Prostate cancer is a kind of commonly diagnosed male malignancy. With the aging population in China, both incidence and mortality of prostate cancer are expected to keep increasing in the future. The methylation of RAR?2 gene promoter is a common molecular event in prostate cancer. Thus, we aimed at establishing a high-performance noninvasive DNA methylation assay based on pyrosequencing for screening of prostate cancer in this article. The assay is designed to detect aberrant promoter methylation of RAR?2 gene in ejaculate samples. The negative and positive control plasmids were constructed with different treatments by direct bisulfite conversion or conversion after Sss I Methylase methylation to establish quality control standard. The ejaculate and tissue samples were collected from patients with histologically confirmed adenocarcinoma of prostate (n = 43) and benign prostatic hyperplasia (n = 40). Significant correlation was observed between prostate cancer and methylation level of RAR?2 gene promoter. In addition, the results of pyrosequencing in ejaculate samples were compared with that of DNA sequencing in tissue samples from the same patients. There is no significant difference in the detection of RAR?2 promotor methylation between these two methods (p < 0.05). In conclusion, we have developed a high-performance noninvasive DNA methylation assay based on pyrosequencing which is more suitable for high-throughput detection of aberrant promoter methylation in ejaculate samples. Moreover, the acceptive degree of this noninvasive method makes it potentially promising for future screening of prostate cancer.
Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.
Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array-based discovery of prognostic serum miRNAs in melanoma. In the current study, we examined the clinical utility of these serum-based miRNAs for prognosis as well as detection of melanoma recurrence.
Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L(-1)) and TCP (0.01, 0.1, 0.5 mg L(-1)), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01-1 mg L(-1)) and TCP (0.01-0.5 mg L(-1)) are both highly toxic to zebrafish.
Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small-molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood-stage malaria. Most of the screening hits inhibited both liver- and blood-stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin-dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual-stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.
Periodontal ligament stem cells (PDLSCs) with bone morphogenic ability are used to treat diseases such as periodontitis. Their treatment potential is increased when used in combination with proteins that induce osteogenic differentiation. For example, bone morphogenetic protein-9 (BMP9) has been found to have potent osteogenic activity. In the present study, PDLSCs were isolated from human periodontal membrane and infected with recombinant adenoviruses expressing BMP9 (Ad-BMP9). Levels of osteogenic markers such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) as well as mineralization ability were measured. The results showed that BMP9 promoted bone formation of PDLSCs. In other experiments, SB203580 and PD98059, which are inhibitors of p38 and ERK1/2, respectively, were used to determine if these kinases are involved in the osteogenic differentiation process. The resulting protein expression profiles and osteogenic markers of PDLSCs revealed that the mitogen-activated protein kinase (MAPK) signaling pathway might play an important role in the process of BMP9-induced osteogenic differentiation of PDLSCs.
In this study, a genetically engineered Escherichia coli strain, HBUT-D (?pflB ?pta ?frdABCD ?adhE ?ald ?cscR), was initially evaluated on a laboratory scale (7 L) in a glucose (130 g L(-1)) mineral salts medium for d-lactic acid fermentation using 6N KOH, Ca(OH)2 or NH4OH as the neutralizing agent. Fermentations neutralized by Ca(OH) 2 achieved a volumetric productivity of 6.35 g L(-1) h(-1), tripling that achieved by KOH (1.71 g L(-1) h(-1)) and NH4OH (1.5 g L(-1) h(-1)). The facilitative effect of Ca(OH)2 neutralization was then demonstrated on a pilot scale (6 ton vessel, 130 kg glucose ton(-1)), resulting in a volumetric productivity of 6 kg ton(-1) h(-1), a titer of 126 kg ton(-1), a yield of 97%, and an optical purity of 99.5%. These results demonstrated that E. coli HBUT-D is a promising strain for large scale d-lactic acid fermentation using mineral salts medium and Ca(OH)2 for neutralization.
In the beginning of the 21st century, the deterioration of water quality in Taihu Lake, China, has caused widespread concern. The primary source of pollution in Taihu Lake is river inflows. Effective pollution load reduction scenarios need to be implemented in these rivers in order to improve the water quality of Taihu Lake. It is important to select appropriate pollution load reduction scenarios for achieving particular goals. The aim of this study was to facilitate the selection of appropriate scenarios. The QUAL2K model for river water quality was used to simulate the effects of a range of pollution load reduction scenarios in the Wujin River, which is one of the major inflow rivers of Taihu Lake. The model was calibrated for the year 2010 and validated for the year 2011. Various pollution load reduction scenarios were assessed using an analytic hierarchy process, and increasing rates of evaluation indicators were predicted using the Delphi method. The results showed that control of pollution from the source is the optimal method for pollution prevention and control, and the method of "Treatment after Pollution" has bad environmental, social and ecological effects. The method applied in this study can assist for environmental managers to select suitable pollution load reduction scenarios for achieving various objectives.
Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer (CRC) and to investigate their anti-metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH-306 significantly inhibited the migration and invasion of CRC cells in a dose-dependent manner. In addition, YH-306 inhibited cell adhesion and protrusion formation of HCT116 and HT-29 CRC cells. Moreover, YH-306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH-306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH-306 suppressed the activation of focal adhesion kinase (FAK), c-Src, paxillin, and phosphatidylinositol 3-kinases (PI3K), Rac1 and the expression of matrix metalloproteases (MMP) 2 and MMP9. Meanwhile, YH-306 also inhibited actin-related protein (Arp2/3) complex-mediated actin polymerization. Taken together, YH-306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway.
Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.
Ionic liquids (ILs) have a great reputation due to their negligible volatility, designability, good stability, and ability to be recycled. They are considered to be "green" solvents and have great promise in many fields. In recent years, the toxicities of ILs have garnered increasing attention as reported by a number of studies. However, previous studies have primarily focused on their lethal toxicities, and data were limited on their toxic effects at nonlethal doses. We performed a study on the toxic effects of 1-octyl-3-methylimidazolium bromide ([Omim]Br) on zebrafish. During a 28-day period, male and female zebrafish were separately exposed to sequential concentrations (0, 5, 10, 20, and 40 mg/L) of [Omim]Br. Fishes were sampled after 7, 14, 21, and 28 days of exposure, and reactive oxygen species (ROS) levels, activities of antioxidant enzymes (superoxide dismutase and catalase), lipid peroxidation (LPO), and DNA damage in fish livers were measured. ROS, LPO, and DNA damage were all induced by the ionic liquid, and antioxidant enzyme activities increased at the beginning and then decreased. These phenomena demonstrate that [Omim]Br can induce oxidative stress and DNA damage in zebrafish.
Patients with triple-negative breast cancer (TNBC) have a high incidence of early relapse and metastasis; however, the molecular basis for recurrence in these individuals remains poorly understood. Here, we demonstrate that RASAL2, which encodes a RAS-GTPase-activating protein (RAS-GAP), is a functional target of anti-invasive microRNA-203 and is overexpressed in a subset of triple-negative or estrogen receptor-negative (ER-negative) breast tumors. As opposed to luminal B ER-positive breast cancers, in which RASAL2 has been shown to act as a RAS-GAP tumor suppressor, we found that RASAL2 is oncogenic in TNBC and drives mesenchymal invasion and metastasis. Moreover, high RASAL2 expression was predictive of poor disease outcomes in patients with TNBC. RASAL2 acted independently of its RAS-GAP catalytic activity in TNBC; however, RASAL2 promoted small GTPase RAC1 signaling, which promotes mesenchymal invasion, through binding and antagonizing the RAC1-GAP protein ARHGAP24. Together, these results indicate that activation of a RASAL2/ARHGAP24/RAC1 module contributes to TNBC tumorigenesis and identify a context-dependent role of RASAL2 in breast cancer.
Ikaros, the product of IKZF1, is a regulator of lymphoid development and polymorphisms in the gene have been associated with the acute lymphoblastic leukemia (ALL). Additionally, IKZF1 deletions and mutations identify high-risk biological subsets of childhood ALL [Georgopoulos et al. Cell 1995;83(2):289-299; Mullighan et al. N Engl J Md 2009;360(5):470-480].
BRAF mutations are frequent in cutaneous melanomas, and BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients. However, acquired drug resistance can occur rapidly and tumor(s) often progresses thereafter. Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial. In this study, we developed BRAFi-resistant melanoma cell lines and found that metastasis-related epithelial to mesenchymal transition properties of BRAFi-resistant cells were enhanced significantly. Upregulation of EGFR was observed in BRAFi-resistant cell lines and patient tumors because of demethylation of EGFR regulatory DNA elements. EGFR induced PI3K/AKT pathway activation in BRAFi-resistant cells through epigenetic regulation. Treatment of EGFR inhibitor was effective in BRAFi-resistant melanoma cell lines. The study demonstrates that EGFR epigenetic activation has important implications in BRAFi resistance in melanoma.Journal of Investigative Dermatology advance online publication, 30 October 2014; doi:10.1038/jid.2014.418.
Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases.
Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPAR?2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.
Azoxystrobin has been widely used in recent years. The present study investigated the oxidative stress and DNA damage effects of azoxystrobin on earthworms (Eisenia fetida). Earthworms were exposed to different azoxystrobin concentrations in an artificial soil (0, 0.1, 1, and 10mg/kg) and sampled on days 7, 14, 21, and 28. Superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione-S-transferase (GST), reactive oxygen species (ROS), and malondialdehyde (MDA) content were measured by an ultraviolet spectrophotometer to determine the antioxidant responses and lipid peroxidation. Single cell gel electrophoresis (SCGE) was used to detect DNA damage in the coelomocytes. Compared with these in the controls, earthworms exposed to azoxystrobin had excess ROS accumulation and greater SOD, POD, and GST activity while the opposite trend occurred for CAT activity. MDA content increased after 14-day exposure, and DNA damage was enhanced with an increase in the concentration of azoxystrobin. In conclusion, azoxystrobin caused oxidative stress leading to lipid peroxidation and DNA damage in earthworms.
BTK is a member of the TEC family of non-receptor tyrosine kinases whose deregulation has been implicated in a variety of B-cell-related diseases. We have used structure-based drug design in conjunction with kinome profiling and cellular assays to develop a potent, selective, and irreversible BTK kinase inhibitor, QL47, which covalently modifies Cys481. QL47 inhibits BTK kinase activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of a downstream effector PLC?2 (Tyr759) with an EC50 of 318 nM. In Ramos cells QL47 induces a G1 cell cycle arrest that is associated with pronounced degradation of BTK protein. QL47 inhibits the proliferation of B-cell lymphoma cancer cell lines at submicromolar concentrations.
The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser(445). We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser(445) is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
A new cyclic tetrapeptide, cyclo-(L-leucyl-trans-4-hydroxy-L-prolyl-D-leucyl-trans-4-hydroxy-L-proline) (1), was isolated from the co-culture broth of two mangrove fungi Phomopsis sp. K38 and Alternaria sp. E33. The structure of 1 was determined by analysis of spectroscopic data and Marfey's analytic method. Primary bioassay demonstrated that compound 1 exhibited moderate to high inhibitory activity against four crop-threatening fungi including Gaeumannomyces graminis, Rhizoctonia cerealis, Helminthosporium sativum and Fusarium graminearum as compared with triadimefon.
Myoporum bontioides A. Gray, an evergreen shrub from the Myoporaceae family, is a commonly used medicinal plant. Many studies have been conducted on the biologically active constituents of whole parts of M. bontioides. However, the endophytes of M. bontioides have not been intensively investigated. A new chlorine-containing isocoumarin, named dichlorodiaportinol A (1) was isolated from the endophytic fungus Trichoderma sp. 09 isolated from the root of M. bontioides. Its cytotoxic activity against human breast cancer (MCF-7) and human liver cancer (HepG2) cell lines was evaluated.
A high-throughput method for the simultaneous determination of 26 mycotoxins in sesame butter was developed by coupling the modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method with ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS). The samples were sequentially extracted using 20 mL (80:20, v/v) and 5 mL (20:80, v/v) acetonitrile aqueous solutions, followed by salting out by the addition of magnesium sulfate and sodium chloride. Finally, the samples were purified using hexane and dispersed C18 solid phase extraction (dSPE). The mycotoxins were further separated using a C18 column and detected by electrospray ionization (ESI) in the multiple reactions monitoring (MRM) mode. Using this detection technique, 16 mycotoxins were detected as positive ions using methanol and water containing 0.1% formic acid as the mobile phase, whereas the other 10 mycotoxins were detected as negative ions using methanol and water as the mobile phase. With the matrix-matched quantification calibration, the developed method showed a good linear dynamic range with regression coefficients of 0.995 or higher. This method allowed for the detection of the 26 mycotoxins at LOQs significantly lower than the available maximum residue levels currently regulated by EU regulations. Additionally, at the three spiking levels examined, the majority of recoveries were within 60-120%, with RSDs within 15%. The method developed herein has the advantages of high sensitivity, accuracy and throughput, and it can be applied to the target screening of mycotoxins in real samples.
As the main optical isomer of metalaxyl, metalaxyl-M has been widely used worldwide in recent years because of its notable effect on the prevention and control of crop diseases. Together with the toxicity and degradation of metalaxyl-M, the chemical has attracted the attention of researchers. The present study examined the toxic effects of metalaxyl-M on earthworms at 0?mg?kg(-1) , 0.1?mg?kg(-1) , 1?mg?kg(-1) , and 3?mg?kg(-1) on days 7, 14, 21 and 28 after exposure. The results showed that metalaxyl-M could cause an obvious increase in the production of reactive oxygen species (ROS) when the concentration was higher than 0.1?mg?kg(-1) , which led to lipid peroxidation in earthworms. Metalaxyl-M can induce DNA damage in earthworms, and the level of DNA damage markedly increased with increasing the concentration of metalaxyl-M. Metalaxyl-M also has a serious influence on the activities of antioxidant enzymes, which results in irreversible oxidative damage in cells. The changes of these indicators all indicated that metalaxyl-M may cause cytotoxic and genotoxic effects on earthworms.
We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.
Many biological experiments are not compatible with the use of immunofluorescence, genetically encoded fluorescent tags, or FRET-based reporters. Conjugation of existing kinase inhibitors to cell-permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well-established dual Src-Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib but that dasatinib-BODIPY largely retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry. It can also be used to track the localization of Src by fixed and live-cell fluorescence microscopy. This strategy could enable generation of additional kinase-specific probes useful in systems not amenable to genetic manipulation or could be used together with fluorescent proteins to enable a multiplexed assay readout.
Three new resveratrol derivatives, namely, resveratrodehydes A-C (1-3), were isolated from the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS, 1D and 2D NMR spectroscopic data. All compounds showed broad-spectrum inhibitory activities against three human cancer cell lines including human breast MDA-MB-435, human liver HepG2, and human colon HCT-116 by MTT assay (IC50 < 50 ?M). Among them, compounds 1 and 2 both exhibited marked cytotoxic activities against MDA-MB-435 and HCT-116 cell lines (IC?? < 10 ?M). Additionally, compounds 1 and 3 showed moderate antioxidant activity by DPPH radical scavenging assay.
Herlyn-Werner-Wunderlich syndrome (HWWS) is a müllerian duct anomaly typically associated with a uterus didelphys with two cervices and two vaginas, one of which is obstructed. A remarkable case of HWWS with contralateral duplex kidneys and duplication of ureters is described, which, to our knowledge, is a rarely reported variant to date. For this congenital anomaly, a strong suspicion and knowledge of HWWS are essential for a precise diagnosis.
Prostatectomy or irradiation is the most common traditional treatments for localized prostate cancer. In the event of recurrence and/or metastasis, androgen ablation therapy has been the mainstay treatment for many years. Although initially effective, the cancer inevitably recurs as androgen-independent PCa, a disease with limited effective treatments. Enhanced predictive biomarkers are needed at the time of diagnosis to better tailor therapies for patients. MicroRNAs are short nucleotide sequences which can complementary bind to and control gene expression at the post-transcriptional level. Recent studies have demonstrated that many miRNAs are variably expressed in cancers vs. normal tissues, including PCa. In this review, we summarize PCa-specific miRNAs that show potential for their utilization as identifiers of aggressive disease and predictors for risk of recurrence. Additionally, we discuss their potential clinical applications as biomarkers and therapeutic targets.
Atrazine is a widely used herbicide with great environmental concern due to its high potential to contaminate soil and waters. An atrazine-degrading bacterial strain HB-6 was isolated from industrial wastewater and the 16S rRNA gene sequencing identified HB-6 as a Bacillus subtilis. PCR assays indicated that HB-6 contained atrazine-degrading genes trzN, atzB and atzC. The strain HB-6 was capable of utilizing atrazine and cyanuric acid as a sole nitrogen source for growth and even cleaved the s-triazine ring and mineralized atrazine. The strain demonstrated a very high efficiency of atrazine biodegradation with a broad optimum pH and temperature ranges and could be enhanced by cooperating with other bacteria, suggesting its huge potential for remediation of atrazine-contaminated sites. To our knowledge, there are few Bacillus subtilis strains reported that can mineralize atrazine, therefore, the present work might provide some new insights on atrazine remediation.
Global microRNA (miRNA) profile may predict prostate cancer (PCa) behaviors. In this study, we examined global miRNA expression by miRNA profiling as well as specific miRNA expression levels in PCa epithelium and stroma by in situ hybridization (ISH) and correlated with various clinicopathological features. We first performed comprehensive miRNA profiling on 27 macrodissected cases of PCa by miRNA microarray. A total of 299 miRNAs were significantly dysregulated in high grade and advanced stage PCa. We demonstrated that PCa can be readily classified into high grade/stage and low-grade/stage groups by its global miRNA expression profile. Next, we examined the expression of several selected dysregulated miRNAs, including let-7c, miR-21, miR-27a, miR-30c, and miR-219, in PCa by ISH. The levels of miRNA expression in epithelial and stromal cells were scored semiquantitatively and compared with clinicopathological features, including age, race, Gleason score, stage, PSA recurrence, metastasis, hormone resistance and survival. We found that the expression of miR-30c and miR-219 were significantly down-regulated in PCa. miR-21 and miR-30c were significantly down-regulated in PCa in African Americans compared to Caucasian Americans. In addition, down-regulation of let-7c, miR-21, miR-30c, and miR-219 are associated with metastatic disease. Furthermore, down-regulation of miR-30c and let-7c are significantly associated with androgen-dependent PCa. In PCa stromal cells, let-7c downregulation is significantly associated with extraprostatic extension. Our data suggest that selected miRNAs may serve as potential biomarkers to predict cancer progression.
Transcriptional intermediary factor 1 gamma (Tif1?) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGF? pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1? interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1? inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1? and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1? resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1? resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1? reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1? results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1? may inhibit tumor invasion. These data suggest that Tif1? might serve as a potential therapeutic target for pancreatic cancer.
Recently there has been an increased interest in the role of tumor-associated stroma in prostate tumorigenesis, but little is known about the respective roles of stomal ER? and ER? in prostate cancer (PCa). This study characterizes the expression patterns of ER? and ER? in tumor-associated stroma in association with various clinicopathological factors of importance in PCa prognosis and treatment.
BackgroundNuclear factor IA (NFIA), a transcription factor and essential regulator in embryonic glial development, is highly expressed in human glioblastoma (GBM) compared with normal brain, but its contribution to GBM and cancer pathogenesis is unknown. Here we demonstrate a novel role for NFIA in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions.MethodsTo determine the role of NFIA in glioma, we examined the effects of NFIA in growth, proliferation, apoptosis, and migration. We used gain-of-function (overexpression) and loss-of-function (shRNA knockdown) of NFIA in primary patient-derived GBM cells and established glioma cell lines in culture and in intracranial xenografts in mouse brains.ResultsKnockdown of native NFIA blocked tumor growth and induced cell death and apoptosis. Complementing this, NFIA overexpression accelerated growth, proliferation, and migration of GBM in cell culture and in mouse brains. These NFIA tumor-promoting effects were mediated via transcriptional repression of p53, p21, and plasminogen activator inhibitor 1 (PAI1) through specific NFIA-recognition sequences in their promoters. Importantly, the effects of NFIA on proliferation and apoptosis were independent of TP53 mutation status, a finding especially relevant for GBM, in which TP53 is frequently mutated.ConclusionNFIA is a modulator of GBM growth and migration, and functions by distinct regulation of critical oncogenic pathways that govern the malignant behavior of GBM.
To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (deltapflB, deltafrdABCD, deltaackA, deltaldhA), resulting in loss of glucose effect in the mutant SZ470P (deltaptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25 g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production.
Fomesafen is a diphenyl ether herbicide that has an important role in the removal of broadleaf weeds in bean and fruit tree fields. However, very little information is known about the effects of this herbicide on soil microbial community structure and activities. In the present study, laboratory experiments were conducted to examine the effects of different concentrations of fomesafen (0, 10, 100, and 500 ?g/kg) on microbial community structure and activities during an exposure period of 60 days, using soil enzyme assays, plate counting, and denaturing gradient gel electrophoresis (DGGE). The results of enzymatic activity experiments showed that fomesafen had different stimulating effects on the activities of acid phosphatase, alkaline phosphatase, and dehydrogenase, with dehydrogenase being most sensitive to fomesafen. On the tenth day, urease activity was inhibited significantly after treatment of different concentrations of fomesafen; this inhibiting effect then gradually disappeared and returned to the control level after 30 days. Plate counting experiments indicated that the number of bacteria and actinomycetes increased in fomesafen-spiked soil relative to the control after 30 days of incubation, while fungal number decreased significantly after only 10 days. The DGGE results revealed that the bacterial community varied in response to the addition of fomesafen, and the intensity of these six bands was greater on day 10. Sequencing and phylogenetic analyses indicated that the six excised DGGE bands were closely related to Emticicia, Bacillus, and uncultured bacteria. After 10 days, the bacterial community exhibited no obvious change compared with the control. Throughout the experiment, we concluded that 0-500 ?g/kg of fomesafen could not produce significant toxic effects on soil microbial community structure and activities.
We describe a droplet-based microfluidic electrochemical sensor using platinum-black (Pt-black) microelectrode. Pt-black microelectrode was generated by electrodeposition of Pt nanoparticles on bare Pt microelectrode. Scanning electron microscope (SEM) image displays a flower-like microstructure of Pt nanoparticels. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) indicate that the Pt-black efficiently decreased the charge transfer resistance and improved the electrocatalytic activity towards oxidation of hydrogen peroxide (H2O2). Compared with bare Pt microelectrode, the current response on Pt-black microelectrode increased 10.2 folds. The effect of applied potential and electrodeposition time has been investigated in detail. The proposed sensor was validated by performing enzyme activity assay in flowing droplets. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of ?-d-glucose to the product H2O2. The enzyme activity of GOx was evaluated by measuring the electrochemical current responding to various glucose concentrations. And the results indicate that this microfluidic sensor holds great potential in fabricating novel glucose sensors with linear response up to 43.5mM. The analytical applications of the droplet-based microfluidic sensor were tested by using human blood serum samples. Reproducibility, interferences, and long-term stability of the modi?ed electrode were also investigated. The present approach shows the feasibility and great potentials in constructing highly sensitive and low-consumption sensors in the field of droplet microfluidics.
Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of <1 year. Alteration in DNA methylation is a major hallmark of tumor progression and metastasis; however, it remains largely unexplored in MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.
MicroRNAs (miRNAs) play important roles in regulating gene expression of plants, animals and viruses. Comprehensive characterization of host and viral miRNA will help uncover the molecular mechanisms that underlie the progression of human cytomegalovirus (HCMV) latent infection. To investigate the miRNA expression profile of HCMV and host cells during latent infection, we performed deep-sequencing analysis of the small RNAs isolated from HCMV-infected and mock-infected human monocytic leukemia cell line, THP-1.
The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the DFG-out conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.
Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest.
Ionic liquids (ILs) are called "green" solvents, which are due to their unique physicochemical properties and potential applications in various areas. However, the toxicity of ILs has attracted increasing attention from scientific researchers. The present paper studied the toxic effects of 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim]PF6) on wheat seedlings at 0, 1, 2, 4, 6, and 8 mg l(-1) on days 7, 10 and 13. The present results showed that the growth of wheat seedlings was seriously inhibited when the concentration was higher than 2 mg l(-1) and the inhibitory effect enhanced with increasing concentration and time. The EC50 values for germination, shoot length, root length and dry weight were 11.104, 5.187, 4.380 and 6.292 mg l(-1), respectively. [C8mim]PF6 could cause an increase in the production of ROS, which led to the oxidative damage and lipid peroxidation. Furthermore, these toxic effects on wheat seedlings were irreversible.
The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure-activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 ?M and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 ?M. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.
In this study, we constructed a recombinant Escherichia coli strain for the production of high-purity L-lactic acid, using a homoethanol fermenting mutant E. coli SZ470 (deltafrdBC deltaldhA deltaackA deltafocA-pflB deltapdhR: :pflBp6-pflBrbs-aceEF-lpd) as the starting strain.
The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.
Follicular helper T (Tfh) cells are dedicated to providing help to B cells and are strongly associated with antibody-mediated autoimmune disease. B cell lymphoma 6 (Bcl-6) is a key transcription factor of Tfh cells, and IL-21 is known to be a critical cytokine produced by Tfh cells. We silenced Bcl-6 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of Bcl-6 short hairpin RNAs (shRNAs) in experimental autoimmune myasthenia gravis (EAMG). Our data demonstrate that CD4(+)CXCR5(+)PD-1(+) Tfh cells, Bcl-6 and IL-21 were significantly increased in EAMG mice, compared with controls. In addition, we found that frequencies of Tfh cells were positively correlated with the levels of serum anti-AChR Ab. In-vivo transduction of lenti-siRNA-Bcl6 ameliorates the severity of ongoing EAMG with decreased Tfh cells, Bcl-6 and IL-21 expression, and leads to decreased anti-AChR antibody levels. Furthermore, we found that siRNA knockdown of Bcl-6 expression increases the expression of Th1(IFN-?, T-bet) and Th2 markers (IL-4 and GATA3), but failed to alter the expression of Th17-related markers (ROR?t, IL-17) and Treg markers (FoxP3). Our study suggests that Tfh cells contribute to the antibody production and could be one of the most important T cell subsets responsible for development and progression of EAMG or MG. Bcl-6 provides a promising therapeutic target for immunotherapy not only for MG, but also for other antibody-mediated autoimmune diseases.
Efforts to improve the clinical outcome of highly aggressive triple-negative breast cancer (TNBC) have been hindered by the lack of effective targeted therapies. Thus, it is important to identify the specific gene targets/pathways driving the invasive phenotype to develop more effective therapeutics. Here we show that ubiquitin-associated and SH3 domain-containing B (UBASH3B), a protein tyrosine phosphatase, is overexpressed in TNBC, where it supports malignant growth, invasion, and metastasis largely through modulating epidermal growth factor receptor (EGFR). We also show that UBASH3B is a functional target of anti-invasive microRNA200a (miR200a) that is down-regulated in TNBC. Importantly, the oncogenic potential of UBASH3B is dependent on its tyrosine phosphatase activity, which targets CBL ubiquitin ligase for dephosphorylation and inactivation, leading to EGFR up-regulation. Thus, UBASH3B may function as a crucial node in bridging multiple invasion-promoting pathways, thereby providing a potential therapeutic target for TNBC.
Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9-induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9-induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre-osteoblast C2C12 cells. BMP9-induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7-induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9-induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2-induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9-induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9s potent osteogenic capability in MSCs.
Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.
The D-lactic acid producing strain, Escherichia coli HBUT-D, was reengineered for L(+)-lactic acid fermentation by replacing the D-lactate dehydrogenase gene (ldhA) with an L(+)-lactate dehydrogenase gene (ldhL) from Pedicoccus acidilactici, followed by adaptive evolution in sucrose. The resulting strain, WYZ-L, has enhanced expression of the sucrose operon (cscA and cscKB). In 100 g L(-1) of sucrose fermentation using mineral salt medium, WYZ-L produced 97 g L(-1) of l(+)-lactic acid, with a yield of 90%, a maximum productivity of 3.17 g L(-1)h(-1) and an optical purity of greater than 99%. In fermentations using sugarcane molasses and corn steep liquor without additional nutrients, WYZ-L produced 75 g L(-1) of l(+)-lactic acid, with a yield of 85%, a maximum productivity of 1.18 g L(-1)h(-1), and greater than 99% optical purity. These results demonstrated that WYZ-L has the potential to use waste molasses and corn steep liquor as a resource for L(+)-lactic acid fermentation.
BMX is a member of the TEC family of nonreceptor tyrosine kinases. We have used structure-based drug design in conjunction with kinome profiling to develop a potent, selective, and irreversible BMX kinase inhibitor, BMX-IN-1, which covalently modifies Cys496. BMX-IN-1 inhibits the proliferation of Tel-BMX-transformed Ba/F3 cells at two digit nanomolar concentrations but requires single digit micromolar concentrations to inhibit the proliferation of prostate cancer cell lines. Using a combinatorial kinase inhibitor screening strategy, we discovered that the allosteric Akt inhibitor, MK2206, is able to potentiate BMX inhibitors antiproliferation efficacy against prostate cancer cells.
Alcaligenes faecalis strain JBW4, a strain of bacteria that is capable of degrading endosulfan, was inoculated into sterilized and natural soils spiked with endosulfan. JBW4 degraded 75.8 and 87.0 % of ?-endosulfan and 58.5 and 69.5 % of ?-endosulfan in sterilized and natural soils, respectively, after 77 days. Endosulfan ether and endosulfan lactone were the major metabolites that were detected by gas chromatography-mass spectrometry. This result suggested that A. faecalis strain JBW4 degrades endosulfan using a non-oxidative pathway in soils. The ability of strain JBW4 to colonize endosulfan-contaminated soils was confirmed by polymerase chain reaction-denaturing gradient gel electrophoresis. This result suggested that strain JBW4 competed with the original inhabitants in the soil to establish a balance and successfully colonize the soils. In addition, the detoxification of endosulfan by strain JBW4 was evaluated using single-cell gel electrophoresis and by determining the soil microbial biomass carbon and enzymatic activities. The results showed that the genotoxicity and ecotoxicity of endosulfan in soil were reduced after degradation. The natural degradation of endosulfan in soil is inadequate; therefore, JBW4 shows potential for the bioremediation of industrial soils that are contaminated with endosulfan residues.
Emerging evidence demonstrates that miRNAs, a new family of key mRNA regulatory molecules, have crucial roles in controlling and modulating immunity. Their contribution to myasthenia gravis (MG), a T cell-dependent, antibody-mediated nervous system autoimmune disease, has not been thoroughly investigated. In the present study, using a highly sensitive microarray-based approach, we identified 11 miRNAs with differential expression between Peripheral Blood Mononuclear Cells (PBMC) from experimental autoimmune MG (EAMG) rats and control rats. miR-145 is one of the most significantly down-regulated miRNAs in PBMC from EAMG rats. Down-regulation of miR-145 expression was confirmed in PBMC and CD4+CD25- T cells (T effector cells) from both EAMG rats and MG patients by real-time PCR. Bioinformatics target prediction identified two crucial immune-related molecules-CD28 and NFATc1, as putative targets of miR-145. Furthermore, miR-145 inhibited CD28 and NFATc1 expression by directly targeting their 3-UTRs, which was abolished by mutation of the miR-145 and CD28/NFATc1 binding sites. In vitro up-regulation of miR-145 in CD4+ T cells can significantly reduce CD28 protein levels accompanied by decreased proliferative response. In a dendritic cell (DC)-T cell coculture system, overexpression of miR-145 in AChR-specific CD4+ T cells suppresses NFATc1 expression and T Helper 17 cells level. Finally, we observed that administration of lentiviral-miR-145 decreased the severity of ongoing, established EAMG with decreased IL-17 production, and also decreased serum anti-AChR IgG levels. Our studies provide an important new insight into the pathogenesis of EAMG and MG, which may open a new perspective for the development of effective gene therapy for EAMG/MG.
The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3?, has also been reported to stimulate osteogenesis by stabilizing ? catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.
Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10?gL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.
Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser-213 (AR-Ser[P]-213) and Ser-650 (AR-Ser[P]-650), in breast cancer and evaluated their association with clinicopathological parameters.
Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose --> 2 acetyl-CoA + 4 NADH --> 2 ethanol) in the engineered strain, Escherichia coli SZ420 (?frdBC ?ldhA ?ackA ?focA-pflB ?pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (YRPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual YRPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.
B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility were shown by reverse transcription and real-time PCR and immunohistochemistry analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double-blind study. B7-H3 messenger RNA expression was shown to be significantly increased with stage of melanoma (P<0.0001) and significantly associated with melanoma-specific survival in both stage III (P<0.0001) and stage IV (P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression of B7-H3 increased migration and invasion, whereas knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to phosphorylated signal transducer and activator of transcription-3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression and events of metastasis.
Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.
Simultaneous determination of bisphenol A, tetrabromobisphenol A, and perfluorooctanoic acid in small household electronics appliances by accelerated solvent extraction-ultra-performance liquid chromatography-tandem mass spectrometry was established. Samples, heated for 5 min, were extracted by toluene/methanol (10:1, v/v) under the pressure 1500 psi at 100°C, and were extracted 3 static cycles with 20 min per cycle. And then 15 mL extractant solvent was used to wash the samples, and at last the sample was purged by nitrogen for 100 s. The partial extractant (10 mL) was concentrated by nitrogen and re-dissolved with 1 mL methanol/water (1:1, v/v). The three compounds were separated by BEH C18 column effectively in 3 min and detected by electrospray ionization mode mass spectrometry. The linear ranges for bisphenol A, perfluorooctanoic acid, and tetrabromobisphenol A were 1-100, 10-1000 ng/mL, and 0.1-10 ?g/mL, respectively. The correlation coefficient was greater than 0.996. The LOD and limit of quantitation for three compounds were 0.1, 10, 1 ng/mL, and 0.5, 50, 5 ng/mL, respectively. And the recoveries were 84-92, 76-82, and 72-74%, respectively, with RSD < 5%. The method was successfully used in determining the real samples. The method and the result were confirmed by liquid chromatography-ion trap-time of flight mass spectrometry.
Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing "white pollution". However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass.
Activation of signal transducers and activators of transcription (STAT) proteins may be critical to their oncogenic functions as demonstrated by the development of B-cell lymphoma/leukemia in transgenic (TG) mice overexpressing a constitutively activated form of Stat5b. However, low incidence of CD8(+) T cell lymphoma was observed in B6 transgenic mice overexpressing a wild-type Stat5b (B6.Stat5b(Tg)) despite of undetectable Stat5b phosphorylation and the rate of lymphomagenesis was markedly enhanced by immunization or the introduction of TCR transgenes . Here, we report that the wild-type Stat5b transgene leads to the acceleration and high incidence (74%) of CD8(+) T cell lymphoblastic lymphomas in the non-obese-diabetic (NOD) background. In contrast to the B6.Stat5b(Tg) mice, Stat5b in transgenic NOD (NOD.Stat5b(Tg)) mice is selectively and progressively phosphorylated in CD8(+) thymocytes. Stat5 phosphorylation also leads to up-regulation of many genes putatively relevant to tumorigenesis. Treatment of NOD.Stat5b(Tg) mice with cancer chemopreventive agents Apigenin and Xanthohumol efficiently blocked lymphomagenesis through reduction of Stat5 phosphorylation and genes up-regulated in the NOD.Stat5b(Tg) mice. These results suggest that NOD genetic background is critical to the Stat5b-mediated lymphomagenesis through regulation of Stat5 hyperactivation. NOD.Stat5b(Tg) mouse is an excellent model for studying the molecular mechanisms underlying lymphomagenesis and testing novel chemoprevention strategies.
Ionic liquids were recently found to be toxic to aquatic organisms. Therefore, the present study investigated the effects of 1-decyl-3-methylimidazolium bromide ([C10mim]Br) on oxidative stress and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to five concentrations of [C10mim]Br (0, 5, 10, 20, and 40 mg L(-1)) and were sampled on days 7, 14, 21 and 28. Compared to control groups, the activities of antioxidant enzymes were significantly decreased at most exposure intervals. This decreased activity resulted in the production of excess reactive oxygen species (ROS) and increased malondialdehyde (MDA) content in zebrafish liver. Additionally, it was noteworthy that a clear dose-response was found for DNA damage. As for sex differences, significant differences in catalase (CAT) and ROS were found on the 7th day. In conclusion, the exposure of [C10mim]Br caused DNA damage, leading to antioxidant responses in zebrafish livers.
Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/?-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ER? in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ER?-mediated synergy can be effectively blocked by ER? antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ER? significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on ?-catenin/Tcf reporter activity. However, ER? expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ER? expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ER? expression and down-regulating ER? expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.
Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer.
The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor ? (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
Progenitor cell-based cardiomyocyte regeneration holds great promise of repairing an injured heart. Although cardiomyogenic differentiation has been reported for a variety of progenitor cell types, the biological factors that regulate effective cardiomyogenesis remain largely undefined. Primary cardiomyogenic progenitors (CPs) have a limited life span in culture, hampering the CPs in vitro and in vivo studies. The objective of this study is to investigate if primary CPs isolated from fetal mouse heart can be reversibly immortalized with SV40 large T and maintain long-term cell proliferation without compromising cardiomyogenic differentiation potential.
Bone morphogenetic proteins (BMPs) are members of the TGF-? superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
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