Fangji Dihuang formulation ameliorated DNCB-induced atopic dermatitis-like skin lesions by IL-17 signaling pathway: integrating network analysis and experimental validation.

Background: The Fangji Dihuang formulation (FJDHF) is a widely recognized Traditional Chinese Medicine (TCM) formula that consists of five plant drugs: Stephaniae Tetrandrae Radix, Cinnamomi Ramulus, Rehmanniae Radix, Saposhnikoviae Radix, and Glycyrrhiza Urensis Fisch. This formulation has been known to exhibit clinical therapeutic effects in the treatment of inflammatory skin diseases. However, there is a lack of pharmacological research on its anti-atopic dermatitis (AD) activity. Methods: To investigate the potential anti-AD activity of FJDHF, DNCB was used to induce AD-like skin inflammation in the back of mice. Following successful modeling, the mice were administered FJDHF orally. The extent of the inflammatory skin lesions was recorded at day 4, 7, 14 and 28. UHPLC-Q-Exactive Orbitrap MS was used to identify and match the compounds present in FJDHF with ITCM, TCMIP and TCMSID. In silico predictions of potential target proteins of the identified compounds were obtained from SwishTargetPrediction, ITCM and TargetNet databases. AD-related genes were identified from GSE32924 data set, and FJDHF anti-AD hub genes were identified by MCODE algorithm. ClueGo enrichment analysis was employed to identify the core pathway of FJDHF's anti-AD effect. To further investigate the anti-AD effect of FJDHF, single-cell RNA sequencing data set (GSE148196) from AD patients was analyzed to determine the target cells and signaling pathways of FJDHF in AD. Finally, rt-PCR, flow cytometry, and mouse back skin RNA sequencing were utilized to validate our findings. Results: FJDHF was found to be effective in improving the degree of the AD-like lesions in the mice. Network pharmacological analysis revealed the core pathway of FJDHF to be the IL-17 signaling pathway, which is interactively associated with cytokines. Single-cell RNA sequencing analysis suggested that FJDHF may play an anti-AD role by influencing dendritic cells. Flow cytometry and rt-PCR results showed that FJDHF can reduce the influence of AD sample of IL-4, IFN-γ and the expression of IL-17. The RNA sequencing of mouse back skin also confirmed our conclusion. Conclusion: FJDHF may inhibit DNCB-induced AD-like skin inflammation in mice by inhibiting the IL-17 signaling pathway. Thus, FJDHF can be considered as a potential therapeutic agent for AD.

Baicalin inhibits hepatocellular carcinoma cell growth and metastasis by suppressing ROCK1 signaling.

Hepatocellular carcinoma (HCC) is a common malignancy affecting many people worldwide. Baicalin is a flavonoid extracted from the dried root of Scutellaria baicalensis Georgi. It can effectively inhibit the occurrence and development of HCC. Nonetheless, the mechanism through which Baicalin inhibits HCC growth and metastasis remain unknown. This work discovered that Baicalin inhibited HCC cell proliferation, invasion, metastasis while inducing cell cycle arrest at the G0/G1 phase and apoptosis. In vivo HCC xenograft results indicated that Baicalin inhibited HCC growth. Western blotting analysis indicated that Baicalin suppressed the expressions of ROCK1, p-GSK-3ß, and ß-catenin, whereas it up-regulated the expressions of GSK-3ß and p-ß-catenin. Baicalin also reduced the expressions of Bcl-2, C-myc, Cyclin D1, MMP-9, and VEGFA, while increasing the expression of Bax. Molecular docking revealed that Baicalin docked in the binding site of the ROCK1 agonist, with a binding energy of -9 kcal/mol between the two. In addition, lentivirus-mediated suppression of ROCK1 expression improved the inhibitory effect of Baicalin on the proliferation, invasion, and metastasis of HCC and the expression of proteins associated with ROCK1/GSK-3ß/ß-catenin signaling pathway. Moreover, restoring ROCK1 expression decreased the anti-HCC efficacy of Baicalin. These findings suggest that Baicalin may decrease HCC proliferation and metastasis by suppressing ROCK1/GSK-3ß/ß-catenin signaling.

New insights on low-temperature storage regulating garlic greening and the accumulation of pigment precursors via glutathione metabolism and energy cycles.

To explore regulation mechanism of temperature on garlic greening and pigment precursors' accumulation, greening capacities, pigment precursors and critical metabolites, enzyme and genes involved in glutathione and NADPH metabolism of garlic stored at five temperatures (4, 8, 16, 24 and 30 ℃) were analyzed. Results showed that garlic pre-stored at 4, 8 and 16 ℃ were more likely to green than ones at 24 and 30 ℃ after pickling. After 25 days, more S-1-propenyl-l-cysteine sulfoxide (1-PeCSO) were detected in garlic stored at 4, 8 and 16 ℃ (753.60, 921.85 and 756.75 mAU, respectively) than that at 24 and 30 ℃ (394.35 and 290.70 mAU). Pigment precursors' accumulation in garlic was mainly realized by glutathione and NADPH metabolism under low-temperature storage, through enhancements of activities or expressions for GR (GSR), GST (GST), γ-GT (GGT1, GGT2), 6PGDH (PGD) and ICDHc (IDH1). This study enriched the mechanism of garlic greening.

Biejiajian pill inhibits progression of hepatocellular carcinoma by downregulating PDGFRß signaling in cancer-associated fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Biejiajian pill (BJJP) is a canonical formula that is clinically used to treat chronic liver disease, especially to decrease the incidence of hepatocellular carcinoma (HCC). However, the mechanisms underlying the prevention of HCC progression by BJJP remain unclear. AIM OF THE STUDY: This study aimed to determine whether BJJP inhibits HCC progression by downregulating platelet-derived growth factor receptor beta (PDGFRß) signaling in cancer-associated fibroblasts (CAFs) in a mouse model of diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced HCC. MATERIALS AND METHODS: C57BL/6 male mice were intraperitoneally injected with DEN 2 weeks after birth, followed by repeated injections of CCl4 weekly from 6 weeks of age onwards, to recapitulate features of HCC. At week 14, BJJP was orally administered to mice. The effects of BJJP on HCC progression were evaluated using histology, immunohistochemistry, and serum biochemical marker levels. Transcriptome analysis, molecular docking, quantitative real-time PCR, and Western blot were used to study the genes targeted by BJJP and the associated signaling pathway. The effects of BJJP on PDGFRß signaling in CAFs and the underlying mechanism were demonstrated. RESULTS: BJJP treatment significantly suppressed carcinogenesis and cancer progression, and it ameliorated liver inflammation in mice with HCC. A total of 176 genes, including PDGFRß, were significantly downregulated after BJJP treatment and five components of BJJP with high binding affinity to PDGFRß were identified. BJJP inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3ß) by suppressing PDGFRß expression in CAFs, and it also downregulated the expression of the downstream proteins hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A). Furthermore, BJJP-containing serum consistently reduced PDGFRß, HGF, and VEGF-A expression levels in HSC-derived CAFs in vitro. Importantly, PDGF-BB induced PDGFRß activation in CAFs and both BJJP and sunitinib (a kinase inhibitor) inhibited PDGF-BB/PDGFRß signaling. CONCLUSION: BJJP inhibits the progression of HCC through suppressing VEGF-A and HGF expression in CAFs by downregulating PDGFRß signaling.

Comparative transcriptome and proteome profiles reveal the regulation mechanism of low temperature on garlic greening.

Garlic stored at low temperature (0-13 ℃) for some times and subsequently crushed and placed at room temperature would turn green, while the one stored at high temperature (30 ℃) would not. In order to elucidate the regulatory mechanism of low temperature on garlic greening, transcriptome and proteome profiles of garlic stored at 4 ℃ and 30 ℃ were explored by RNA-seq and iTRAQ techniques. Principal component analysis showed that garlic at different storage temperatures were of significant differences on both gene and protein levels. 14,381 and 861 differential expression genes (DEGs) and proteins (DEPs) were identified respectively, in which 268 factors were shared according to their joint analysis, including 186 (144) up-regulated genes (proteins) and 82 (124) down-regulated genes (proteins) in comparing garlic stored at 4 ℃ with ones at 30 ℃. These 268 factors were mainly attributed to biological process (metabolic process) and molecular function (catalytic activity, binding) categories by Gene Ontology classification. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways enrichment of DEGs and DEPs revealed that GSSG production, GSH degradation, amino acid biosynthesis (cysteine and methionine) and energy metabolism (TCA and HMP cycles) were promoted by low-temperature storage to responding to oxidative stress and prepared for pigment synthesis in garlic. These results provide valuable information for the regulation of garlic greening during processing.

Low frequency ultrasound treatment enhances antibrowning effect of ascorbic acid in fresh-cut potato slices.

Ascorbic acid (AsA) is generally used as an antibrowning agent for fresh-cut potato (FCP). However, its browning inhibitory effect is temporary because of its rapid consumption during redox processes. In this study, the effect of browning inhibition in FCP slices was evaluated using low frequency ultrasound (LFU; 40 kHz, 200 W, 3 min) and AsA (0.2%, w/v) treatments alone or in combination. The results showed that LFU combined with AsA (LFU-AsA) treatment could achieve a better antibrowning effect than either treatment alone (higher L*, lower a* and ΔE*), and there was no adverse effect on texture properties. LFU-AsA treatment not only inhibited the activity of PPO more effectively than AsA treatment, but also enhanced the antibrowning effect by retaining higher AsA content. Moreover, it also maintained membrane integrity by limiting lipid peroxidation and solute migration during storage. Overall, LFU-AsA treatment would be a promising method in food preservation industries.

Biejiajian Pill Promotes the Infiltration of CD8+ T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5.

Tumor-infiltrating CD8+T lymphocytes are mostly associated with a favorable prognosis in numerous cancers, including hepatocellular carcinoma (HCC). Biejiajian Pill (BJJP) is a common type of traditional Chinese medicine that is widely used in the treatment of HCC in China. Previous studies showed that BJJP suppressed the growth of HCC cells both in vivo and in vitro, by exerting direct cytotoxic effects on tumor cells. The present study demonstrated that in addition to direct cytotoxicity, BJJP inhibits the growth of tumor cells by promoting the infiltration of CD8+T cells into the tumor in H22-bearing mice. Mechanistically, chemokine ligand 5 (CCL5) was identified as one of the most highly expressed chemokines by tumor cells in vivo after treatment with BJJP. Additionally, CCL5 was knocked down in H22 cells and the results showed that knockdown of the gene significantly impaired the infiltration of CD8+T cells in vivo. Furthermore, the effects of BJJP on human HCC cell lines were assessed in vitro. Similarly, cells treated with BJJP had higher expression of CCL5 mRNA, which was consistent with increased levels of CCL5 protein in human tumor cells. These findings provide new insights into the anticancer effects of BJJP, which regulated the expression of CCL5 and the infiltration of CD8+T cells. The results, therefore, suggest that BJJP has great potential application in clinical practice.

Sinisan Protects Primary Hippocampal Neurons Against Corticosterone by Inhibiting Autophagy via the PI3K/Akt/mTOR Pathway.

Objective: Corticosterone causes significant neurotoxicity in primary hippocampal neurons which is associated with depression. Dysfunctional autophagy is implicated in cognitive impairment and depressive-like behavior. The traditional Chinese medicine Sinisan (SNS) is highly effective in clinical treatment of depression. However, the molecular mechanisms underlying therapeutic effects of SNS are unknown. Purpose: The aim of this study was to elucidate the protective effect of SNS and the underlying mechanisms against corticosterone-induced neuronal damage. Study Design: The effects of serum derived from rats containing SNS (or untreated controls) on the expression of autophagy-related molecules in primary rat hippocampal neurons exposed to different concentrations of corticosterone for different intervals were explored. Methods: CCK-8 assay, LDH assay were used to analyze cell viability and LDH activity. Western blot, qRT-PCR, and immunofluorescence assays were used to determine protein and mRNA expression levels of molecules such as LC3, p62, Beclin1, ULK1, PI3K, p-PI3K, Akt p-Akt, mTOR, p-mTOR, p70S6, p-p70S6, 4ebp1 and p-4ebp1. Results: Corticosterone induced a dose- and time-dependent reduction in cellular viability. Moreover, corticosterone (100-400 µM) treatment for 24 h increased LC3-II/LC3-I protein ratio, increased Beclin1 and ULK1 protein expression levels, and decreased p62, PI3K, p-PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein expression levels. Notably, SNS-containing serum reversed corticosterone-induced reduction of neuronal viability, and increased p62, PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein and mRNA expression levels. In addition, SNS-containing serum decreased LC3-II/LC3-I protein ratio, and downregulated Beclin1, and ULK1 protein and mRNA expression in primary hippocampal neurons. Conclusion: SNS protects primary hippocampal neurons against corticosterone-induced neurotoxicity by preventing excessive autophagy through activation of PI3K/AKT/mTOR pathway.

Urinary metabolomics analysis of the protective effects of Daming capsule on hyperlipidemia rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Hyperlipidemia is recognized as one of the most important risk factors for morbidity and mortality due to cardiovascular diseases. Daming capsule, a Chinese patent medicine, has shown definitive efficacy in patients with hyperlipidemia. In this study, serum biochemistry and histopathology assessment were used to investigate the lipid-lowering effect of Daming capsule. Furthermore, urinary metabolomics based on ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was conducted to identify the urinary biomarkers associated with hyperlipidemia and discover the underlying mechanisms of the antihyperlipidemic action of Daming capsule. After 10 weeks of treatment, Daming capsule significantly lowered serum lipid levels and ameliorated hepatic steatosis induced by a high-fat diet. A total of 33 potential biomarkers associated with hyperlipidemia were identified, among which 26 were robustly restored to normal levels after administration of Daming capsule. Pathway analysis revealed that the lipid-lowering effect of Daming capsule is related to the regulation of multiple metabolic pathways including vitamin B and amino acid metabolism, tricarboxylic acid cycle, and pentose phosphate pathway. Notably, the study demonstrates that metabolomics is a powerful tool to elucidate the multitarget mechanism of traditional Chinese medicines, thereby promoting their research and development.

Biejiajian Pill Inhibits Carcinogenesis and Metastasis via the Akt/GSK-3ß/Snail Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is among the most usual cancers globally. In China, Biejiajian pill (BJJP), Traditional Chinese Medicine clinical prescription, is broadly utilized for the prevention and therapy of HCC. However, the mechanisms by which BJJP exerts its effects on the prevention of tumor invasion and metastasis are still largely unknown. In this study, in vitro multiple hepatic cancer cell lines and an in vivo xenograft mice model were used to validate the preventive effects and molecular mechanisms of BJJP in HCC. We established that BJJP significantly repressed the proliferation, metastasis and infiltration of HCC cells. Furthermore, BJJP remarkably suppressed HCC cell migration, as well as invasion via epithelial-mesenchymal transition (EMT) by modulating Snail expression, which was associated with the repression of Akt/GSK-3ß/Snail signaling axis activation. In vivo HCC xenograft results indicated that BJJP delayed HCC development and efficiently inhibited lung metastasis. Taken together, BJJP was shown to be an effective therapeutic agent against HCC through repression of the Akt/GSK-3ß/Snail signaling cascade and EMT.

A new fusicoccane diterpene and a new polyene from the plant endophytic fungus Talaromyces pinophilus and their antimicrobial activities.

A new fusicoccane diterpene, pinophicin A (1), and a new polyene, pinophol A (2), were isolated from the plant endophytic fungus Talaromyces pinophilus obtained from the aerial parts of Salvia miltiorrhiza. The structures and relative configurations of 1-2 were determined by the analysis of extensive spectroscopic data, chemical method, and comparison with known compounds. Compound 2 exhibited weak antibacterial activity against Bacterium paratyphosum B with an MIC value of 50 µg/mL.

Two novel eremophylane acetophenone conjugates from Colletotrichum gloeosporioides, an endophytic fungus in Salvia miltiorrhiza.

Two novel eremophylane acetophenone conjugates, colletotricholides A (1) and B (2), were isolated from the solid fermentation cultures of an endophytic fungus Colletotrichum gloeosporioides XL1200 isolated from the aerial parts of Salvia miltiorrhiza. The chemical structures of 1-2 were characterized by extensive spectroscopic methods and single-crystal X-ray crystallography. Structurally, compounds 1-2 are two unusual eremophylane acetophenone conjugates originating from the hybrid pathways of polyketide synthase and sesquiterpene synthase. In addition, compounds 1-2 were inactive against tested pathogens.

(±)-Preisomide: A new alkaloid featuring a rare naturally occurring tetrahydro-2H-1,2-oxazin skeleton from an endophytic fungus Preussia isomera by using OSMAC strategy.

A new alkaloid, named (±)-preisomide (1), together with five known polyketides (2-6), were isolated from an endophytic fungus Preussia isomera in Panax notoginseng by using one strain-many compounds (OSMAC) strategy. Their structures were identified by extensive spectroscopic experiments and comparison with literature data. Structurally, compound 1 possessed a rare naturally occurring tetrahydro-2H-1,2-oxazin ring. Compound 6 displayed significant antibacterial activity against multidrug-resistant Enterococcus faecium, methicinllin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with an MIC value of 25 µg/mL, as well as moderate antifungal activity against Gibberella saubinetii with an MIC value of 50 µg/mL.

Membrane curvature sensing of the lipid-anchored K-Ras small GTPase.

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras G12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras G12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras G12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

Docking- and pharmacophore-based virtual screening for the identification of novel Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitor with a thiobarbiturate scaffold.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an important virulence factor for Mtb that contributes to survival of the bacteria in macrophages. The absence of a human ortholog makes MptpB an attractive target for new therapeutics to treat tuberculosis. MptpB inhibitors could be an effective treatment to overcome emerging TB drug resistance. Adopting a structure-based virtual screening strategy, we successfully identified thiobarbiturate-based drug-like MptpB inhibitor 15 with an IC50 of 22.4 µM, and as a non-competitive inhibitor with a Ki of 24.7 µM. Importantly, not only did it exhibit moderate cell membrane permeability, compound 15 also displayed potent inhibition of intracellular TB growth in the macrophage, making it an excellent lead compound for anti-TB drug discovery. To the best of our knowledge, this novel thiobarbiturate is the first class of MptpB inhibitor reported so far that leveraged docking- and pharmacophore-based virtual screening approaches. The results of preliminary structure-activity relationship demonstrated that compound 15 identified herein was not a singleton and may inspire the design of novel selective and drug-like MptpB inhibitors.

Upregulation of miRNA-155 expression by OxLDL in dendritic cells involves JAK1/2 kinase and transcription factors YY1 and MYB.

Dendritic cells (DCs) have been implicated in the pathogenesis of atherosclerosis (AS). Whereas the pathogenic role of oxidized low-density lipoprotein (oxLDL) in the development and progression of AS has been recognized previously, the contribution of microRNA-155 (miR-155) to AS was previously not fully understood. It had also been noted that miR-155 levels were upregulated by oxLDL in various cell types under different (patho)physiological conditions, but its underlying mechanisms had not been examiend in detail. Thus, in the present study, we observed that oxLDL treatment increased miR-155 expression in DCs, and transfecting DCs with siRNA against scavenger receptor A (SRA) revealed that repression of SRA attenuated this upregulation. We also noted that miR-155 negatively regulated SRA expression by suppressing the JNK pathway. Furthermore, we noted that Yin Yang 1 (YY1) and V-Myb avian myeloblastosis viral oncogene homolog (MYB), which were also upregulated by oxLDL, directly bound to the cognate sequences of the promoter region of miR-155 to activate its transcription. In addition, using SP600125, a specific inhibitor for c-Jun N-terminal kinase (JNK) signaling, we demonstrated that JNK signaling was involved in the miR­155-mediated suppression of SRA expression. Thus, in the present study we uncovered the molecular mechanism through which miR-155 expression is regulated by oxLDL, and we also identified a negative feedback loop, miR­155-JNK-SRA-miR-155. Our findings thus provide novel insights into the regulatory network underlying the expression and activity of miR-155 in DCs.

A simplified procedure for the determination of organochlorine pesticides and polychlorobiphenyls in edible vegetable oils.

A one-step extraction-purification multiresidue method for the determination of 14 organochlorine pesticides (OCPs) and 7 polychlorinated biphenyls (PCBs) in edible vegetable oils based on matrix solid-phase dispersion (MSPD) has been developed. The experimental parameters affecting the recoveries and the efficiency of the cleanup procedure were thoroughly evaluated. Under an optimised condition, 0.5 g of oil sample was blended with 3.5 g of sulfuric acid-impregnated silica and 0.8 g of silica gel was used as co-column absorbent. The PCBs and OCPs were eluted by 10 mL of n-hexane/dichloromethane (70:30, v/v) and determined by gas chromatography equipped with an electron capture detector (GC-ECD). Good recoveries were obtained in the range of 69.6-105.3% with relative standard deviations (RSD) values below 15% in most cases. The limits of detection (LOD), based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.04-0.74 ng/g.

Conducting nanosponge electroporation for affordable and high-efficiency disinfection of bacteria and viruses in water.

High-efficiency, affordable, and low energy water disinfection methods are in great need to prevent diarrheal illness, which is one of the top five leading causes of death over the world. Traditional water disinfection methods have drawbacks including carcinogenic disinfection byproducts formation, energy and time intensiveness, and pathogen recovery. Here, we report an innovative method that achieves high-efficiency water disinfection by introducing nanomaterial-assisted electroporation implemented by a conducting nanosponge filtration device. The use of one-dimensional (1D) nanomaterials allows electroporation to occur at only several volts, which is 2 to 3 orders of magnitude lower than that in traditional electroporation applications. The disinfection mechanism of electroporation prevents harmful byproduct formation and ensures a fast treatment speed of 15,000 L/(h·m(2)), which is equal to a contact time of 1 s. The conducting nanosponge made from low-cost polyurethane sponge coated with carbon nanotubes and silver nanowires ensures the device's affordability. This method achieves more than 6 log (99.9999%) removal of four model bacteria, including Escherichia coli, Salmonella enterica Typhimirium, Enterococcus faecalis, and Bacillus subtilis, and more than 2 log (99%) removal of one model virus, bacteriophage MS2, with a low energy consumption of only 100 J/L.

A microsystem compatible strategy for viable Escherichia coli detection.

This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.