COWID: an efficient cloud-based genomics workflow for scalable identification of SARS-COV-2.

Implementing a specific cloud resource to analyze extensive genomic data on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a challenge when resources are limited. To overcome this, we repurposed a cloud platform initially designed for use in research on cancer genomics (https://cgc.sbgenomics.com) to enable its use in research on SARS-CoV-2 to build Cloud Workflow for Viral and Variant Identification (COWID). COWID is a workflow based on the Common Workflow Language that realizes the full potential of sequencing technology for use in reliable SARS-CoV-2 identification and leverages cloud computing to achieve efficient parallelization. COWID outperformed other contemporary methods for identification by offering scalable identification and reliable variant findings with no false-positive results. COWID typically processed each sample of raw sequencing data within 5 min at a cost of only US$0.01. The COWID source code is publicly available (https://github.com/hendrick0403/COWID) and can be accessed on any computer with Internet access. COWID is designed to be user-friendly; it can be implemented without prior programming knowledge. Therefore, COWID is a time-efficient tool that can be used during a pandemic.

Tumoricidal Activity of Simvastatin in Synergy with RhoA Inactivation in Antimigration of Clear Cell Renal Cell Carcinoma Cells.

Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic ccRCC drastically declines even with intensive treatment. We examined the efficacy of simvastatin, a lipid-lowering drug with reduced mevalonate synthesis, in ccRCC treatment. Simvastatin was found to reduce cell viability and increase autophagy induction and apoptosis. In addition, it reduced cell metastasis and lipid accumulation, the target proteins of which can be reversed through mevalonate supplementation. Moreover, simvastatin suppressed cholesterol synthesis and protein prenylation that is essential for RhoA activation. Simvastatin might also reduce cancer metastasis by suppressing the RhoA pathway. A gene set enrichment analysis (GSEA) of the human ccRCC GSE53757 data set revealed that the RhoA and lipogenesis pathways are activated. In simvastatin-treated ccRCC cells, although RhoA was upregulated, it was mainly restrained in the cytosolic fraction and concomitantly reduced Rho-associated protein kinase activity. RhoA upregulation might be a negative feedback effect owing to the loss of RhoA activity caused by simvastatin, which can be restored by mevalonate. RhoA inactivation by simvastatin was correlated with decreased cell metastasis in the transwell assay, which was mimicked in dominantly negative RhoA-overexpressing cells. Thus, owing to the increased RhoA activation and cell metastasis in the human ccRCC dataset analysis, simvastatin-mediated Rho inactivation might serve as a therapeutic target for ccRCC patients. Altogether, simvastatin suppressed the cell viability and metastasis of ccRCC cells; thus, it is a potentially effective ccRCC adjunct therapy after clinical validation for ccRCC treatment.

Exerting the Appropriate Application of Methylprednisolone in Acute Spinal Cord Injury Based on Time Course Transcriptomics Analysis.

Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.

RBM4-SRSF3-MAP4K4 splicing cascade modulates the metastatic signature of colorectal cancer cell.

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.

3-Methylcholanthrene/Aryl-Hydrocarbon Receptor-Mediated Hypertension Through eNOS Inactivation.

Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and Western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC-mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. J. Cell. Physiol. 232: 1020-1029, 2017. © 2016 Wiley Periodicals, Inc.

Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, is involved in tumorigenesis. However, no systematic research has demonstrated the cell-transforming activity of PRMT5. We investigated the involvement of PRMT5 in tumor formation. First, we showed that PRMT5 was associated with many human cancers, through statistical analysis of microarray data in the NCBI GEO database. Overexpression of ectopic PRMT5 per se or its specific shRNA enhanced or reduced cell growth under conditions of normal or low concentrations of serum, low cell density, and poor cell attachment. A stable clone that expressed exogenous PRMT5 formed tumors in nude mice, which demonstrated that PRMT5 is a potential oncoprotein. PRMT5 accelerated cell cycle progression through G1 phase and modulated regulators of G1; for example, it upregulated cyclin-dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 and E1, and inactivated retinoblastoma protein (Rb). Moreover, PRMT5 activated phosphoinositide 3-kinase (PI3K)/AKT and suppressed c-Jun N-terminal kinase (JNK)/c-Jun signaling cascades. However, only inhibition of PI3K activity, and not overexpression of JNK, blocked PRMT5-induced cell proliferation. Further analysis of PRMT5 expression in 64 samples of human lung cancer tissues by microarray and western blot analysis revealed a tight association of PRMT5 with lung cancer. Knockdown of PRMT5 retarded cell growth of lung cancer cell lines A549 and H1299. In conclusion, to the best of our knowledge, we have characterized the cell-transforming activity of PRMT5 and delineated its underlying mechanisms for the first time.

Robust Mutation Profiling of SARS-CoV-2 Variants from Multiple Raw Illumina Sequencing Data with Cloud Workflow.

Several variants of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging all over the world. Variant surveillance from genome sequencing has become crucial to determine if mutations in these variants are rendering the virus more infectious, potent, or resistant to existing vaccines and therapeutics. Meanwhile, analyzing many raw sequencing data repeatedly with currently available code-based bioinformatics tools is tremendously challenging to be implemented in this unprecedented pandemic time due to the fact of limited experts and computational resources. Therefore, in order to hasten variant surveillance efforts, we developed an installation-free cloud workflow for robust mutation profiling of SARS-CoV-2 variants from multiple Illumina sequencing data. Herein, 55 raw sequencing data representing four early SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, and Delta) from an open-access database were used to test our workflow performance. As a result, our workflow could automatically identify mutated sites of the variants along with reliable annotation of the protein-coding genes at cost-effective and timely manner for all by harnessing parallel cloud computing in one execution under resource-limitation settings. In addition, our workflow can also generate a consensus genome sequence which can be shared with others in public data repositories to support global variant surveillance efforts.

L-Carnitine reduces reactive oxygen species/endoplasmic reticulum stress and maintains mitochondrial function during autophagy-mediated cell apoptosis in perfluorooctanesulfonate-treated renal tubular cells.

We previously reported that perfluorooctanesulfonate (PFOS) causes autophagy-induced apoptosis in renal tubular cells (RTCs) through a mechanism dependent on reactive oxygen species (ROS)/extracellular signal-regulated kinase. This study extended our findings and determined the therapeutic potency of L-Carnitine in PFOS-treated RTCs. L-Carnitine (10 mM) reversed the effects of PFOS (100 µM) on autophagy induction and impaired autophagy flux. Furthermore, it downregulated the protein level of p47Phox, which is partly related to PFOS-induced increased cytosolic ROS in RTCs. Moreover, L-Carnitine reduced ROS production in mitochondria and restored PFOS-impeded mitochondrial function, leading to sustained normal adenosine triphosphate synthesis and oxygen consumption and reduced proton leakage in a Seahorse XF stress test. The increased inositol-requiring enzyme 1α expression by PFOS, which indicated endoplasmic reticulum (ER) stress activation, was associated with PFOS-mediated autophagy activation that could be attenuated through 4-phenylbutyrate (5 mM, an ER stress inhibitor) and L-Carnitine pretreatment. Therefore, by reducing the level of IRE1α, L-Carnitine reduced the levels of Beclin and LC3BII, consequently reducing the level of apoptotic biomarkers including Bax and cleaving PARP and caspase 3. Collectively, these results indicate that through the elimination of oxidative stress, extracellular signal-regulated kinase activation, and ER stress, L-Carnitine reduced cell autophagy/apoptosis and concomitantly increased cell viability in RTCs. This study clarified the potential mechanism of PFOS-mediated RTC apoptosis and provided a new strategy for using L-Carnitine to prevent and treat PFOS-induced RTC apoptosis.

Humoral Immunogenicity and Reactogenicity of the Standard ChAdOx1 nCoV-19 Vaccination in Taiwan.

BACKGROUND: The ChAdOx1 nCoV-19 vaccine has been widely administered against SARS-CoV-2 infection; however, data regarding its immunogenicity, reactogenicity, and potential differences in responses among Asian populations remain scarce. METHODS: 270 participants without prior COVID-19 were enrolled to receive ChAdOx1 nCoV-19 vaccination with a prime-boost interval of 8-9 weeks. Their specific SARS-CoV-2 antibodies, neutralizing antibody titers (NT50), platelet counts, and D-dimer levels were analyzed before and after vaccination. RESULTS: The seroconversion rates of anti-RBD and anti-spike IgG at day 28 after a boost vaccination (BD28) were 100% and 95.19%, respectively. Anti-RBD and anti-spike IgG levels were highly correlated (r = 0.7891), which were 172.9 ± 170.4 and 179.3 ± 76.88 BAU/mL at BD28, respectively. The geometric mean concentrations (GMCs) of NT50 for all participants increased to 132.9 IU/mL (95% CI 120.0-147.1) at BD28 and were highly correlated with anti-RBD and anti-spike IgG levels (r = 0.8248 and 0.7474, respectively). Body weight index was statistically significantly associated with anti-RBD IgG levels (p = 0.035), while female recipients had higher anti-spike IgG levels (p = 0.038). The GMCs of NT50 declined with age (p = 0.0163) and were significantly different across age groups (159.7 IU/mL for 20-29 years, 99.4 IU/mL for ≥50 years, p = 0.0026). Injection-site pain, fever, and fatigue were the major reactogenicity, which were more pronounced after prime vaccination and in younger participants (<50 years). Platelet counts decreased and D-dimer levels increased after vaccination but were not clinically relevant. No serious adverse events or deaths were observed. CONCLUSION: The vaccine is well-tolerated and elicited robust humoral immunity against SARS-CoV-2 after standard prime-boost vaccination in Taiwanese recipients.

Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer.

BACKGROUND: A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers. METHODS: Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients. RESULTS: A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p < 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p < 0.01). CONCLUSIONS: In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.

Orchestrating an Optimized Next-Generation Sequencing-Based Cloud Workflow for Robust Viral Identification during Pandemics.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently become a novel pandemic event following the swine flu that occurred in 2009, which was caused by the influenza A virus (H1N1 subtype). The accurate identification of the huge number of samples during a pandemic still remains a challenge. In this study, we integrate two technologies, next-generation sequencing and cloud computing, into an optimized workflow version that uses a specific identification algorithm on the designated cloud platform. We use 182 samples (92 for COVID-19 and 90 for swine flu) with short-read sequencing data from two open-access datasets to represent each pandemic and evaluate our workflow performance based on an index specifically created for SARS-CoV-2 or H1N1. Results show that our workflow could differentiate cases between the two pandemics with a higher accuracy depending on the index used, especially when the index that exclusively represented each dataset was used. Our workflow substantially outperforms the original complete identification workflow available on the same platform in terms of time and cost by preserving essential tools internally. Our workflow can serve as a powerful tool for the robust identification of cases and, thus, aid in controlling the current and future pandemics.

Perfluorooctane sulfonate induces autophagy-associated apoptosis through oxidative stress and the activation of extracellular signal-regulated kinases in renal tubular cells.

Perfluorooctane sulfonate (PFOS) is among the most abundant organic pollutants and is widely distributed in the environment, wildlife, and humans. Its toxic effects and biological hazards are associated with its long elimination half-life in humans. However, how it affects renal tubular cells (RTCs) remains unclear. In this study, PFOS was observed to mediate the increase in reactive oxygen species (ROS) generation, followed by the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, which induced autophagy in RTCs. Although PFOS treatment induced autophagy after 6 h, prolonged treatment (24 h) reduced the autophagic flux by increasing lysosomal membrane permeability (LMP), leading to increased p62 protein accumulation and subsequent apoptosis. The increase in LMP was visualized through increased green fluorescence with acridine orange staining, and this was attenuated by 3-methyladenine, an autophagy inhibitor. N-acetyl cysteine and an inhibitor of the mitogen-activated protein kinase kinases (U0126) attenuated autophagy and apoptosis. Taken together, these results indicate that ROS activation and ROS-mediated phosphorylated ERK1/2 activation are essential to activate autophagy, resulting in the apoptosis of PFOS-treated RTCs. Our findings provide insight into the mechanism of PFOS-mediated renal toxicity.

Altered expressions and splicing profiles of Acin1 transcripts differentially modulate brown adipogenesis through an alternative splicing mechanism.

Apoptotic chromatin condensation inducer in the nucleus (also referred as Acin1) was first characterized as an RNA-binding protein involved in apoptosis. In later reports, Acin1 was identified as an auxiliary component of the exon junction complex (EJC) which is assembled throughout pre-messenger RNA splicing. In this study, results of whole-transcriptome analyses revealed reduced expressions and reprogrammed splicing profiles of Acin1 transcripts throughout development of brown adipose tissues (BATs) that execute non-shivering thermogenesis in small rodents and infants by consuming lipids. Depletion of endogenous Acin1 isoforms led to activation of brown adipogenic signatures in mouse C3H10T1/2 fibroblasts. Nevertheless, overexpressions of the Acin1-L or Acin1-S isoform exerted discriminative influences on brown adipogenesis and reprogramming of the expression of serine/arginine-rich splicing factor 3 (SRSF3) through an alternative splicing-coupled nonsense-mediated decay mechanism in a sequence-specific manner. Moreover, the Acin1-SRSF3 axis constitutes a regulatory pathway that participates in the brown adipocyte-related splicing network. Taken together, the interplay between accessory EJC components and splicing regulators constitutes an emerging mechanism for differentially manipulating the activity of brown adipogenesis via alternative splicing network.

Author Correction: Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Cross-Platform Comparison of Affymetrix, Agilent, and Illumina Microarray Reveals Functional Genomics in Colorectal Cancer Progression.

Colorectal cancer is one of the most common cancers with the second highest mortality rate in the world. The microarray can be used to collect gene expression alteration information from many tissue samples that will be useful to understand colorectal cancer from the molecular level. However, the mechanism behind the progression from normal to cancer is not fully understood. Here, a cross-platform comparison among three common microarray platforms (Affymetrix, Agilent, and Illumina) was applied. As results, we found a significant correlation of purine metabolism and p53 signaling pathway role in colorectal cancer progression. Purine metabolism can control the regulation of cell proliferation which involve hydro-lyase activity on organelle lumen. Meanwhile, genetic alterations in p53 signaling pathways could control some hallmarks of cancer. These two terms might play important roles in inducing normal colorectal cells into cancer.

Berberine Promotes Beige Adipogenic Signatures of 3T3-L1 Cells by Regulating Post-transcriptional Events.

Induced brown adipocytes (also referred to as beige cells) execute thermogenesis, as do the classical adipocytes by consuming stored lipids, being related to metabolic homeostasis. Treatment of phytochemicals, including berberine (BBR), was reported to induce conversion from white adipocytes to beige cells. In this study, results of microRNA (miRNA)-seq analyses revealed a decrease in miR-92a, of which the transcription is driven by the c13orf25 promoter in BBR-treated 3T3-L1 cells. BBR treatment manipulated the expressions of SP1 and MYC, in turn, reducing the activity of the c13orf25 promoter. A decrease in miR-92a led to an increase in RNA-binding motif protein 4a (RBM4a) expression, which facilitated the beige adipogenesis. Overexpression of miR-92a or depletion of RBM4a reversely interfered with the impact of BBR treatment on the beige adipogenic signatures, gene expressions, and splicing events in 3T3-L1 cells. Our findings demonstrated that BBR treatment enhanced beige adipogenesis of 3T3-L1 cells through transcription-coupled post-transcriptional regulation.

Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation.

The therapeutic effects of simvastatin for renal cell carcinoma (RCC) are controversial. In this study, the effects of simvastatin on the carcinogenic properties of 3-methylcholanthrene (3MC; an aryl-hydrocarbon receptor [AhR] agonist) in human renal epithelial cells (hRECs) were investigated. We exposed in vitro and in vivo models to 3MC to induce RCC onset. 3MC upregulated the epithelial-mesenchymal transition (EMT) and tumor biomarkers; the models exhibited the reciprocal expression of histone deacetylase 1 (HDAC1) and RhoA, namely increased HDAC1 and decreased RhoA expression, through hypoxia-inducible-factor (HIF)- and AhR-dependent mechanisms. In addition to inducing EMT biomarkers, 3MC decreased von Hippel-Lindau protein levels (a risk factor for RCC) and increased CD44 expression in hRECs, which were reversed by digoxin (a HIF inhibitor) and HDAC inhibitors (suberoylanilide hydroxamic acid and trichostatin A [TSA]). Simvastatin abolished the detrimental effects of 3MC by reducing HDAC1 expression, with resulting RhoA upregulation, and reactivating RhoA in vitro and in vivo. Notably, the protective effects of simvastatin were negated by an HDAC activator (ITSA) through TSA suppression. The crucial role of RhoA in RCC carcinogenesis was verified by the overexpression of constitutively active RhoA. Collectively, these results demonstrate that simvastatin restores RhoA function through HDAC1 inhibition; therefore, simvastatin might serve as adjunct therapy for RCC induced by 3MC.

Synergistic effects of cAMP-dependent protein kinase A and AMP-activated protein kinase on lipolysis in kinsenoside-treated C3H10T1/2 adipocytes.

BACKGROUND: We previously showed that 3-O-ß-D-glucopyranosyl-(3R)-hydroxybutanolide (kinsenoside), a major compound of Anoectochilus formosanus, increased lipolysis through an AMP-activated protein kinase (AMPK)-dependent pathway. PURPOSE: To extend our previous finding, we investigated the in vivo and in vitro effects of kinsenoside on lipolysis and the involvement of cyclic AMP (cAMP)-dependent protein kinase A (PKA) and AMPK in kinsenoside-mediated lipolysis. STUDY DESIGN/METHODS: Mice were fed a high-fat diet for six weeks to induce lipid deposition and then treated with 50 and 100  mg/kg kinsenoside for two weeks. The coordination of PKA and AMPK activation in lipolysis in C3H10T1/2 adipocytes was evaluated in vitro by using PKA and AMPK's corresponding inhibitors, oil-red O staining, a glycerol production assay, and Western blot analysis. RESULTS: Kinsenoside reduced body weight, fat pad mass, and hepatic lipid accumulation in obese mice, and concurrently increased the induction and activation of hormone-sensitive lipase (HSL), perilipin, adipose triglyceride lipase (ATGL), and carnitine palmitoyltransferase I (CPT1). Kinsenoside concentration-dependently increased PKA activation by increasing the phosphorylation of Ser/Thr-PKA substrates in vitro. These increases were accompanied by a reduction in fat accumulation. Using H89 and Rp-8-Br-cAMPs to inhibit PKA reduced the release of glycerol but did not alter the activation of peroxisome proliferator-activated receptor alpha or the expression of CPT1 or ATGL. By contrast, compound C, an AMPK inhibitor, inhibited CPT1 and ATGL expression in kinsenoside-treated C3H10T1/2 adipocytes. In addition, H89 caused the reactivation of AMPK downstream targets by increasing the levels of the active form of pAMPK-Thr172, suggesting that PKA negatively modulates AMPK activity. CONCLUSION: Kinsenoside increased HSL activation through PKA-mediated phosphorylation at Ser660/563 and concomitantly increased perilipin activation in lipolysis. These lipolytic effects of kinsenoside were validated using 6-Bnz-cAMPs, a PKA agonist. In this study, we demonstrated that in addition to AMPK, PKA also plays a crucial role in kinsenoside-mediated lipolysis.

Phosphorylation and stabilization of HURP by Aurora-A: implication of HURP as a transforming target of Aurora-A.

Aurora-A, a mitotic serine/threonine kinase with oncogene characteristics, has recently drawn intense attention because of its association with the development of human cancers and its relationship with mitotic progression. Using the gene expression profiles of Aurora-A as a template to search for and compare transcriptome expression profiles in publicly accessible microarray data sets, we identified HURP (encodes hepatoma upregulated protein) as one of the best Aurora-A-correlated genes. Empirical validation indicates that HURP has several characteristics in common with Aurora-A. These two genes have similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression and across a variety of tissues and cell lines. Moreover, Aurora-A phosphorylated HURP in vitro and in vivo. Ectopic expression of either the catalytically inactive form of Aurora-A or the HURP-4P mutant, in which the Aurora-A phosphorylation sites were replaced with Ala, resulted in HURP instability and complex disassembly. In addition, HURP-wild-type stable transfectants were capable of growing in low-serum environments whereas HURP-4P grew poorly under low-serum conditions and failed to proliferate. These studies together support the view that the ability to integrate evidence derived from microarray studies into biochemical analyses may ultimately augment our predictive power when analyzing the potential role of poorly characterized proteins. While this combined approach was simply an initial attempt to answer a range of complex biological questions, our findings do suggest that HURP is a potential oncogenic target of Aurora-A.

Identification of trophinin as an enhancer for cell invasion and a prognostic factor for early stage lung cancer.

The explosion of microarray studies has promised to shed light on the identification of disease markers. To find novel prognostic factors, we used the expression profile of a poor prognostic factor of lung cancer, survivin (BIRC5), as a template to search for and compare transcriptome expression profiles in a lung adenocarcinoma microarray dataset. Trophinin (TRO) was identified as one of the best-correlated genes. The trophinin expression in lung cancer specimens was examined by immunohistochemical staining. The role of trophinin in cancer metastasis was further investigated by approaches of overexpression and knock down with small interfering RNA (siRNA). For stage I lung adenocarcinoma, the patients without trophinin expression had a better overall and disease-free survival. Overexpression of trophinin increases cell invasion ability and knock down with siRNA inhibits cell invasion. Through a combination of data mining and biochemical assays, we identified trophinin, which could enhance cell invasion, as a novel prognostic factor for early stage lung cancer.