Inhibiting Delta-6 Desaturase Activity Suppresses Tumor Growth in Mice

Recent studies have shown that a tumor-supportive microenvironment is characterized by high levels of pro-inflammatory and pro-angiogenic eicosanoids derived from omega-6 (n−6) arachidonic acid (AA). Although the metabolic pathways (COX, LOX, and P450) that generate these n−6 AA eicosanoids have been targeted, the role of endogenous AA production in tumorigenesis remains unexplored. Delta-6 desaturase (D6D) is the rate-limiting enzyme responsible for the synthesis of n−6 AA and increased D6D activity can lead to enhanced n−6 AA production. Here, we show that D6D activity is upregulated during melanoma and lung tumor growth and that suppressing D6D activity, either by RNAi knockdown or a specific D6D inhibitor, dramatically reduces tumor growth. Accordingly, the content of AA and AA-derived tumor-promoting metabolites is significantly decreased. Angiogenesis and inflammatory status are also reduced. These results identify D6D as a key factor for tumor growth and as a potential target for cancer therapy and prevention

Klinička primjena RT-PCR u otkrivanju DNK tuberkuloze u kombinaciji s TB-IGRA u dijagnostici plućne tuberkuloze s negativnim nalazom sputuma

The aim was to investigate detection of pulmonary alveolar lavage fluid tuberculosis DNA by real-time fluorescent polymerase chain reaction (RT-PCR) combined with clinical application of the sputum smear-negative pulmonary tuberculosis diagnosis with TB interferon-γ release assay (TB-IGRA). From October 2014 to October 2015, 632 outpatients and inpatients treated in our hospital were randomly selected, of which 459 patients as the research group managed with RT-PCR detection combined with TB-IGRA and 173 patients as the control group undergoing electronic bronchoscopy alveolar lavage fluid detection, with detection results statistically evaluated. The positive rate in the research group was 96.51%, i.e. significantly higher than that in the control group (66.47%), yielding a statistically significant difference (χ2=109.68, p=0.00). The true positive rate was 97.7% in the research group and 67.92% in the control group; the true positive rate was significantly higher in the research group patients as compared with the control group, yielding a statistically significant difference (χ2=112.04, p=0.00). The sensitivity and specificity, as well as Youden index were significantly higher in the research group as compared with the control group. In conclusion, TB DNA detection by RT-PCR combined with TB-IGRA is a very good method of diagnosing tuberculosis, and it can be implemented in clinical diagnosis of pulmonary tuberculosis.Cilj istraživanja bio je ispitati otkrivanje DNK tuberkuloze u plućnom alveolarnom ispirku pomoću lančane reakcije polimeraze u stvarnom vremenu s fluorescentnim bojama (RT-PCR) u kombinaciji s kliničkom primjenom dijagnostike plućne tuberkuloze s negativnim nalazom sputuma pomoću testa otpuštanja TB interferona-γ (TB-IGRA). Od listopada 2014. do listopada 2015. godine nasumce su odabrane 632 osobe bolnički i izvanbolnički liječene u našoj bolnici, od kojih je 459 uključeno u ispitnu skupinu podvrgnutu otkrivanju pomoću RT-PCR u kombinaciji s testom TB-IGRA, a 173 u kontrolnu skupinu podvrgnutu elektroničkom otkrivanju u bronhoskopskom alveolarnom ispirku; rezultati su statistički obrađeni. Stopa pozitivnih nalaza bila je 96,51% u ispitnoj skupini, odnosno značajno viša od one u kontrolnoj skupini (66,47%), uz statistički značajnu razliku (χ2=109,68; p=0,00). Stopa stvarno pozitivnih nalaza bila je 97,7% u ispitnoj skupini i 67,92% u kontrolnoj skupini, dakle značajno viša kod bolesnika ispitne skupine u usporedbi s kontrolnom skupinom, uz statistički značajnu razliku (χ2=112,04; p=0,00). Osjetljivost i specifičnost, kao i Youdenov indeks bili su značajno viši u ispitnoj skupini u usporedbi s kontrolnom skupinom. U zaključku, otkrivanje TB DNK pomoću RT-PCR u kombinaciji s TB-IGRA vrlo je dobra metoda za dijagnosticiranje tuberkuloze, koja se može primjenjivati u kliničkoj dijagnostici plućne tuberkuloze

Discovery of Coumarin as Microtubule Affinity-Regulating Kinase 4 Inhibitor That Sensitize Hepatocellular Carcinoma to Paclitaxel

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Nowadays, pharmacological therapy for HCC is in urgent needs. Paclitaxel is an effective drug against diverse solid tumors, but commonly resisted in HCC patients. We recently have disclosed that microtubule affinity-regulating kinase 4 (MARK4) increases the microtubule dynamics and confers paclitaxel resistance in HCC, suggesting MARK4 as an attractive target to overcome paclitaxel resistance. Herein, we synthesized and identified coumarin derivatives 50 as a novel MARK4 inhibitor. Biological evaluation indicated compound 50 directly interacted with MARK4 and inhibited its activity in vitro, suppressed cell viability and induced apoptosis of HCC cells in a MARK4-dependent manner. Importantly, compound 50 significantly increased the drug response of paclitaxel treatment to HCC cells, providing a promise strategy to HCC treatment and broadening the application of paclitaxel in cancer therapy

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ

Antitumor agents. 271: Total synthesis and evaluation of brazilein and analogs as anti-inflammatory and cytotoxic agents

The first total synthesis of the naturally occurring tetracyclic homoisoflavonoid brazilein (1) and 14 new analogs (1a–n) is reported. Target compounds and intermediates were assayed for anti-inflammatory effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB, and for cytotoxic activity against nasopharyngeal (KB), vincristine-resistant nasopharyngeal (KBvin), lung (A549) and prostate (DU-145) human cancer cell lines. The most active compound 1b showed potent effects on superoxide anion generation and elastase release with IC50 values of 1.2 and 1.9 µM, respectively, and was 65 times more potent than phenylmethylsulfonyl fluoride (PMSF), the positive control, in the latter assay. Additionally, 1b exhibited broad spectrum in vitro anticancer activity with IC50 values of 6–11 µM against the four tested cancer cell lines

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data