Engineered Bacillus subtilis as oral probiotics to target circulating lactic acid.

Lactic acid or lactate, a key byproduct of anaerobic glycolysis, plays pivotal roles in routine metabolism. An increase in lactic acid is observed in various pathological conditions such as cancer, diabetes, genetic mitochondrial disease, and aging. While several groups have proposed small molecule inhibitors to reduce circulating lactic acid, there are few clinically relevant ways to manage acute or chronic elevations in lactic acid in patients. In addition, recent evidence suggests that lactic acid exchanges between the gut, blood, and peripheral tissues, and professional marathon runners harbor specific gut microbial species that more efficiently metabolize lactic acid. Inspired by these findings, this work sought to engineer probiotic B. subtilis strains to express lactate oxidase that could increase circulating lactic acid catabolism after delivery to the gut. After optimization, oral administration of engineered B. subtilis to the gut of mice reduced the elevation in blood lactic acid levels after exogenous lactic acid challenge without affecting normal gut microbiota composition, inflammation or liver enzymes. Taken together, through the oral delivery of engineered probiotics to the gastrointestinal tract, our proof-of-concept study offers a new opportunity to therapeutically target diseases where blood lactic acid is elevated, and provides a new approach to "knocking down" metabolites to help understand the roles of metabolites in host physiological and pathological processes.

Germination ecology of Chenopodium album L. and implications for weed management.

Chenopodium album L. is a troublesome annual species in various cropping systems, and a sound knowledge of the ecological response of C. album germination to environmental factors would suggest suitable management strategies for inhibiting its spread. Preliminary laboratory-based research was conducted to investigate germination and emergence requirements of C. album under various environmental conditions (e.g., photoperiods, constant temperature, salinity, moisture, soil pH, burial depth, and oat crop residue). Results showed C. album seeds were found to be photoblastic, with only 13% germination in darkness. The maximum germination (94%) of C. album occurred at an optimal temperature of 25°C, and the depressive effect of other temperatures on germination was more severe at lower rather than higher temperatures. Seed germination was suitably tolerant of salinity and osmotic potential, with germination observed at 200 mM NaCl (37.0%) and -0.8 MPa (20%), respectively. Germination was relatively uniform (88-92%) at pH levels ranging from 4 to 10. The maximum germination of C. album was observed on the soil surface, with no or rare emergence of seeds at a burial depth of 2 cm or under 7000 kg ha-1 oat straw cover, respectively. Information provided by this study will help to develop more sustainable and effective integrated weed management strategies for the control of C. album, including (i) a shallow-tillage procedures to bury weed seeds in conventional-tillage systems and (ii) oat residue retention or coverage on the soil surface in no-tillage systems.

Effect of Light Intensity on Morphology, Photosynthesis and Carbon Metabolism of Alfalfa (Medicago sativa) Seedlings.

To understand how light intensity influences plant morphology and photosynthesis in the forage crop alfalfa (Medicago sativa L. cv. Zhongmu 1), we investigated changes in leaf angle orientation, chlorophyll fluorescence, parameters of photosynthesis and expression of genes related to enzymes involved in photosynthesis, the Calvin cycle and carbon metabolism in alfalfa seedlings exposed to five light intensities (100, 200, 300, 400 and 500 µmol m-2 s-1) under hydroponic conditions. Seedlings grown under low light intensities had significantly increased plant height, leaf hyponasty, specific leaf area, photosynthetic pigments, leaf nitrogen content and maximal PSII quantum yield, but the increased light-capturing capacity generated a carbon resource cost (e.g., decreased carbohydrates and biomass accumulation). Increased light intensity significantly improved leaf orientation toward the sun and upregulated the genes for Calvin cycle enzymes, thereby increasing photosynthetic capacity. Furthermore, high light (400 and 500 µmol m-2 s-1) significantly enhanced carbohydrate accumulation, accompanied by gene upregulation and increased activity of sucrose and starch-synthesis-related enzymes and those involved in carbon metabolism. Together, these results advance our understanding of morphological and physiological regulation in shade avoidance in alfalfa, which would guide the identification of suitable spatial planting patterns in the agricultural system.

Ginsenoside Rg5 Inhibits Human Osteosarcoma Cell Proliferation and Induces Cell Apoptosis through PI3K/Akt/mTORC1-Related LC3 Autophagy Pathway.

The function and mechanism underlying the suppression of human osteosarcoma cells by ginsenoside-Rg5 (Rg5) was investigated in the present study. MG-63, HOS, and U2OS cell proliferation was determined by MTT assay after Rg5 treatment for 24 h. Rg5 inhibited human osteosarcoma cell proliferation effectively in a dose-dependent manner. The range of effective inhibitory concentrations was 160-1280 nM. Annexin V-FITC and PI double-staining assay revealed that Rg5 induced human osteosarcoma cell apoptosis. Western blotting, qRT-PCR, and FACS experiments revealed that Rg5 inhibited human osteosarcoma cells via caspase-3 activity which was related to the LC3-mediated autophagy pathway. Rg5 decreased the phosphorylation of PI3K, Akt, and mTORC1 activation. In contrast, LC3-mediated autophagy and caspase-3 activity increased significantly. A PI3K/AKT stimulator, IGF-1, reversed Rg5-induced cell autophagy and apoptosis in MG-63 cells. Collectively, the current study demonstrated that Rg5 induced human osteosarcoma cell apoptosis through the LC3-mediated autophagy pathway. Under physiological conditions, activation of PI3K/AKT/mTORC1 inhibits LC3 activity and caspase-3-related cell apoptosis. However, Rg5 activated LC3 activity by inhibiting the activation of PI3K/AKT/mTORC1. The present study indicated that Rg5 could be a promising candidate as a chemotherapeutic agent against human osteosarcoma.

Pharmacological activities of ginsenoside Rg5 (Review).

Ginseng, a perennial plant belonging to genus Panax, has been widely used in traditional herbal medicine in East Asia and North America. Ginsenosides are the most important pharmacological component of ginseng. Variabilities in attached positions, inner and outer residues and types of sugar moieties may be associated with the specific pharmacological activities of each ginsenoside. Ginsenoside Rg5 (Rg5) is a minor ginsenoside synthesized during ginseng steaming treatment that exhibits superior pharmaceutical activity compared with major ginsenosides. With high safety and various biological functions, Rg5 may act as a potential therapeutic candidate for diverse diseases. To date, there have been no systematic studies on the activity of Rg5. Therefore, in this review, all available literature was reviewed and discussed to facilitate further research on Rg5.

Disorders Associated With Diverse, Recurrent Deletions and Duplications at 1q21.1.

The subchromosomal region 1q21.1 is one of the hotspots in the human genome for deletions and reciprocal duplications, owing to the existence of hundreds of segmental duplications. Recurrent deletions and duplications in this region are thought to be causative in patients with variable clinical manifestations. Based on the genomic locations, deletions and duplications at the 1q21.1 locus have been associated with distinguishable syndromes: chromosome 1q21.1 deletion syndrome, chromosome 1q21.1 duplication syndrome, and thrombocytopenia-absent radius (TAR) syndrome, which is partially due to deletions at the proximal 1q21.1 region. We report here diverse, recurrent deletions and duplications at the 1q21.1 locus in 36 patients from a cohort of 5,200 individuals. Among the 36 patients, 18 patients carry 1q21.1 deletions, nine individuals have reciprocal duplications at 1q21.1, two patients share an identical short deletion, and the remaining seven possess variable sizes of duplications at the proximal 1q21.1 region. Furthermore, we provide cytogenetic characterization and detailed clinical features for each patient. Notably, duplications at the proximal 1q21.1 region have not been associated with a defined disorder in publications. However, recurrent duplications at the proximal 1q21.1 region among the seven patients strongly suggested that the variants are likely pathogenic. The common phenotypical features of those disorders are also summarized to facilitate clinical diagnoses and genetic counseling.

Bronchiolitis obliterans murine model induced by nitric acid aerosol inhalation: An economical and reproducible model.

PURPOSE: Bronchiolitis obliterans (BO) is a highly debilitative and fatal syndrome associated with a series of severe lower airway disorders. The pathogenesis of BO is complicated and not entirely understood. An appropriate animal model of BO may aid research into its pathogenesis. Here, we establish a mouse model of BO to provide insight into this disease. MATERIALS AND METHODS: 6-8 week old BABL/c mice were exposed to 5% nitric acid (NA) aerosol through a nebulizer for 3 hours, and controls were exposed to distilled water instead. Symptoms, airway resistance and pathological process were observed dynamically. The levels of matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), 8-isoprostane and myeloperoxidase (MPO) in lung tissue and bronchoalveolar lavage fluids (BLAF) were determined by ELISA on day 3, 7, 14, 28 and 56 after the aerosol nebulization. RESULTS: Typical BO lesions were observed in NA nebulized mice characterized histologically by initial necrotizing bronchiolitis and final airway fibrosis at day 28 after the aerosol nebulization. NA nebulized mice also exhibited labored breathing and significantly increased airway resistance. Expression of MMP-2, MMP-9, TIMP-1, 8-isoprostane and MPO were significantly elevated in NA nebulized mice in different time frame. CONCLUSION: A murine BO model was established by NA aerosol inhalation. It provides an easy, economic, and reproducible mice model for BO research.

Association of imbalance of effector T cells and regulatory cells with the severity of asthma and allergic rhinitis in children.

PURPOSE: This study investigated the frequency and absolute numbers of different subsets of peripheral blood cells, including CD4+ T cells and CD19+ interleukin (IL) 10+ B regulatory cells (Breg) and their potential association with clinical laboratory measurements in children diagnosed with simple asthma or asthma plus allergic rhinitis (AR). METHOD: The frequency and numbers of peripheral blood CD4+ interferon (IFN) gamma+ T-helper (Th) type 1, CD4+ IL-4+ Th2, CD4+ IL-17A+ Th17, CD4+CD25+ Forkhead Box P3+ regulatory T cells (Treg) and CD19+ IL-10+ Bregs in 22 children with asthma, 17 children with asthma and AR, and 25 healthy controls were determined by flow cytometry. The levels of serum cytokines were determined by enzyme-linked immunosorbent assay. RESULTS: In comparison with healthy controls, patients with asthma alone had significantly increased numbers of Th1, Th2, and Th17 cells, and their cytokines but decreased numbers of Tregs and Bregs, and the cytokines IL-10 and transforming growth factor beta 1. This imbalance between effector and regulatory cells and their cytokines further increased in patients with asthma and AR. The ratios of percentage effector T cells (Th1 + Th2 + Th17) to regulatory cells (Treg + Breg) were positively correlated with fractional exhaled nitric oxide but negatively correlated with forced expiratory volume in 1 second in patients with asthma or asthma plus AR. CONCLUSION: The imbalance of effector T cells and regulatory cells contributed to the pathogenesis of airway inflammation of asthma and AR in children.

Potential factors involved in the causation of rhabdomyolysis following status asthmaticus.

Rhabdomyolysis is a rare but potentially fatal complication of status asthmaticus. Since the first case was reported in 1978, only a few dozen cases have been described till date. We performed a literature review with the aim to characterize the pathophysiological basis of the occurrence of rhabdomyolysis in patients with status asthmaticus. Excessive exertion of respiratory muscles, hypoxia and acidosis, electrolyte imbalance, infections, some drugs used for asthma control, use of mechanical ventilation, prolonged cardiopulmonary resuscitation, higher age of the patient and some underlying diseases or genetic factors appear to be involved in its causation. In patients with status asthmaticus, it is important to pay more attention to these factors and to closely monitor creatine kinase levels in blood so as to ensure early detection of rhabdomyolysis.

Andrographolide plays an important role in bleomycin-induced pulmonary fibrosis treatment.

Pulmonary fibrosis (PF) leads to chronic inflammation and accumulation of macrophages, neutrophils, and lymphocytes in the alveoli. The factors involved in the development of PF include reactive oxygen species and tissue remodelling regulators. The present study demonstrates the effect of andrographolide on bleomycin (BLM)-induced PF in Sprague-Dawley rats. We investigated the total bronchoalveolar lavage fluid protein (BALF) and hydroxyproline (HYP) content along with the level of oxidative stress markers like malondialdehyde (MDA) and GSH/GSSG ratio. In addition, the levels of MMP-1 and TIMP-1 were also analysed. The results revealed an increase in BALF protein, HYP, and MDA contents and decrease in GSH/GSSG ratio of the lungs in animals treated with BLM. However, andrographolide treatment caused a reversal of the BLM induced changes after 20 or 40 days. Treatment with andrographolide suppressed oxidative stress with the decrease of MDA and the increase of the GSH/GSSG ratio. Andrographolide also improved the BLM mediated changes in the MMP-1/TIMP-1 ratio. Therefore, andrographolide has a potential therapeutic effect in the prevention of PF.

Early-Life Exposure to Clostridium leptum Causes Pulmonary Immunosuppression.

INTRODUCTION: Low Clostridium leptum levels are a risk factor for the development of asthma. C. leptum deficiency exacerbates asthma; however, the impact of early-life C. leptum exposure on cesarean-delivered mice remains unclear. This study is to determine the effects of early-life C. leptum exposure on asthma development in infant mice. METHODS: We exposed infant mice to C. leptum (fed-CL) and then induced asthma using the allergen ovalbumin (OVA). RESULTS: Fed-CL increased regulatory T (Treg) cells in cesarean-delivered mice compared with vaginally delivered mice. Compared with OVA-exposed mice, mice exposed to C. leptum + OVA did not develop the typical asthma phenotype, which includes airway hyper-responsiveness, cell infiltration, and T helper cell subset (Th1, Th2, Th9, Th17) inflammation. Early-life C. leptum exposure induced an immunosuppressive environment in the lung concurrent with increased Treg cells, resulting in the inhibition of Th1, Th2, Th9, and Th17 cell responses. CONCLUSION: These findings demonstrate a mechanism whereby C. leptum exposure modulates adaptive immunity and leads to failure to develop asthma upon OVA sensitization later in life.

Involvement of Kruppel-like factor 9 in bleomycin-induced pulmonary toxicity.

Oxidative stress or the production of reactive oxygen species (ROS) has been implicated as an important factor in the development of bleomycin (BLM)-induced pulmonary toxicity; however, the mechanism behind the toxicity remains to be elucidated. The present study aimed to investigate the key factor involved in BLM­induced toxicity. The study was conducted in human pulmonary fibroblast (HPF) cells and in a mouse model. The ROS level, cell death assay, protein and gene expression levels of Kruppel­like factor 9 (Klf9) and other associated factors were assessed. A dose­dependent increase in ROS, lipid peroxidation, cell death, and protein and mRNA expression levels of NF­E2­related transcription factor 2 (Nrf2) and Klf9 were observed in BLM­treated cells. However, the expression levels of the other antioxidant proteins assessed, including catalase, super oxide dismutase, glutathione reductase and thioredoxin reductase 2, were decreased. The expression levels of Nrf2 were decreased in cells treated with a higher concentration (>200 µM) of BLM. These results suggested that in response to increased intracellular levels of ROS, above a critical threshold, Nrf2 stimulates the expression of Klf9, resulting in a further increase in Klf9­mediated ROS production and subsequent cell death. Furthermore, the data suggested that Klf9 may be considered as an adjunctive therapeutic target for BLM-induced pulmonary toxicity.