Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously ...that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3′-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3′-phosphate–containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3′s role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP’s activity can be a promising therapeutic strategy for SCA3.
In so doing, we can learn valuable lessons from evidence-based movements in other sectors such as evidence-based management, which has provided new insights into the use of evidence for ...implementation decision making.2 The scientific community has made progress in building and applying implementation science to generate the research evidence needed for improving implementation decision making and practice. The Commissioners include a diverse group of pioneers in evidence-based implementation, balanced by gender and geographical region, with broad expertise in implementation science. Financial support for the Commission has been provided by the Bill & Melinda Gates Foundation, the Sall Family Foundation, and the University of North Carolina at Chapel Hill.
Microorganisms in wastewater treatment plants (WWTPs) are essential for water purification to protect public and environmental health. However, the diversity of microorganisms and the factors that ...control it are poorly understood. Using a systematic global-sampling effort, we analysed the 16S ribosomal RNA gene sequences from ~1,200 activated sludge samples taken from 269 WWTPs in 23 countries on 6 continents. Our analyses revealed that the global activated sludge bacterial communities contain ~1 billion bacterial phylotypes with a Poisson lognormal diversity distribution. Despite this high diversity, activated sludge has a small, global core bacterial community (n = 28 operational taxonomic units) that is strongly linked to activated sludge performance. Meta-analyses with global datasets associate the activated sludge microbiomes most closely to freshwater populations. In contrast to macroorganism diversity, activated sludge bacterial communities show no latitudinal gradient. Furthermore, their spatial turnover is scale-dependent and appears to be largely driven by stochastic processes (dispersal and drift), although deterministic factors (temperature and organic input) are also important. Our findings enhance our mechanistic understanding of the global diversity and biogeography of activated sludge bacterial communities within a theoretical ecology framework and have important implications for microbial ecology and wastewater treatment processes.
Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-
-methyl-1'-cyano-7-deaza-adenosine
-nucleotide ...analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (
) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.
Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) ...infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.
Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) ...infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.
A low-energy electronic recoil calibration of XENON1T, a dual-phase xenon time projection chamber, with an internal 37Ar source was performed. This calibration source features a 35-day half-life and ...provides two mono-energetic lines at 2.82 keV and 0.27 keV. The photon yield and electron yield at 2.82 keV are measured to be (32.3±0.3) photons/keV and (40.6±0.5) electrons/keV, respectively, in agreement with other measurements and with NEST predictions. The electron yield at 0.27 keV is also measured and it is (68.0+6.3−3.7) electrons/keV. The 37Ar calibration confirms that the detector is well-understood in the energy region close to the detection threshold, with the 2.82 keV line reconstructed at (2.83±0.02) keV, which further validates the model used to interpret the low-energy electronic recoil excess previously reported by XENON1T. The ability to efficiently remove argon with cryogenic distillation after the calibration proves that 37Ar can be considered as a regular calibration source for multi-tonne xenon detectors.
Little is known about the year-round occurrence of blue whales in Atlantic Canadian waters. We used passive acoustic monitoring to investigate blue whale call presence and increase our understanding ...of year-round blue whale occurrence along the eastern edge of the Scotian Shelf, Nova Scotia, Canada. Blue whale calls were recorded at 3 deep water sites within and near the Gully Marine Protected Area from October 2012 to September 2014 using bottom-mounted passive acoustic recorders. Calls were categorized as either tonal or downsweeping. At all 3 sites, tonal calls occurred most often from November through January in both years and in August 2014, while the majority of downsweeping calls occurred during May through September 2014. There was no discernible diel pattern in the occurrence of tonal calls. Downsweeping calls occurred most often during evening twilight, with a dramatic peak in occurrence during the hour of 19:00 Atlantic Standard Time at all 3 sites. The peak in downsweeping call occurrence during evening twilight may have been related to blue whale foraging ecology. This study sheds light on the poorly understood year-round occurrence of blue whales off Atlantic Canada. Understanding when and where blue whales occur will help to inform management decisions for the protection and conservation of this endangered population.
A low-energy electronic recoil calibration of XENON1T, a dual-phase xenon time projection chamber, with an internal
37
Ar source was performed. This calibration source features a 35-day half-life and ...provides two mono-energetic lines at 2.82 keV and 0.27 keV. The photon yield and electron yield at 2.82 keV are measured to be (
32.3
±
0.3
) photons/keV and (
40.6
±
0.5
) electrons/keV, respectively, in agreement with other measurements and with NEST predictions. The electron yield at 0.27 keV is also measured and it is (
68
.
0
-
3.7
+
6.3
) electrons/keV. The
37
Ar calibration confirms that the detector is well-understood in the energy region close to the detection threshold, with the 2.82 keV line reconstructed at (
2.83
±
0.02
) keV, which further validates the model used to interpret the low-energy electronic recoil excess previously reported by XENON1T. The ability to efficiently remove argon with cryogenic distillation after the calibration proves that
37
Ar can be considered as a regular calibration source for multi-tonne xenon detectors.
Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such ...diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3' untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.
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