Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles.

Extracellular vesicles (EVs) are tiny lipid bilayer-enclosed membrane particles released from a variety of cell types into the surrounding environment. These EVs have massive participated in cell-to-cell communication and interspecies communication. In recent years, plant-derived extracellular vesicles (PDEVs) and "exosome-like" EVs populations found in distinct plants have attracted widespread attention. Especially, research on medicinal plant-derived extracellular vesicles (MPDEVs) are increasing, which are considered a kind of promising natural compound. This review summarizes current knowledge on MPDEVs in terms of bioactive compounds, including small RNA, protein, lipid, and metabolite, have been found on the surface and/or in the lumen of MPDEVs. Moreover, both in vitro and in vivo experiments have shown that MPDEVs exert broad biomedical functions, such as anti-inflammatory, anticancer, antioxidant, modulate microbiota, etc. MPDEVs may be a better substitute than animal-derived extracellular vesicles (ADEVs) because of safety and biocompatibility in the future.

Calcium-mediated modulation of Pseudomonas fluorescens biofilm formation.

Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca2+) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca2+ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca2+ significantly reduced the swarming motility of P. fluorescens strains (P.F2, P.F4, and P.F17), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca2+. By the supplementation of Ca2+, complex structures with more cell clusters glued together in P. fluorescens P.F4 biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F4 biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F4 showed a transcriptional response to the supplementation of 10 mM Ca2+, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca2+ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.

Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear. AIM OF THE STUDY: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl4)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis. MATERIALS AND METHODS: In this study, CCl4-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting. RESULTS: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl4 and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis. CONCLUSION: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.

RNA-sequencing analysis reveals the co-biodegradation performance of crude oil by marine Chlorella vulgaris under norfloxacin stress.

In this study, the microalgal growth and crude oil (CRO) biodegradation by marine Chlorella vulgaris (C. vulgaris) were assessed under norfloxacin (NFX) stress. The presence of NFX negatively affected the bio-removal of CRO within 5 days, as the NFX concentration increased from 100 to 1600 µg/L, due to its toxicity as an antibiotic. However, its negative impact on the final degradation capabilities of C. vulgaris was less significant (P-value <0.05). After 9 days of cultivation, CRO bio-removal efficiencies still exceeded 90 %, while NFX bio-removal efficiencies maintained over 47 %. RNA-seq analysis revealed that the degradation of CRO and NFX was attributed to the combined action of functional genes involved in scavenging reactive oxygen species. The production of pigments and the bio-removal performance of C. vulgaris in CRO, NFX, and CRO & NFX coexistence media were consistent with the changes in the number of differentially expressed genes in these samples.

Tandem Expression of a Mobile RNA and Its RNA-Binding Protein(s) Enhances Tuber Productivity in Potato.

A significant number of discoveries in past two decades have established the importance of long-distance signaling in controlling plant growth, development, and biotic and abiotic stress responses. Numerous mobile signals, such as mRNAs, proteins, including RNA-binding proteins, small RNAs, sugars, and phytohormones, are shown to regulate various agronomic traits such as flowering, fruit, seed development, and tuberization. Potato is a classic model tuber crop, and several mobile signals are known to govern tuber development. However, it is unknown if these mobile signals have any synergistic effects on potato crop improvement. Here, we employed a simple innovative strategy to test the cumulative effects of a key mobile RNA, StBEL5, and its RNA-binding proteins, StPTB1, and -6 on tuber productivity of two potato cultivars, Solanum tuberosum cv. Désirée and subspecies andigena, using a multi-gene stacking approach. In this approach, the coding sequences of StBEL5 and StPTB1/6 are driven by their respective native promoters to efficiently achieve targeted expression in phloem for monitoring tuber productivity. We demonstrate that this strategy resulted in earliness for tuberization and enhanced tuber productivity by 2-4 folds under growth chamber, greenhouse, and field conditions. This multi-gene stacking approach could be adopted to other crops, whose agronomic traits are governed by mobile macromolecules, expanding the possibilities to develop crops with improved traits and enhanced yields.

Inhibition of checkpoint kinase prevents human oocyte apoptosis induced by chemotherapy and allows enhanced tumour chemotherapeutic efficacy.

STUDY QUESTION: Could inhibition of the checkpoint kinase (CHEK) pathway protect human oocytes and even enhance the anti-tumour effects, during chemotherapy? SUMMARY ANSWER: CHEK inhibitors prevented apoptosis of human oocytes induced by chemotherapy and even enhanced the anti-tumour effects. WHAT IS KNOWN ALREADY: CHEK inhibitors showed ovarian protective effects in mice during chemotherapy, while their role in human oocytes is unclear. STUDY DESIGN, SIZE, DURATION: This experimental study evaluated the ovarian reserve of young patients (120 patients) with cancer, exposed or not exposed to taxane and platinum (TP)-combined chemotherapy. Single RNA-sequencing analysis of human primordial oocytes from 10 patients was performed to explore the mechanism of oocyte apoptosis induced by TP chemotherapy. The damaging effects of paclitaxel (PTX) and cisplatin on human oocytes were also evaluated by culturing human ovaries in vitro. A new mouse model that combines human ovarian xenotransplantation and patient-derived tumour xenografts was developed to explore adjuvant therapies for ovarian protection. The mice were randomly allocated to four groups (10 mice for each group): control, cisplatin, cisplatin + CK1 (CHEK1 inhibitor, SCH 900776), and cisplatin + CK2 (CHEK2 inhibitor, BML277). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the prospective cohort study, human ovarian follicles were counted and serum AMH levels were evaluated. RNA-sequencing analysis was conducted, and staining for follicular damage (phosphorylated H2AX histone; γH2AX), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays and assessments of apoptotic biomarkers (western blot and immunofluorescence) were conducted in human ovaries. After the treatments, histological analysis was performed on human ovarian samples to investigate follicular populations, and oocyte damage was measured by γH2AX staining, BAX staining, and TUNEL assays. At the same time, the tumours were evaluated for volume, weight, and apoptosis levels. MAIN RESULTS AND THE ROLE OF CHANCE: Patients who received TP chemotherapy showed decreased ovarian reserves. Single RNA-sequencing analysis of human primordial oocytes indicated that TP chemotherapy induced apoptosis of human primordial oocytes by causing CHEK-mediated TAp63α phosphorylation. In vitro culture of human ovaries showed greater damaging effects on oocytes after cisplatin treatment compared with that after PTX treatment. Using the new animal model, CHEK1/2 inhibitors prevented the apoptosis of human oocytes induced by cisplatin and even enhanced its anti-tumour effects. This protective effect appeared to be mediated by inhibiting DNA damage via the CHEK-TAp63α pathway and by generation of anti-apoptotic signals in the oocytes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preclinical study performed with human ovarian samples, and clinical research is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the therapeutic potential of CHEK1/2 inhibitors as a complementary strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the National Natural Science Foundation of China (nos. 82001514 and 81902669) and the Fundamental Research Funds for the Central Universities (2021yjsCXCY087). The authors declare no conflict of interest.

METTL14-Mediated m6A Modification of TNFAIP3 Involved in Inflammation in Patients With Active Rheumatoid Arthritis.

OBJECTIVE: The aim of the study was to investigate the role of N6 -methyladenosine (m6A) modification in the progression of rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were collected. The expression of m6A modification-related proteins and m6A levels were detected using polymerase chain reaction (PCR), western blot, and m6A enzyme-linked immunosorbent assay (ELISA). The roles of methyltransferase-like 14 (METTL14) in the regulation of inflammation in RA was explored using methylated RNA immunoprecipitation (MeRIP) sequencing and RNA immunoprecipitation assays. Collagen antibody-induced arthritis (CAIA) mice were used as an in vivo model to study the role of METTL14 in the inflammation progression of RA. RESULTS: We found that m6A writer METTL14 and m6A levels were decreased in PBMCs of patients with active RA and correlated negatively with the disease activity score using 28 joint counts (DAS28). Knockdown of METTL14 downregulated m6A and promoted the secretion of inflammatory cytokines interleukin 6 (IL-6) and IL-17 in PBMCs of patients with RA. Consistently, METTL14 knockdown promoted joint inflammation accompanied by upregulation of IL-6 and IL-17 in CAIA mice. MeRIP sequencing and functional studies confirmed that tumor necrosis factor α induced protein 3 (TNFAIP3), a key suppressor of the nuclear factor-κB inflammatory pathway, was involved in m6A-regulated PBMCs. Mechanistic investigations revealed that m6A affected TNFAIP3 expression by regulation of messenger RNA stability and translocation in TNFAIP3 protein coding sequence. CONCLUSIONS: Our study highlights the critical roles of m6A on regulation of inflammation in RA progression. Treatment strategies targeting m6A modification may represent a new option for management of RA.

METTL16 promotes glycolytic metabolism reprogramming and colorectal cancer progression.

BACKGROUND: Glycolysis is the key hallmark of cancer and maintains malignant tumor initiation and progression. The role of N6-methyladenosine (m6A) modification in glycolysis is largely unknown. This study explored the biological function of m6A methyltransferase METTL16 in glycolytic metabolism and revealed a new mechanism for the progression of Colorectal cancer (CRC). METHODS: The expression and prognostic value of METTL16 was evaluated using bioinformatics and immunohistochemistry (IHC) assays. The biological functions of METTL16 in CRC progression was analyzed in vivo and in vitro. Glycolytic metabolism assays were used to verify the biological function of METTL16 and Suppressor of glucose by autophagy (SOGA1). The protein/RNA stability, RNA immunoprecipitation (RIP), Co-immunoprecipitation (Co-IP) and RNA pull-down assays were used to explore the potential molecular mechanisms. RESULTS: SOGA1 is a direct downstream target of METTL16 and involved in METTL16 mediated glycolysis and CRC progression. METTL16 significantly enhances SOGA1 expression and mRNA stability via binding the "reader" protein insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1). Subsequently, SOGA1 promotes AMP-activated protein kinase (AMPK) complex ubiquitination, inhibits its expression and phosphorylation, thus upregulates pyruvate dehydrogenase kinase 4 (PDK4), a crucial protein controlling glucose metabolism. Moreover, Yin Yang 1 (YY1) can transcriptionally inhibit the expression of METTL16 in CRC cells by directly binding to its promoter. Clinical data showed that METTL16 expression is positively correlated to SOGA1 and PDK4, and is associated with poor prognosis of CRC patients. CONCLUSIONS: Our findings suggest that METTL16/SOGA1/PDK4 axis might be promising therapeutic targets for CRC.

A modified Fangji Huangqi decoction ameliorates pulmonary artery hypertension via phosphatidylinositide 3-kinases/protein kinase B-mediated regulation of proliferation and apoptosis of smooth muscle cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary artery hypertension (PAH) is a progressive and fatal lung disease of multifactorial etiology, which arouses an enhanced interest in PAH disease therapy. Modified Fangji Huangqi decoction (MFJHQ), a traditional Chinese medicine (TCM) formula, has a crucial role in the treatment of PAH. However, the pharmacological roles and mechanisms of MFJHQ on PAH remain unknown. AIM OF THE STUDY: To investigate the effects and potential mechanism of MFJHQ on pulmonary vascular remodeling in PAH. MATERIAL AND METHODS: Ultra-performance liquid chromatography (UPLC) was employed to quantitate the principal components in MFJHQ. Rats were treated with MFJHQ by gavage for final 2 weeks in monocrotaline (MCT)-induced PAH rats. RNA-sequencing and network pharmacology analysis were performed to explore the potential mechanism. The primary rat pulmonary artery smooth muscle cells (PASMCs) were utilized to evaluate the regulatory effect of MFJHQ in vitro. RESULTS: Seven active components from MFJHQ were quantitated by UPLC. In rats with MCT-induced PAH, MFJHQ treatment significantly improved hemodynamic parameters, right ventricular hypertrophy index, lung function, and attenuated pulmonary vascular remodeling. Mechanistically, we further confirmed that MFJHQ inhibits MCT-induced phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) pathway predicated by network pharmacology and RNA-sequencing analysis to reduce the proliferation of pulmonary arteries and promote pulmonary artery apoptosis in lung tissues. Additionally, MFJHQ hindered the proliferation and migration, and accelerated apoptosis in PDGF-BB-induced PASMCs in vitro, which can be enhanced by the presence of the PI3K inhibitor LY294002. CONCLUSIONS: Our results indicated that MFJHQ inhibited MCT-induced pulmonary vascular remodeling by decreasing proliferation and migration of PASMCs and promoting PASMC apoptosis through PI3K/Akt pathway, which provides a novel treatment option for PAH with multi-targeting mechanisms inspired by TCM theory.

RNA Methylome Reveals the m6A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering.

The epitranscriptomic mark N6-methyladenosine (m6A), which is the predominant internal modification in RNA, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m6A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m6A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea (Camellia sinensis) leaves under solar-withering conditions. Dynamic changes in global m6A level in tea leaves were mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m6A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.

Targeting Synovial Lymphatic Function as a Novel Therapeutic Intervention for Age-Related Osteoarthritis in Mice.

OBJECTIVE: The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. METHODS: SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. RESULTS: Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[µW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P = 0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004). CONCLUSION: SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.

Ganoderma lucidum polysaccharide inhibits HSC activation and liver fibrosis via targeting inflammation, apoptosis, cell cycle, and ECM-receptor interaction mediated by TGF-ß/Smad signaling.

BACKGROUND: Ganoderma lucidum polysaccharide (GLP) has many biological properties, however, the anti-fibrosis effect of GLP is unknown at present. PURPOSE: This study aimed to examine the anti-fibrogenic effect of GLP and its underlying molecular mechanisms in vivo and in vitro. STUDY DESIGN: Both CCl4-induced mouse and TGF-ß1-induced HSC-T6 cellular models of fibrosis were established to examine the anti-fibrogenic effect of a water-soluble GLP (25 kDa) extracted from the sporoderm-removed spores of G. lucidum.. METHOD: Serum markers of liver injury, histology and fibrosis of liver tissues, and collagen formation were examined using an automatic biochemical analyzer, H&E staining, Sirius red staining, immunohistochemistry, immunofluorescence, ELISA, Western blotting, and qRT-PCR. RNA-sequencing, enrichment pathway analysis, Western blotting, qRT-PCR, and flow cytometry were employed to identify the potential molecular targets and signaling pathways that are responsible for the anti-fibrotic effect of GLP. RESULTS: We showed that GLP (150 and 300 mg/kg) significantly inhibited hepatic fibrogenesis and inflammation in CCl4-treated mice as mediated by the TLR4/NF-κB/MyD88 signaling pathway. We further demonstrated that GLP significantly inhibited hepatic stellate cell (HSCs) activation in mice and in TGF-ß1-induced HSC-T6 cells as manifested by reduced collagen I and a-SMA expressions. RNA-sequencing uncovered inflammation, apoptosis, cell cycle, ECM-receptor interaction, TLR4/NF-κB, and TGF-ß/Smad signalings as major pathways suppressed by GLP administration. Further studies demonstrated that GLP elicits anti-fibrotic actions that are associated with a novel dual effect on apoptosis in vivo (inhibit) or in vitro (promote), suppression of cell cycle in vivo, induction of S phase arrest in vitro, and attenuation of ECM-receptor interaction-associated molecule expressions including integrins ITGA6 and ITGA8. Furthermore, GLP significantly inhibited the TGF-ß/Smad signaling in mice, and reduced TGF-ß1 or its agonist SRI-011381-induced Smad2 and Smad3 phosphorylations, but increased Samd7 expression in HSC-T6 cells. CONCLUSION: This study provides the first evidence that GLP could be a promising dietary strategy for treating liver fibrosis, which protects against liver fibrosis and HSC activation through targeting inflammation, apoptosis, cell cycle, and ECM-receptor interactions that are mediated by TGF-ß/Smad signaling.

Alterations in energy metabolism, total protein, uric and nucleic acids in African sharptooth catfish (Clarias gariepinus Burchell) exposed to crude oil and fractions.

Water contamination by crude oil is a growing challenge and little is known about the probabilistic and non-probabilistic ecosystem and species consequences. Therefore, research aimed at understanding species survival strategy in crude oil-contaminated environments with focus on cellular metabolic alterations and dynamics is vital. This study assessed the alterations in lactate dehydrogenase (LDH), glucose (GLU), glucose-6-phosphate dehydrogenase (G-6-PDH), total protein (TP), uric and nucleic acids (UA, RNA, and DNA) in the liver, heart, kidney, blood supernatants, and muscle homogenates of African sharptooth catfish ([ASC] Clarias gariepinus) exposed to varying bonny-light crude oil concentrations to understand the underlying cause of their delayed development as well as potential health and wellbeing. Three concentrations (20, 50, and 100 mg/L) of diluted whole bonny-light crude oil (DWC), water-soluble (WSF), and water-insoluble (WIF) fractions of bonny-light crude oil were used to grow ASC for 9 weeks at room temperature. Biochemical assessments revealed significant (at p < 0.05) elevations in heart LDH (48.57 ± 4.67 to 3011.34 ± 4.67 U/L) and blood G-6-PDH activities (54.86 ± 0.00 to 128 ± 18.29 mU/mL), GLU (0.22 ± 0.01 to 0.77 ± 0.01 mg/dL), TP (5.15 ± 0.14 to 22.33 ± 0.21 g/L), UA (0.29 ± 0.05 to 10.05 ± 0.27 mg/dL), as well as liver DNA (0.38 ± 0.02 to 2.33 ± 0.09 µg/mL) and RNA (12.52 ± 0.05 to 30.44 ± 0.02 µg/mL) levels for laboratory-grown ASC in DWC, WSF, WIF, and oil-impacted Ubeji river collected ASC relative to the control. Due to greater levels of cellular metabolic alterations in oil-impacted Ubeji River collected ASC, it is evident that bonny-light contamination levels in the river is greater than 100 mg/L. In conclusion, bonny-light crude oil is toxic to ASC and induces stress response. The ecological changes caused by bonny-light crude oil contamination may ultimately affect niche functioning and the development of organs in ASC.

Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures.

We have developed methods to achieve efficient CRISPR-Cas9-mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the ß1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell-matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.

Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Exploring the RNA Editing Events and Their Potential Regulatory Roles in Tea Plant (Camellia sinensis L.).

RNA editing is a post-transcriptional modification process that alters the RNA sequence relative to the genomic blueprint. In plant organelles (namely, mitochondria and chloroplasts), the most common type is C-to-U, and the absence of C-to-U RNA editing results in abnormal plant development, such as etiolation and albino leaves, aborted embryonic development and retarded seedling growth. Here, through PREP, RES-Scanner, PCR and RT-PCR analyses, 38 and 139 RNA editing sites were identified from the chloroplast and mitochondrial genomes of Camellia sinensis, respectively. Analysis of the base preference around the RNA editing sites showed that in the -1 position of the edited C had more frequent occurrences of T whereas rare occurrences of G. Three conserved motifs were identified at 25 bases upstream of the RNA editing site. Structural analyses indicated that the RNA secondary structure of 32 genes, protein secondary structure of 37 genes and the three-dimensional structure of 5 proteins were altered due to RNA editing. The editing level analysis of matK and ndhD in six tea cultivars indicated that matK-701 might be involved in the color change of tea leaves. Furthermore, 218 PLS-CsPPR proteins were predicted to interact with the identified RNA editing sites. In conclusion, this study provides comprehensive insight into RNA editing events, which will facilitate further study of the RNA editing phenomenon of the tea plant.

Inhibition of GABAAR or Application of Lactobacillus casei Zhang Alleviates Ulcerative Colitis in Mice: GABAAR as a Potential Target for Intestinal Epithelial Renewal and Repair.

Emerging evidence indicates that the gamma-aminobutyric acid type A receptor (GABAAR) and Lactobacillus casei Zhang regulate colitis in a variety of ways, such as by participating in host immune and inflammatory responses, altering the gut microbiota, and influencing intestinal barrier function. However, not much is known about the mechanisms by which GABAAR and L. casei affect colon epithelial cell renewal and the interaction between GABAAR and L. casei during this process. To elucidate this, we established a dextran sulfate sodium (DSS)-induced model and measured the mouse body weights, colon length, the disease activity index (DAI), and histological scores. Our results indicated that inhibition of GABAAR alleviated the DSS-induced colitis symptoms, resulting in less weight loss and more intact colon tissue. Moreover, treatment with bicuculline (Bic, a GABAAR inhibitor) increased the levels of PCNA, ß-catenin, and TCF4 in mice with colitis. Interestingly, open field test performances showed that inhibition of GABAAR also attenuated colitis-related anxiety-like behavior. By 16S RNA gene sequencing analysis, we showed that inhibition of GABAAR partially reversed the gut dysbacteriosis of DSS-induced mice and increased the abundance of beneficial bacteria. Additionally, L. casei Zhang supplementation inhibited the expression of GABAAR in mice with colitis, promoted the proliferation and renewal of colon epithelial cells, and alleviated anxiety-like behavior and intestinal microflora disorder in mice. Thus, GABAAR plays a key role in the beneficial effects of L. casei on DSS-induced colitis in mice.

Antiproliferative effects of dried Moringa oleifera leaf extract on human Wharton's Jelly mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have seen an elevated use in clinical works like regenerative medicine. Its potential therapeutic properties increases when used in tandem with complementary agents like bio-based materials. Therefore, the present study is the first to investigate the cytotoxicity of a highly valued medicinal plant, Moringa oleifera, on human Wharton's Jelly mesenchymal stem cells (hWJMSCs) and its effects on the cells' gene expression when used as a pre-treatment agent in vitro. M. oleifera leaves (MOL) were dried and subjected to UHPLC-QTOF/MS analysis, revealing several major compounds like apigenin, kaempferol, and quercetin in the MOL, with various biological activities like antioxidant and anti-cancer properties. We then treated the hWJMSCs with MOL and noticed a dose-dependant inhibition on the cells' proliferation. RNA-sequencing was performed to explain the possible mechanism of action and revealed genes like PPP1R1C, SULT2B1, CDKN1A, mir-154 and CCNB1, whose expression patterns were closely associated with the negative cell cycle regulation and cell cycle arrest process. This is also evident from gene set enrichment analysis where the GO and KEGG terms for down-regulated pathways were closely related to the cell cycle regulation. The Ingenuity pathway analysis (IPA) software further predicted the significant activation of (p < 0.05, z-score > 2) of the G2/M DNA damage checkpoint regulation pathway. The present study suggests that MOL exhibits an antiproliferative effect on hWJMSCs via cell cycle arrest and apoptotic pathways. We believe that this study provides an important baseline reference for future works involving MOL's potential to accompany MSCs for clinical works. Future works can take advantage of the cell's strong anti-cancer gene expression found in this study, and evaluate our MOL treatment on various cancer cell lines.

Ginger Root Extract Improves GI Health in Diabetic Rats by Improving Intestinal Integrity and Mitochondrial Function.

Background Emerging research suggests hyperglycemia can increase intestinal permeability. Ginger and its bioactive compounds have been reported to benefit diabetic animals due to their anti-inflammatory and antioxidant properties. In this study, we revealed the beneficial effect of gingerol-enriched ginger (GEG) on intestinal health (i.e., barrier function, mitochondrial function, and anti-inflammation) in diabetic rats. Methods Thirty-three male Sprague Dawley rats were assigned to three groups: low-fat diet (control group), high-fat-diet (HFD) + streptozotocin (single low dose 35 mg/kg body weight (BW) after 2 weeks of HFD feeding) (DM group), and HFD + streptozotocin + 0.75% GEG in diet (GEG group) for 42 days. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted at baseline and prior to sample collection. Total pancreatic insulin content was determined by ELISA. Total RNA of intestinal tissues was extracted for mRNA expression using qRT-PCR. Results Compared to the DM group, the GEG group had improved glucose tolerance and increased pancreatic insulin content. Compared to those without GEG (DM group), GEG supplementation (GEG group) increased the gene expression of tight junction (Claudin-3) and antioxidant capacity (SOD1), while it decreased the gene expression for mitochondrial fusion (MFN1), fission (FIS1), biogenesis (PGC-1α, TFAM), mitophagy (LC3B, P62, PINK1), and inflammation (NF-κB). Conclusions Ginger root extract improved glucose homeostasis in diabetic rats, in part, via improving intestinal integrity and mitochondrial dysfunction of GI health.