Inhibition of Janus Kinase 1 synergizes docetaxel sensitivity in prostate cancer cells

Prostate cancer (PCa) is the second most common malignancy and is the fifth leading cause of cancer mortality among men globally. Docetaxel-based therapy remains the first-line treatment for metastatic castration-resistant prostate cancer. However, dose-limiting toxicity including neutropenia, myelosuppression and neurotoxicity is the major reason for docetaxel dose reductions and fewer cycles administered, despite a recent study showing a clear survival benefit with increased total number of docetaxel cycles in PCa patients. Although previous studies have attempted to improve the efficacy and reduce docetaxel toxicity through drug combination, no drug has yet demonstrated improved overall survival in clinical trial, highlighting the challenges of improving the activity of docetaxel monotherapy in PCa. Herein, we identified 15 lethality hits for which inhibition could enhance docetaxel sensitivity in PCa cells via a high-throughput kinome-wide loss-of-function screen. Further drug-gene interactions analyses identified Janus kinase 1 (JAK1) as a viable druggable target with existing experimental inhibitors and FDA-approved drugs. We demonstrated that depletion of endogenous JAK1 enhanced docetaxel-induced apoptosis in PCa cells. Furthermore, inhibition of JAK1/2 by baricitinib and ruxolitinib synergizes docetaxel sensitivity in both androgen receptor (AR)–negative DU145 and PC3 cells, but not in the AR-positive LNCaP cells. In contrast, no synergistic effects were observed in cells treated with JAK2-specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.     

Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.     

Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.     

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.     

Direct electrochemistry of myoglobin based on ionic liquid-clay composite films

The direct electron transfer of myoglobin (Mb) was realized by immobilizing Mb onto ionic liquid (1-butyl-3-methyl imidazolium tetrafluoraborate, [bmim][BF(4)])-clay composite film modified glassy carbon electrode. A pair of well-defined redox peaks of Mb with a formal potential (E(o)') of -0.297 V (vs. Ag/AgCl) was observed in 0.1M phosphate buffer solution (pH 6.0). The ionic liquid-clay composite film showed good biocompatibility and an obvious promotion capability for the direct electron transfer between Mb and electrode. The electron transfer rate constant (k(s)) of Mb was calculated to be (3.58+/-0.12)s(-1). UV-vis spectrum suggested that Mb retained its native conformation in the ionic liquid-clay system. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process, in ionic liquid and clay during the drying process of the modification, and the ionic liquid played the key role for promotion of the direct electron transfer between Mb and the ionic liquid-clay composite film modified electrode. The biocatalytic activity of Mb in the composite film was exemplified by the reduction of hydrogen peroxide. Under the optimal conditions, the reduction peak currents of Mb increased linearly with the concentration of H(2)O(2) in the range of 3.90 x 10(-6) to 2.59 x 10(-4)M, with a detection limit of 7.33 x 10(-7)M. The kinetic parameter I(max) and the apparent Michaelis constant (K(m)) for the electrocatalytic reactions were 3.87 x 10(-8)A and 17.6 microM, respectively. The proposed method would be valuable for the construction of a new third-generation H(2)O(2) sensor.     

Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection

Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.     

Pectinases in Aqueous Extracts of Ditylenchus dipsaci

Aqueous extracts of a population of Ditylenchus dipsaci isolated from onion and maintained monoxenically on onion callus contained endo-polygalacturonase (endo-PG) and endo-pectinmethyltranseliminase (endo-PMTE). In viscometric tests pH 4.2 and 4.0 were optimal for degradation of sodium polypectate and pectin N.F., respectively, by endo-PG. Endo-PMTE reduced viscosity of pectin N.F. optimally at pH 8.5 or above. Activity was dependent on CaCl₂. Pectinmethylesterase activity was not detected in water, NaCl, or sucrose extracts of these nematodes. The extracts macerated potato tuber tissue, onion cotyledonary tissue, and strips of onion epidermis from the ventral surface of onion bulb scales at pH 4.2, 5.3, and 6.2. Pectin could not be localized with hydroxylamine-ferric chloride reagent in macerated tissues treated for 24 hr with active extract.     

Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysms

PurposeTo test the hypothesis that ruptured abdominal aortic aneurysms (AAA) are globally weaker than unruptured ones.MethodsFour ruptured and seven unruptured AAA specimens were harvested whole from fresh cadavers during autopsies performed over an 18-month period. Multiple regionally distributed longitudinally oriented rectangular strips were cut from each AAA specimen for a total of 77 specimen strips. Strips were subjected to uniaxial extension until failure. Sections from approximately the strongest and weakest specimen strips were studied histologically and histochemically. From the load-extension data, failure tension, failure stress and failure strain were calculated. Rupture site characteristics such as location, arc length of rupture and orientation of rupture were also documented.ResultsThe failure tension, a measure of the tissue mechanical caliber was remarkably similar between ruptured and unruptured AAA (group mean±standard deviation of within-subject means: 11.2±2.3 versus 11.6±3.6 N/cm; p=0.866 by mixed model ANOVA). In post-hoc analysis, there was little difference between the groups in other measures of tissue mechanical caliber as well such as failure stress (95±28 versus 98±23 N/cm²; p=0.870), failure strain (0.39±0.09 versus 0.36±0.09; p=0.705), wall thickness (1.7±0.4 versus 1.5±0.4 mm; p=0.470) , and % coverage of collagen within tissue cross section (49.6±12.9% versus 60.8±9.6%; p=0.133). In the four ruptured AAA, primary rupture sites were on the lateral quadrants (two on left; one on left-posterior; one on right). Remarkably, all rupture lines had a longitudinal orientation and ranged from 1 to 6 cm in length.ConclusionThe findings are not consistent with the hypothesis that ruptured aortic aneurysms are globally weaker than unruptured ones.     

Reporting Quality of Systematic Reviews/Meta-Analyses of Acupuncture

Background

The QUOROM and PRISMA statements were published in 1999 and 2009, respectively, to improve the consistency of reporting systematic reviews (SRs)/meta-analyses (MAs) of clinical trials. However, not all SRs/MAs adhere completely to these important standards. In particular, it is not clear how well SRs/MAs of acupuncture studies adhere to reporting standards and which reporting criteria are generally ignored in these analyses.

Objectives

To evaluate reporting quality in SRs/MAs of acupuncture studies.

Methods

We performed a literature search for studies published prior to 2014 using the following public archives: PubMed, EMBASE, Web of Science, the Cochrane Database of Systematic Reviews (CDSR), the Chinese Biomedical Literature Database (CBM), the Traditional Chinese Medicine (TCM) database, the Chinese Journal Full-text Database (CJFD), the Chinese Scientific Journal Full-text Database (CSJD), and the Wanfang database. Data were extracted into pre-prepared Excel data-extraction forms. Reporting quality was assessed based on the PRISMA checklist (27 items).

Results

Of 476 appropriate SRs/MAs identified in our search, 203, 227, and 46 were published in Chinese journals, international journals, and the Cochrane Database, respectively. In 476 SRs/MAs, only 3 reported the information completely. By contrast, approximately 4.93% (1/203), 8.81% (2/227) and 0.00% (0/46) SRs/Mas reported less than 10 items in Chinese journals, international journals and CDSR, respectively. In general, the least frequently reported items (reported≤50%) in SRs/MAs were “protocol and registration”, “risk of bias across studies”, and “additional analyses” in both methods and results sections.

Conclusions

SRs/MAs of acupuncture studies have not comprehensively reported information recommended in the PRISMA statement. Our study underscores that, in addition to focusing on careful study design and performance, attention should be paid to comprehensive reporting standards in SRs/MAs on acupuncture studies.     

Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

A psychrotolerant and H₂O₂-resistant bacterium, Exiguobacterium oxidotolerans T-2-2^T, exhibits extraordinary H₂O₂ resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.