 promotes the biosynthesis of caryophyllene in Toona sinensis tender leaves.

Introduction: The strong aromatic characteristics of the tender leaves of Toona sinensis determine their quality and economic value. Methods and results: Here, GC-MS analysis revealed that caryophyllene is a key volatile compound in the tender leaves of two different T. sinensis varieties, however, the transcriptional mechanisms controlling its gene expression are unknown. Comparative transcriptome analysis revealed significant enrichment of terpenoid synthesis pathway genes, suggesting that the regulation of terpenoid synthesis-related gene expression is an important factor leading to differences in aroma between the two varieties. Further analysis of expression levels and genetic evolution revealed that TsTPS18 is a caryophyllene synthase, which was confirmed by transient overexpression in T. sinensis and Nicotiana benthamiana leaves. Furthermore, we screened an AP2/ERF transcriptional factor ERF-IX member, TsERF66, for the potential regulation of caryophyllene synthesis. The TsERF66 had a similar expression trend to that of TsTPS18 and was highly expressed in high-aroma varieties and tender leaves. Exogenous spraying of MeJA also induced the expression of TsERF66 and TsTPS18 and promoted the biosynthesis of caryophyllene. Transient overexpression of TsERF66 in T. sinensis significantly promoted TsTPS18 expression and caryophyllene biosynthesis. Discussion: Our results showed that TsERF66 promoted the expression of TsTPS18 and the biosynthesis of caryophyllene in T. sinensis leaves, providing a strategy for improving the aroma of tender leaves.

Novel strategy of S. mutans gcrR gene over-expression plus antibacterial dimethylaminohexadecyl methacrylate suppresses biofilm acids and reduces dental caries in rats.

OBJECTIVE: Streptococcus mutans (S. mutans) is a major contributor to dental caries, with its ability to synthesize extracellular polysaccharides (EPS) and biofilms. The gcrR gene is a regulator of EPS synthesis and biofilm formation. The objectives of this study were to investigate a novel strategy of combining gcrR gene over-expression with dimethylaminohexadecyl methacrylate (DMAHDM), and to determine their in vivo efficacy in reducing caries in rats for the first time. METHODS: Two types of S. mutans were tested: Parent S. mutans; and gcrR gene over-expressed S. mutans (gcrR OE S. mutans). Bacterial minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were measured with DMAHDM and chlorhexidine (CHX). Biofilm biomass, polysaccharide, lactic acid production, live/dead staining, colony-forming units (CFUs), and metabolic activity (MTT) were evaluated. A Sprague-Dawley rat model was used with parent S. mutans and gcrR OE S. mutans colonization to determine caries-inhibition in vivo. RESULTS: Drug-susceptibility of gcrR OE S. mutans to DMAHDM or CHX was 2-fold higher than that of parent S. mutans. DMAHDM reduced biofilm CFU by 3-4 logs. Importantly, the combined gcrR OE S. mutans+ DMAHDM dual strategy reduced biofilm CFU by 5 logs. In the rat model, the parent S. mutans group had a higher cariogenicity in dentinal (Dm) and extensive dentinal (Dx) regions. The DMAHDM + gcrR OE group reduced the Dm and Dx caries to only 20 % and 0 %, those of parent S. mutans + PBS control group (p < 0.05). The total caries severity of gcrR OE + DMAHDM group was decreased to 51 % that of parent S. mutans control (p < 0.05). SIGNIFICANCE: The strategy of combining S. mutans gcrR over-expression with antibacterial monomer reducing biofilm acids by 97 %, and reduced in vivo total caries in rats by 48 %. The gcrR over-expression + DMAHDM strategy is promising for a wide range of dental applications to inhibit caries and protect tooth structures.