TALE-based organellar genome editing and gene expression in plants.

KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Analysis of the microbial community diversity in various regions of the healthy oral cavity.

BACKGROUND: Microbiomics offers new methods for conducting epidemiological surveys of oral microbiota in large populations. Compared to curette sampling, swab sampling is more convenient and less technically sensitive, making it more suitable for such surveys. To verify the feasibility of using swabs for buccal mucosa sampling in large-scale studies, we collected samples from the buccal mucosa and tooth surfaces of healthy individuals using both swabs and curettes. Microbiomics was employed to analyze and compare microbial abundance and diversity between these two methods. METHODS: Four sites were assessed: the buccal mucosa on both sides and the buccal surfaces of the left and right mandibular first molars. Two sampling methods, swab and curette, were used to collect bacterial communities from healthy individuals. Specifically, buccal mucosa samples (n = 10) and tooth surface samples (n = 20) were analyzed using 16 S rDNA gene sequencing. Bacterial signals were detected through fluorescence in situ hybridization (FISH), targeting the bacterial 16 S rDNA gene. Metastats analysis and Wilcoxon test were used. RESULTS: A total of 383 OTUs were detected in the 30 samples, which belonged to 1 kingdom (bacteria), 11 phyla, 23 classes, 40 orders, 75 families, 143 genus, and 312 species. Among them, 223 OTUs were found on both the buccal mucosa and tooth surfaces. The statistics suggest that although there were no significant differences in colony composition, there were differences in the abundance and distribution of colonies on the dental and buccal mucosal surfaces. When detecting oral disease-causing pathogens such as Enterococcus faecalis and Porphyromonas gingivalis, the efficiency of detection is higher when using curette sampling. Compared to right tooth sampling with a curette, the swab sampling group had higher levels of Firmicutes, while Fusobacteria and Bacteroidetes were more prevalent in the curette tissues. CONCLUSIONS: In oral health individuals, there is no difference in the bacterial composition of the oral buccal mucosa and the dental surface, differing only in abundance. Thus, the buccal mucosa can act as a substitute for the teeth in epidemiological investigations exploring the bacterial composition of the oral cavity.

Inactivation of EGFR/ERK/NF-κB signalling associates with radiosensitizing effect of 18ß-glycyrrhetinic acid on progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is recognized as the fifth most common cancer and the third most common cause of death in Asian population. Studies reported that HCC is relatively insensitive to radiotherapy (RT); thus, considering how to sensitize HCC to RT is worth to be elucidated. Epidermal growth factor receptor (EGFR)-mediated signalling transduction plays the important role in regulating treatment efficacy of HCC. An active compound, 18beta-glycyrrhetinic acid (18ß-GA), has been reported to own anti-tumour effect. However, whether 18ß-GA possess RT sensitization ability in HCC remains unclear. Here, we used RNA data from TCGA-LIHC (Liver hepatocellular carcinoma) to identify the role between EGFR/ERK/nuclear factor kappa B (NF-κB) signalling and RT by radiosensitivity index (RSI) analysis. We suggested that patients with activated NF-κB signalling may show resistance to RT treatment, whereas combining 18ß-GA may reinforce RT efficacy in a Hep3B-bearing animal model. 18ß-GA combined with RT showed superior tumour inhibition capacity as compared to monotherapy and even reached similar efficacy as erlotinib combined with RT. Treatment promotion of RT by 18ß-GA in HCC is not only through diminishing RT-induced EGFR/ERK/NF-κB signalling but also promoting RT-induced apoptosis pathways. 18ß-GA may act as radiosensitizer through inactivating EGFR-mediated HCC progression and inducing caspase-dependent apoptosis signalling.

Advances in the study of exosomes derived from mesenchymal stem cells and cardiac cells for the treatment of myocardial infarction.

Acute myocardial infarction has long been the leading cause of death in coronary heart disease, which is characterized by irreversible cardiomyocyte death and restricted blood supply. Conventional reperfusion therapy can further aggravate myocardial injury. Stem cell therapy, especially with mesenchymal stem cells (MSCs), has emerged as a promising approach to promote cardiac repair and improve cardiac function. MSCs may induce these effects by secreting exosomes containing therapeutically active RNA, proteins and lipids. Notably, normal cardiac function depends on intracardiac paracrine signaling via exosomes, and exosomes secreted by cardiac cells can partially reflect changes in the heart during disease, so analyzing these vesicles may provide valuable insights into the pathology of myocardial infarction as well as guide the development of new treatments. The present review examines how exosomes produced by MSCs and cardiac cells may influence injury after myocardial infarction and serve as therapies against such injury. Video Abstract.

Periodontitis and NAFLD-related diseases: A bidirectional two-sample Mendelian randomization study.

BACKGROUND: Epidemiological studies have shown an association between periodontitis and nonalcoholic fatty liver disease (NAFLD)-related diseases. However, a causal relationship between these two diseases remains unclear. To examine the causal relationship between these two diseases, we conducted a bidirectional two-sample Mendelian randomization (MR) analysis using genetic markers as proxies. METHODS: Statistical summary was obtained from a large genome-wide association study (GWAS) on NAFLD (N = 342,499), nonalcoholic steatohepatitis (NASH, N = 342,499), fibrosis (N = 339,081), cirrhosis (N = 342,499), fibrosis/cirrhosis (N = 334,553), and periodontitis (N = 34,615) in the European ancestry. The inverse variance weighted (IVW) method was used as the main method to estimate the bidirectional association. Sensitivity analysis was performed to evaluate the rigidity of the results. RESULTS: Limited evidence indicated positive causal associations between genetically predicted NAFLD and periodontitis (IVW odds ratio [OR], 1.094; 95% confidence interval [CI], 1.006-1.189; p = 0.036) and between cirrhosis and periodontitis (IVW OR, 1.138; 95% CI, 1.001-1.294; p = 0.048). However, the opposite trend did not indicate a causative effect of periodontitis on NAFLD-related diseases. The sensitivity analysis revealed no obvious pleiotropy or heterogeneity. CONCLUSIONS: Our MR analysis provides new evidence in favor of the moderate causal impact of NAFLD on periodontitis. The causal effects of periodontitis on NAFLD-related diseases warrant further investigation.

Association between chronic pain and acute coronary syndrome in the older population: a nationwide population-based cohort study.

BACKGROUND: Chronic pain (CP) may increase the risk of acute coronary syndrome (ACS); however, this issue in the older population remains unclear. Therefore, this study was conducted to clarify it. METHODS: We used the Taiwan National Health Insurance Research Database to identify older patients with CP between 2001 and 2005 as the study cohort. Comparison cohort was the older patients without CP by matching age, sex, and index date at 1:1 ratio with the study cohort in the same period. We also included common underlying comorbidities in the analyses. The risk of ACS was compared between the two cohorts by following up until 2015. RESULTS: A total of 17241 older patients with CP and 17241 older patients without CP were included in this study. In both cohorts, the mean age (± standard deviation) and female percentage were 73.5 (± 5.7) years and 55.4%, respectively. Spinal disorders (31.9%) and osteoarthritis (27.0%) were the most common causes of CP. Older patients with CP had an increased risk for ACS compared to those without CP after adjusting for all underlying comorbidities (adjusted sub-distribution hazard ratio [sHR] 1.18; 95% confidence interval: 1.07-1.30). The increasement of risk of ACS was more when the follow-up period was longer (adjusted sHR of < 3 years: 1.8 vs. <2 years: 1.75 vs. <1 year: 1.55). CONCLUSIONS: CP was associated with an increased risk of ACS in the older population, and the association was more prominent when the follow-up period was longer. Early detection and intervention for CP are suggested in this population.

An Artificial In Vitro Metabolism to Angiopterlactone B Inspired by Traditional Retrosynthesis.

Nature's way to construct highly complex molecular entities as part of biosynthetic pathways is unmatched by any chemical synthesis. Yet, relying on a cascade of native enzymatic transformations to achieve a certain target structure, biosynthesis is also significantly limited in its scope. In this study, non-natural biocatalytic modules, a peroxidase-mediated Achmatowicz rearrangement and a dehydrogenase-catalyzed borrowing-hydrogen-type isomerization were successfully incorporated into an artificial metabolism, combining the benefits of traditional retrosynthesis with the elegance and efficacy of biosynthetic networks. In a highly streamlined process, the total synthesis of tricyclic angiopterlactone B was achieved in two steps operating entirely in an aqueous environment while relying mainly on enzymes as key reaction mediators.

Polymorphisms of RIG-I-like receptor influence HBV clearance in Chinese Han population.

Retinoic acid-inducible gene I-like receptors (RLRs) play an essential role in human innate immune, which may influence the spontaneous clearance of hepatitis B virus (HBV) infection. We aimed to investigate whether the SNPs in RLR family were associated with HBV spontaneous clearance. The current study included 82 participants with spontaneous clearance, 601 asymptomatic hepatitis B surface antigen (HBsAg) carriers, and 168 participants with chronic hepatitis B (CHB). Six SNPs (DDX58 rs3824456, rs3205166, DHX58 rs2074160, rs2074158, IFIH1 rs2111485, rs3747517) were genotyped to explore their association with HBV spontaneous clearance. Patients carrying the mutant allele C at rs3824456 or A at rs2074160 were more likely to achieve spontaneous clearance compared with asymptomatic HBsAg carriers (additive model: odds ratio [OR] = 0.69, 95% confidence interval [CI] = 0.49-0.97; dominant model: OR = 0.54, 95% CI = 0.31-0.95, respectively). In addition, patients carrying the mutant allele G at rs2111485 were more likely to achieve spontaneous clearance compared with CHB (dominant model: OR = 0.47, 95% CI = 0.25-0.87). The mutations were protective factors for HBV spontaneous clearance. These results suggest the DDX58 rs3824456, DHX58 s2074160, IFIH1 rs2111485 were associated with spontaneous clearance of HBV, which may be predictive markers in the Chinese Han population of HBV.

Overexpression of a multifunctional ß-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter.

The ß-glucosidase, which hydrolyzes the ß(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of ß-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including ß-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2-4. We overexpressed the ß-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with ß-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of ß-glucosidase in plants could further increase. The plant-expressed ß-glucosidase had optimal activity at 80 °C and pH 5-6. In addition, the plant-expressed ß-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with ß-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial ß-glucosidase gene as a reporter in plants based on alternative ß-galactosidase activity.

Respiratory Muscle Training Improves Strength and Decreases the Risk of Respiratory Complications in Stroke Survivors: A Systematic Review and Meta-analysis.

OBJECTIVE: To evaluate the effects of respiratory muscle training in a population of stroke patients. DATA SOURCES: The following databases were searched for clinical trials through December 2019: PubMed, EMBASE, Cochrane Library, CINAHL, and China National Knowledge Infrastructure. STUDY SELECTION: Randomized controlled trials (N=9) published in English met the inclusion criteria. DATA EXTRACTION: Data were extracted and assessed for accuracy by 2 reviewers. Any disagreements were resolved after discussions with an independent third reviewer. The quality of the included randomized controlled trials was assessed using the Cochrane bias tool. DATA SYNTHESIS: The meta-analysis showed increased maximal inspiratory pressure (standardized mean difference [SMD], 0.88; 95% confidence interval [CI], 0.62-1.15; P<.001; 12-wk follow-up period: SMD, 0.94; 95% CI, 0.42-1.45; P<.001), maximal expiratory pressure (SMD, 0.83; 95% CI, 0.15-1.52; P=.017; 12-wk follow-up period: SMD, 0.99; 95% CI, 0.47-1.51; P<.001), forced expiratory volume in 1 second (SMD, 1.41; 95% CI, 0.57-2.24; P=.001), forced vital capacity (SMD, 1.36; 95% CI, 0.55-2.16; P<.001), peak expiratory flow (SMD, 0.74; 95% CI, 0.16-1.32; P=.013), 6-minute walk test (SMD, 0.67; 95% CI, 0.11-1.23; P=.020), and decreased respiratory complications (odds ratio, 0.55; 95% CI, 0.30-1.00; P=.050) compared with no respiratory intervention or a sham intervention. CONCLUSIONS: Respiratory muscle training improved poststroke muscle strength and the benefits were carried over for up to 12 weeks, including improved lung function, walking capacity, and a reduced risk of respiratory impediments.

Fluoxetine Induces Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits ERK/NF-κB-Modulated Anti-Apoptotic and Invasive Potential in Hepatocellular Carcinoma Cells In Vitro.

The aim of the present study was to verify the effects of fluoxetine on dysregulation of apoptosis and invasive potential in human hepatocellular carcinoma (HCC) SK-Hep1 and Hep3B cells. Cells were treated with different concentrations of fluoxetine for different times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used for testing the effects of fluoxetine on cell viability. The regulation of apoptosis signaling, and anti-apoptotic, proliferation, and metastasis-associated proteins after fluoxetine treatment were assayed by flow cytometry and Western blotting assay. The detection of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation after fluoxetine treatment was performed by NF-κB reporter gene assay. The results demonstrated that fluoxetine significantly reduced cell viability, cell migration/invasion, NF-κB, extracellular signal-regulated kinases (ERK) activation, and expression of anti-apoptotic (Cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein (C-FLIP), Myeloid cell leukemia-1 (MCL-1), X-Linked inhibitor of apoptosis protein (XAIP), and Survivin), proliferation (Cyclin-D1), angiogenesis (vascular endothelial growth factor (VEGF)), and metastasis-associated proteins (matrix metalloproteinase-9 (MMP-9)). Fluoxetine also significantly induced apoptosis, unregulated extrinsic (activation of first apoptosis signal protein and ligand (Fas/FasL), and caspase-8) and intrinsic (loss of mitochondrial membrane potential (ΔΨm) pathways and increased Bcl-2 homologous antagonist killer (BAK) apoptosis signaling. Taken together, these results demonstrated that fluoxetine induced apoptosis through extrinsic/intrinsic pathways and diminished ERK/NF-κB-modulated anti-apoptotic and invasive potential in HCC cells in vitro.

Enantioconvergent Biocatalytic Redox Isomerization.

Alcohol dehydrogenases can act as powerful catalysts in the preparation of optically pure γ-hydroxy-δ-lactones by means of an enantioconvergent dynamic redox isomerization of readily available Achmatowicz-type pyranones. Imitating the traditionally metal-mediated "borrowing hydrogen" approach to shuffle hydrides across molecular architectures and interconvert functional groups, this chemoinspired and purely biocatalytic interpretation effectively expands the enzymatic toolbox and provides new opportunities in the assembly of multienzyme cascades and tailor-made cellular factories.

Enzymatic cascades for the stereo-complementary epimerisation of in situ generated epoxy alcohols.

The synthesis of optically pure secondary epoxy alcohols from racemic allylic alcohols using a single whole-cell biocatalyst of recombinant Escherichia coli coexpressing three oxidoreductases is described. The cascade involves the concurrent action of a styrene monooxygenase that catalyzes the formation of the chiral epoxy group, and two alcohol dehydrogenases that fulfil the epimerisation of the hydroxy group. Two sets of alcohol dehydrogenases were each applied to couple with styrene monooxygenase in order to realize the epimerisation in a stereo-complementary manner. Excellent enantio- and diastereo-selectivities were achieved for most of the 12 substrates.