Exploration in the Mechanism of Ginsenoside Rg5 for the Treatment of Osteosarcoma by Network Pharmacology and Molecular Docking.

OBJECTIVE: Osteosarcoma is a primary malignancy originating from mesenchymal tissue characterized by rapid growth, early metastasis and poor prognosis. Ginsenoside Rg5 (G-Rg5) is a minor ginsenoside extracted from Panax ginseng C.A. Meyer which has been discovered to possess anti-tumor properties. The objective of current study was to explore the mechanism of G-Rg5 in the treatment of osteosarcoma by network pharmacology and molecular docking technology. METHODS: Pharmmapper, SwissTargetPrediction and similarity ensemble approach databases were used to obtain the pharmacological targets of G-Rg5. Related genes of osteosarcoma were searched for in the GeneCards, OMIM and DrugBank databases. The targets of G-Rg5 and the related genes of osteosarcoma were intersected to obtain the potential target genes of G-Rg5 in the treatment of osteosarccoma. The STRING database and Cytoscape 3.8.2 software were used to construct the protein-protein interaction (PPI) network, and the Database for Annotation, Visualization and Integrated Discovery (DAVID) platform was used to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. AutoDock vina software was used to perform molecular docking between G-Rg5 and hub targets. The hub genes were imported into the Kaplan-Meier Plotter online database for survival analysis. RESULTS: A total of 61 overlapping targets were obtained. The related signaling pathways mainly included PI3K-Akt signaling pathway, Proteoglycans in cancer, Lipid and atherosclerosis and Kaposi sarcoma-associated herpesvirus infection. Six hub targets including PIK3CA, SRC, TP53, MAPK1, EGFR, and VEGFA were obtained through PPI network and targets-pathways network analyses. The results of molecular docking showed that the binding energies were all less than -7 kcal/mol. And the results of survival analysis showed TP53 and VEGFA affect the prognosis of sarcoma patients. CONCLUSION: This study explored the possible mechanism of G-Rg5 in the treatment of osteosarcoma using network pharmacology method, suggesting that G-Rg5 has the characteristics of multi-targets and multi-pathways in the treatment of osteosarcoma, which lays a foundation for the follow-up experimental and clinical researches on the therapeutic effects of G-Rg5 on osteosarcoma.

High-throughput synchronous erythrocyte cellular antioxidant activity and protection screening of phenolic-rich extracts: Protocol validation and applications.

Oxidative/nitrosative damage takes part in chronic disease development, which generates an urgent need for intervention and better therapies to manage them. The scientific community has demanded easy-to-run, cheap, and reliable methods for cellular antioxidant activity assays. This work standardised and validated an erythrocyte cellular antioxidant activity and membrane protection/injury (HERYCA-P) protocol to study food-derive extracts. The method measures intracellular reactive oxygen species (ROS) generation, lipoperoxidation, and haemolysis induced by 2,2'-azobis(2-amidinopropane) dihydrochloride. Quercetin decreased ROS generation by 50.4% and haemolysis by 2.2%, while ascorbic acid inhibited lipid peroxidation by 40.1%. Total phenolic contents of teas were correlated with decreased ROS generation (r = -0.924), lipoperoxidation (r = -0.951), and haemolysis (r = -0.869). The erythrocyte ROS generation and lipoperoxidation were also associated with CUPRAC (r = -0.925; r = -0.951) and hydroxyl radical scavenging activity (r = -0.936; r = -0.949). The precision rates of antioxidant standards and tea samples were below 15%. HERYCA-P is feasible as a complementary antioxidant assay for food matrices.

Purification and characterization of the low molecular weight xylanase from Bacillus cereus L-1.

Xylanase is widely used in various industries such as food processing, paper, textiles, and leather tanning. In this study, Bacillus cereus L-1 strain was isolated and identified as capable of producing low molecular weight xylanase through 16 s rRNA sequencing. Maximum xylanase yield of 15.51 ± 2.08 U/mL was achieved under optimal fermentation conditions (5% inoculum, 20 g/L xylan, pH 6.0, for 24 h). After purification via ammonium sulfate precipitation and High-S ion exchange chromatography, electrophoretic purity xylanase was obtained with a 28-fold purification and specific activity of 244.97 U/mg. Xylanase had an optimal pH of 6.5 and temperature of 60 °C and displayed thermostability at 30 °C and 40 °C with 48.56% and 45.97% remaining activity after 180 min, respectively. The xylanase retained more than 82.97% of its activity after incubation for 24 h at pH 5.0 and was sensitive to metal ions, especially Mg2+ and Li+. Purified xylanase showed a molecular weight of 23 kDa on SDS-PAGE, and partial peptide sequencing revealed homology to the endo-1,4-beta-xylanase with a molecular weight of 23.3 kDa through LC/MS-MS (liquid chromatography-tandem mass spectrometry). This study suggests that the purified xylanase is easier to purify and enriches low molecular weight xylanases from bacteria source.

Frequently mutated genes in predicting the relapse of stage I lung adenocarcinoma.

PURPOSE: Approximately, 45-65% stage I non-small cell lung cancer (NSCLC) patients with surgical resection relapse within 5 years. Therefore, it is urgent to identify the predictors involved in the relapse of stage I NSCLC. METHODS/PATIENTS: Targeted sequencing was used to examine the mutation of tumor tissues and matched adjacent normal tissues from 35 patients with stage I lung adenocarcinoma (LUAD). Then, tissue microarrays containing tumor tissues from 149 stage I LUAD patients were used to assess protein expression of frequently mutated genes by immunohistochemistry. COX regression model was used to evaluate the impacts of frequently mutated genes and their protein expression on relapse-free survival (RFS) in stage I LUAD. RESULTS AND CONCLUSIONS: Three hundred and twenty-nine non-synonymous somatic variants were identified in 161 genes among these 35 patients. EGFR, TP53, LRP1B, RBM10, KRAS, NTRK3, RB1, ALK, APC, FAT2, KEAP1, MED12 and MLL3 were described as frequently mutated genes with prevalence more than 10%. Patients harboring KRAS mutation had more relapse in 1 year after surgical resection. For the expression of these frequently mutated genes in 149 stage I patients, multivariate Cox regression analyses showed that the expression of RBM10 was positively associated with RFS in all patients (HR 0.40, 95% CI 0.15-1.0, p = 0.052), and the expression of APC was negative associated with RFS in patients with EGFR mutations (HR 3.10, 95% CI 1.54-6.26, p = 0.002). Stage I LUAD patients with KRAS mutation or low RBM10 expression are inclined to receive more positive intervention rather than just disease surveillance.

Extraction, characterization, and biological activities of exopolysaccharides from plant root soil fungus Fusarium merismoides A6.

The exploration of polysaccharides from microorganisms is of great importance. In this study, a new type of exopolysaccharide excreted by Fusarium merismoides A6 (FM-EPS) was isolated, and the extraction conditions were optimized using a response surface methodology (RSM). The extraction temperature at 0 °C, a precipitation time of 7.83 h, and an ethanol precipitation concentration of 77.64% were predicted and proved to be the best extraction conditions with the maximum extraction yield of 0.74 g/mL. Then, two fractions of F. merismoides A6 exopolysaccharides (FM-EPS1 and FM-EPS2) were obtained through DEAE Sepharose fast flow column chromatography. As indicated by monosaccharide composition analysis, both fractions mainly consisted of mannose, glucose, galactose, and ribose, with an average molecular weight of 5.14 × 104 and 6.50 × 104 g/mol, respectively. FT-IR and NMR spectroscopy indicated the FM-EPSs had both α- and ß-glycosidic bonds. Moreover, the determination of antioxidant and antiproliferative activities in vitro proved that FM-EPSs had good antioxidant activities and antiproliferation activities. FM-EPS1 showed stronger antioxidant activities than FM-EPS2. FM-EPS2 showed antiproliferation activities on HeLa and HepG2 cells, while FM-EPS1 had no obvious antiproliferative activity. Therefore, FM-EPSs could be explored as potential antioxidant and anticancer agent applied in food, feed, nutraceutical, pharmaceutical, cosmetics, and chemical industries.

Pharmacological Effects of Resveratrol in Intervertebral Disc Degeneration: A Literature Review.

Intervertebral disc degeneration (IDD) is a high incidence disease of musculoskeletal system that often leads to stenosis, instability, pain and even deformity of the spinal segments. IDD is an important cause of discogenic lower back pain and often leads to large economic burden to families and society. Currently, the treatment of IDD is aimed at alleviating symptoms rather than blocking or reversing pathological progression of the damaged intervertebral disc. Resveratrol (RSV) is a polyphenol phytoalexin first extracted from the Veratrum grandiflflorum O. Loes and can be found in various plants and red wine. Owing to the in-depth study of pharmacological mechanisms, the therapeutic potential of RSV in various diseases such as osteoarthritis, neurodegenerative diseases, cardiovascular diseases and diabetes have attracted the attention of many researchers. RSV has anti-apoptotic, anti-senescent, anti-inflammatory, anti-oxidative, and anabolic activities, which can prevent further degeneration of intervertebral disc cells and enhance their regeneration. With high safety and various biological functions, RSV might be a promising candidate for the treatment of IDD. This review summarizes the biological functions of RSV in the treatment of IDD and to facilitate further research.

Purple tea (Camellia sinensis var. assamica) leaves as a potential functional ingredient: From extraction of phenolic compounds to cell-based antioxidant/biological activities.

A statistical optimization study was used to maximize the extraction of bioactive compounds and antioxidant activity from green tea derived from purple leaves of Camellia sinensis var. assamica. Simultaneous optimization was applied, and a combination of 60 °C, 15 min, and a mass-solvent ratio of 1 g of dehydrated purple leaves to 62.3 mL of an ethanol/citric acid solution, were determined as the ideal extraction conditions. The optimized extract of purple tea leaves (OEPL) contained showed stability in relation to variations in pH, and lyophilized OEPL exerted cytotoxic and antiproliferative effects against cancerous cells (A549 and HCT8), demonstrated antimicrobial activity towards Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 13565) and Staphylococcus epidermidis (ATCC 12288), inhibition of α-amylase and α-glycosidase enzymes and reduced the release of pro-inflammatory cytokines (TNF-α, CXCL2/MIP-2, and IL-6) in lipopolysaccharides-stimulated RAW 264.7 macrophages. Thus, our results provide a broad assessment of the bioactivity of "green" extracts obtained by a simple and low-cost process using non-toxic solvents, and they have the potential to be used for technological applications.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis

Abstract Objectives: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

Response surface optimization of phenolic compounds from jabuticaba (Myrciaria cauliflora [Mart.] O.Berg) seeds: Antioxidant, antimicrobial, antihyperglycemic, antihypertensive and cytotoxic assessments.

The aim of this study was to evaluate the effects of different solvents and maximize the extraction of bioactive compounds from jabuticaba (Myrciaria cauliflora) seeds. In general, the solvent system composed of water and propanone (52:48 v/v) modified the extract polarity and increased extraction yield of bioactive compounds. The optimized extract presented antioxidant capacity measured by different chemical and biological assays. The optimized extract exerted antiproliferative and cytotoxic effects against A549 and HCT8 cells, antimicrobial and antihemolytic effects, inhibited α-amylase/α-glucosidase activities and presented in vitro antihypertensive effect. Nonetheless, the optimized extract showed no cytotoxicity in a human cell model (IMR90). Vescalagin, castalagin and ellagic acid were the major phenolic compounds in the optimized extract. Our results show that jabuticaba seed may be a potential ingredient for the development of potentially functional foods.

Association between bile acid metabolism and bone mineral density in postmenopausal women.

OBJECTIVES: Previous studies have not shown any correlation between bile acid metabolism and bone mineral density (BMD) in women with postmenopausal osteoporosis. Thus, the current study evaluated the association between bile acid levels as well as BMD and bone turnover marker levels in this group of women. METHODS: This single-center cross-sectional study included 150 postmenopausal Chinese women. According to BMD, the participants were divided into three groups: osteoporosis group, osteopenia group, and healthy control group. Serum bile acid, fibroblast growth factor 19 (FGF19), and bone turnover biomarker levels were assessed. Moreover, the concentrations of parathyroid hormone, 25-hydroxy vitamin D [25(OH)D], procollagen type I N-peptide (P1NP), and beta-CrossLaps of type I collagen containing cross-linked C-terminal telopeptide (ß-CTX) were evaluated. The BMD of the lumbar spine and proximal femur were examined via dual-energy X-ray absorptiometry. RESULTS: The serum total bile acid levels in the osteoporosis and osteopenia groups (5.28±1.56 and 5.31±1.56 umol/L, respectively) were significantly lower than that in the healthy control group (6.33±2.04 umol/L; p=0.002 and 0.018, respectively). Serum bile acid level was positively associated with the BMD of the lumbar spine, femoral neck, and total hip. However, it negatively correlated with ß-CTX concentration. Moreover, no correlation was observed between bile acid and P1NP levels, and the levels of the other biomarkers that were measured did not differ between the groups. CONCLUSION: Serum bile acid was positively correlated with BMD and negatively correlated with bone turnover biomarkers reflecting bone absorption in postmenopausal women. Thus, bile acid may play an important role in bone metabolism.

Camu-camu seed (Myrciaria dubia) - From side stream to anantioxidant, antihyperglycemic, antiproliferative, antimicrobial, antihemolytic, anti-inflammatory, and antihypertensive ingredient.

Camu-camu (Myrciaria dubia) seeds are discarded without recovering the bioactive compounds. The main aim of the present work was to optimise the solvent mixture to extract higher total phenolic content and antioxidant capacity of camu-camu seeds. The optimised solvent system increased the extraction of phenolic compounds, in which vescalagin and castalagin were the main compounds. The optimised extract displayed antioxidant capacity measured by different chemical and biological assays, exerted antiproliferative and cytotoxic effects against A549 and HCT8 cancer cells, antimicrobial effects, protected human erythrocytes against hemolysis, inhibited α-amylase and α-glucosidase enzymes and presented in vitro antihypertensive effect. Additionally, the optimized extract inhibited human LDL copper-induced oxidation in vitro and reduced the TNF-α release and NF-κB activation in macrophages cell culture. Thus, the use of camu-camu seed showed to be a sustainable way to recover bioactive compounds with in vitro functional properties.

Association between bile acid metabolism and bone mineral density in postmenopausal women

OBJECTIVES: Previous studies have not shown any correlation between bile acid metabolism and bone mineral density (BMD) in women with postmenopausal osteoporosis. Thus, the current study evaluated the association between bile acid levels as well as BMD and bone turnover marker levels in this group of women. METHODS: This single-center cross-sectional study included 150 postmenopausal Chinese women. According to BMD, the participants were divided into three groups: osteoporosis group, osteopenia group, and healthy control group. Serum bile acid, fibroblast growth factor 19 (FGF19), and bone turnover biomarker levels were assessed. Moreover, the concentrations of parathyroid hormone, 25-hydroxy vitamin D [25(OH)D], procollagen type I N-peptide (P1NP), and beta-CrossLaps of type I collagen containing cross-linked C-terminal telopeptide (β-CTX) were evaluated. The BMD of the lumbar spine and proximal femur were examined via dual-energy X-ray absorptiometry. RESULTS: The serum total bile acid levels in the osteoporosis and osteopenia groups (5.28±1.56 and 5.31±1.56 umol/L, respectively) were significantly lower than that in the healthy control group (6.33±2.04 umol/L; p=0.002 and 0.018, respectively). Serum bile acid level was positively associated with the BMD of the lumbar spine, femoral neck, and total hip. However, it negatively correlated with β-CTX concentration. Moreover, no correlation was observed between bile acid and P1NP levels, and the levels of the other biomarkers that were measured did not differ between the groups. CONCLUSION: Serum bile acid was positively correlated with BMD and negatively correlated with bone turnover biomarkers reflecting bone absorption in postmenopausal women. Thus, bile acid may play an important role in bone metabolism.

Flaxleaf Fleabane Leaves (Conyza bonariensis), A New Functional Nonconventional Edible Plant?

This work aimed to investigate the phytochemical composition, nutritional value, antioxidant, antihemolytic, antihyperglycemic, and antiproliferative activities of flaxleaf fleabane (Conyza bonariensis) leaves. Different concentrations of water and ethanol (0:100, 25:75, 50:50, 75:25, and 100:0 v/v) were used in the extraction process and results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolics, ortho-diphenolics, Folin-Ciocalteu reducing capacity, FRAP, and Fe2+ chelating ability values. Flaxleaf fleabane leaves (FFL) contained 19.6 g/100 g of fibers and 26 g/100 g of proteins. Ellagic acid, procyanidin A2, caffeic, rosmarinic, gallic, and 2,5-dihydroxybenzoic acids were the main phenolics. This phenolic-rich extract inhibited the lipid oxidation of Wistar rat brain (IC50 = 863.0 mg GAE/L), inhibited α-glucosidase activity (IC50 = 435.4 µg/mL), protected human erythrocytes against mechanical hemolysis at different osmolarity conditions, and showed cytotoxic/antiproliferative effects against human ileocecal adenocarcinoma cells (HCT8; IC50 = 552.6 µg/mL) but no cytotoxicity toward noncancerous human lung fibroblast (IMR90). Overall, FFL showed potential to be explored by food companies to be a source of proteins, natural color substances, and phenolic compounds. PRACTICAL APPLICATION: Flaxleaf fleabane leaves (FFL) are usually burnt or partially given to cattle, without a proper utilization as a source of nutrients for human nutrition. Here, we studied the nutritional composition, phenolic composition, and toxicological aspects of FFL using different biological protocols. FFL was proven to be a rich source of proteins and dietary fibers and showed antioxidant activity measured by chemical and in vitro biological assays. Additionally, as it did protected human red cells and did not show cytotoxicity, we assume FFL has relative safety to be consumed as a nonconventional edible plant.

From the Field to the Pot: Phytochemical and Functional Analyses of Calendula officinalis L. Flower for Incorporation in an Organic Yogurt.

Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of water and ethyl alcohol, and the lyophilized extract with higher content of antioxidant compounds was incorporated into an organic yogurt. Results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolic content (TPC), flavonoids, and antioxidant activity (ferric reducing antioxidant power (FRAP), total reducing capacity (TRC), and Cu2+/Fe2+ chelating ability). Phenolic acids and flavonoids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract (LME) also inhibited 12% of Wistar rat's brain lipid oxidation in vitro, inhibited α-amylase, and α-glucosidase activities, but showed no cytotoxicity towards cancerous cells (HCT8 and A549). However, marigold flower extract protected human erythrocytes against mechanical stress. When added into an organic yogurt model (0 to 1.5%), LME increased TPC and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and TRC), and the sensory analysis showed that the organic yogurt had an acceptance of 80.4%. Our results show that the use of LME may be a technological strategy to increase the content of bioactive compounds in yogurts.

Antioxidants-rich ice cream containing herbal extracts and fructooligossaccharides: manufacture, functional and sensory properties.

This work aimed to optimize an aqueous extract rich in phenolic compounds and potential functional properties made of Ilex paraguariensis, Melissa officinalis, and Cymbopogon citratus. The lyophilized extract was used for the development of an ice cream. Total phenolics, FRAP, DPPH, Folin-Ciocalteu's reducing capacity, and total reducing capacity of different combinations of herbal extracts were tested and modeled using response surface methodology. Simultaneous optimisation was employed to maximize the bioactive compounds in the extract and the lyophilized optimum combination was added to ice cream. The lyophilized extract contained quercetin-3-rutinoside, hesperidin, isoquercetin, caffeic acid, and 5,7-dihydroxyflavone. The optimised extract, which showed antihypertensive, antidiabetic, and antioxidant activity using in vitro protocols, increased total phenolics and antioxidant activity in comparison to the control ice cream. The ice cream presented a sensory acceptance index of 83%. After 72 days of storage (-18 °C), total phenolics and antioxidant activity significantly decreased.

Optimized Exon-Exon Junction Library and its Application on Rodents' Brain Transcriptome Analysis

ABSTRACT Background: Alternative splicing (AS), which plays an important role in gene expression and functional regulation, has been analyzed on genome-scale by various bioinformatic approaches based on RNA-seq data. Compared with the huge number of studies on mouse, the AS researches approaching the rat, whose genome is intermedia between mouse and human, were still limited. To enrich the knowledge on AS events in rodents' brain, we perfomed a comprehensive analysis on four transcriptome libraries (mouse cerebrum, mouse cerebellum, rat cerebrum, and rat cerebellum), recruiting high-throughput sequencing technology. An optimized exon-exon junction library approach was introduced to adapt the longer RNA-seq reads and to improve mapping efficiency. Results: In total, 7,106 mouse genes and 2,734 rat genes were differentially expressed between cerebrum and cerebellum, while 7,125 mouse genes and 1,795 rat genes exhibited varieties on transcript variant level. Only half of the differentially expressed exon-exon junctions could be reflected at gene expression level. Functional cluster analysis showed that 32 pathways in mouse and 9 pathways in rat were significantly enriched, and 6 of them were in both. Interestingly, some differentially expressed transcript variants did not show difference on gene expression level, such as PLCβ1 and Kcnma1. Conclusion: Our work provided a case study of a novel exon-exon junction strategy to analyze the expression of genes and isoforms, helping us understand which transcript contributes to the overall expression and further functional change.

The effect of 5-substitution on the electrochemical behavior and antitubercular activity of PA-824.

Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves reduction of the nitroimidazole ring and subsequent nitric oxide release. The importance of this compound and its unique activity prompted us to explore how substitution of the nitroimidazole ring would affect electrochemical reduction and antitubercular activity. We prepared analogs of PA-824 with bromo, chloro, cyano, and amino substituents in the 5-position of the aromatic ring. We found that substitution of the imidazole ring greatly influences reduction and the stability of the corresponding nitro radical anion. Further, the antitubercular activities of the bromo and chloro analogs may indicate that an alternate nitroreductase pathway within Mycobacterium tuberculosis exists.