Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

A Bayesian joint model for multivariate longitudinal and time-to-event data with application to ALL maintenance studies.

The most common type of cancer diagnosed among children is the Acute Lymphocytic Leukemia (ALL). A study was conducted by Tata Translational Cancer Research Center (TTCRC) Kolkata, in which 236 children (diagnosed as ALL patients) were treated for the first two years (approximately) with two standard drugs (6MP and MTx) and were then followed nearly for the next 3 years. The goal is to identify the longitudinal biomarkers that are associated with time-to-relapse, and also to assess the effectiveness of the drugs. We develop a Bayesian joint model in which a linear mixed model is used to jointly model three biomarkers (i.e. white blood cell count, neutrophil count, and platelet count) and a semi-parametric proportional hazards model is used to model the time-to-relapse. Our proposed joint model can assess the effects of different covariates on the progression of the biomarkers, and the effects of the biomarkers (and the covariates) on time-to-relapse. In addition, the proposed joint model can impute the missing longitudinal biomarkers efficiently. Our analysis shows that the white blood cell (WBC) count is not associated with time-to-relapse, but the neutrophil count and the platelet count are significantly associated with it. We also infer that a lower dose of 6MP and a higher dose of MTx jointly result in a lower relapse probability in the follow-up period. Interestingly, we find that relapse probability is the lowest for the patients classified into the "high-risk" group at presentation. The effectiveness of the proposed joint model is assessed through the extensive simulation studies.

A Bayesian joint model for multivariate longitudinal and time-to-event data with application to ALL maintenance studies.

The most common type of cancer diagnosed among children is the acute lymphocytic leukemia (ALL). A study was conducted by Tata Translational Cancer Research Center (TTCRC) Kolkata, in which 236 children (diagnosed as ALL patients) were treated for the first two years (approximately) with two standard drugs (6MP and MTx) and were then followed nearly for the next three years. The goal is to identify the longitudinal biomarkers that are associated with time-to-relapse, and also to assess the effectiveness of the drugs. We develop a Bayesian joint model in which a linear mixed model is used to jointly model three biomarkers (i.e. white blood cell count, neutrophil count, and platelet count) and a semi-parametric proportional hazards model is used to model the time-to-relapse. Our proposed joint model can assess the effects of different covariates on the progression of the biomarkers, and the effects of the biomarkers (and the covariates) on time-to-relapse. In addition, the proposed joint model can impute the missing longitudinal biomarkers efficiently. Our analysis shows that the white blood cell (WBC) count is not associated with time-to-relapse, but the neutrophil count and the platelet count are significantly associated with it. We also infer that a lower dose of 6MP and a higher dose of MTx jointly result in a lower relapse probability in the follow-up period. Interestingly, we find that relapse probability is the lowest for the patients classified into the "high-risk" group at presentation. The effectiveness of the proposed joint model is assessed through the extensive simulation studies.

Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repair Defects in Huntington's Disease.

Emerging evidence suggests that DNA repair deficiency and genome instability may be the impending signs of many neurological diseases. Genome-wide association (GWAS) studies have established a strong correlation between genes that play a role in DNA damage repair and many neurodegenerative diseases, including Huntington's disease (HD), and several other trinucleotides repeat expansion-related hereditary ataxias. Recently, many reports have documented a significant role played by the DNA repair processes in aging and in modifying many neurodegenerative diseases, early during their progression. Studies from our lab and others have now begun to understand the mechanisms that cause defective DNA repair in HD and surprisingly, many proteins that have a strong link to known neurodegenerative diseases seem to be important players in these cellular pathways. Mutations in huntingtin (HTT) gene that lead to polyglutamine repeat expansion at the N-terminal of HTT protein has been shown to disrupt transcription-coupled DNA repair process, a specialized DNA repair process associated with transcription. Due to the recent progress made in understanding the mechanisms of DNA repair in relation to HD, in this review, we will mainly focus on the mechanisms by which the wild-type huntingtin (HTT) protein helps in DNA repair during transcription, and the how polyglutamine expansions in HTT impedes this process in HD. Further studies that identify new players in DNA repair will help in our understanding of this process in neurons. Furthermore, it should help us understand how various DNA repair mechanism(s) coordinate to maintain the normal physiology of neurons, and provide insights for the development of novel drugs at prodromal stages of these neurodegenerative diseases.

A scalable metal-organic framework as a durable physisorbent for carbon dioxide capture.

Metal-organic frameworks (MOFs) as solid sorbents for carbon dioxide (CO2) capture face the challenge of merging efficient capture with economical regeneration in a durable, scalable material. Zinc-based Calgary Framework 20 (CALF-20) physisorbs CO2 with high capacity but is also selective over water. Competitive separations on structured CALF-20 show not just preferential CO2 physisorption below 40% relative humidity but also suppression of water sorption by CO2, which was corroborated by computational modeling. CALF-20 has a low enthalpic regeneration penalty and shows durability to steam (>450,000 cycles) and wet acid gases. It can be prepared in one step, formed as composite materials, and its synthesis can be scaled to multikilogram batches.

DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases.

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Deficiency in classical nonhomologous end-joining-mediated repair of transcribed genes is linked to SCA3 pathogenesis.

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.

Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription.

How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences.

Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.

Development of X-ray CCD camera based X-ray micro-CT system.

Availability of microfocus X-ray sources and high resolution X-ray area detectors has made it possible for high resolution microtomography studies to be performed outside the purview of synchrotron. In this paper, we present the work towards the use of an external shutter on a high resolution microtomography system using X-ray CCD camera as a detector. During micro computed tomography experiments, the X-ray source is continuously ON and owing to the readout mechanism of the CCD detector electronics, the detector registers photons reaching it during the read-out period too. This introduces a shadow like pattern in the image known as smear whose direction is defined by the vertical shift register. To resolve this issue, the developed system has been incorporated with a synchronized shutter just in front of the X-ray source. This is positioned in the X-ray beam path during the image readout period and out of the beam path during the image acquisition period. This technique has resulted in improved data quality and hence the same is reflected in the reconstructed images.

Glucocorticoids increase skeletal muscle NF-κB inducing kinase (NIK): links to muscle atrophy.

Glucocorticoids (GC) are a frontline therapy for numerous acute and chronic diseases because of their demonstrated efficacy at reducing systemic inflammation. An unintended side effect of GC therapy is the stimulation of skeletal muscle atrophy. Pathophysiological mechanisms responsible for GC-induced skeletal muscle atrophy have been extensively investigated, and the ability to treat patients with GC without unintended muscle atrophy has yet to be realized. We have reported that a single, standard-of-care dose of Methylprednisolone increases in vivo expression of NF-κB-inducing kinase (NIK), an important upstream regulatory kinase controlling NF-κB activation, along with other key muscle catabolic regulators such as Atrogin-1 and MuRF1 that induce skeletal muscle proteolysis. Here, we provide experimental evidence that overexpressing NIK by intramuscular injection of recombinant human NIK via adenoviral vector in mouse tibialis anterior muscle induces a 30% decrease in the average fiber cross-sectional area that is associated with increases in mRNA expression of skeletal muscle atrophy biomarkers MuRF1, Atrogin-1, myostatin and Gadd45. A single injection of GC induced NIK mRNA and protein within 2 h, with the increased NIK localized to nuclear and sarcolemmal locations within muscle fibers. Daily GC injections induced skeletal muscle fore limb weakness as early as 3 days with similar atrophy of muscle fibers as observed with NIK overexpression. NIK overexpression in primary human skeletal muscle myotubes increased skeletal muscle atrophy biomarkers, while NIK knockdown significantly attenuated GC-induced increases in NIK and Atrogin-1. These results suggest that NIK may be a novel, previously unrecognized mediator of GC-induced skeletal muscle atrophy.

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes.

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

Anode-Engineered Protonic Ceramic Fuel Cell with Excellent Performance and Fuel Compatibility.

Directly utilizing hydrocarbon fuels, particularly methane, is advantageous yet challenging in high-performance protonic ceramic fuel cells. In this work, this technological hurdle is well addressed by selective deposition of secondary electrocatalysts within the porous Ni-cermet anode. This novel strategy sheds light on the development of multifunctional porous structures for energy and catalysis applications.

Tailoring the Microstructure of a Solid Oxide Fuel Cell Anode Support by Calcination and Milling of YSZ.

In this study, the effects of calcination and milling of 8YSZ (8 mol% yttria stabilized zirconia) used in the nickel-YSZ anode on the performance of anode supported tubular fuel cells were investigated. For this purpose, two different types of cells were prepared based on a Ni-YSZ/YSZ/Nd2NiO4+δ-YSZ configuration. For the anode preparation, a suspension was prepared by mixing NiO and YSZ in a ratio of 65:35 wt% (Ni:YSZ 50:50 vol.%) with 30 vol.% graphite as the pore former. As received Tosoh YSZ or its calcined form (heated at 1500 °C for 3 hours) was used in the anode support as the YSZ source. Electrochemical results showed that optimization of the fuel electrode microstructure is essential for the optimal distribution of gas within the support of the cell, especially under electrolysis operation where the performance for an optimized cell (calcined YSZ) was enhanced by a factor of two. In comparison with a standard cell (containing as received YSZ), at 1.5 V and 800 °C the measured current density was -1380 mA cm(-2) and -690 mA cm(-2) for the cells containing calcined and as received YSZ, respectively. The present study suggests that the anode porosity for improved cell performance under SOEC is more critical than SOFC mode due to more complex gas diffusion under electrolysis mode where large amount of steam needs to be transfered into the cell.

Design of a bioactive small molecule that targets r(AUUCU) repeats in spinocerebellar ataxia 10.

RNA is an important target for chemical probes of function and lead therapeutics; however, it is difficult to target with small molecules. One approach to tackle this problem is to identify compounds that target RNA structures and utilize them to multivalently target RNA. Here we show that small molecules can be identified to selectively bind RNA base pairs by probing a library of RNA-focused small molecules. A small molecule that selectively binds AU base pairs informed design of a dimeric compound (2AU-2) that targets the pathogenic RNA, expanded r(AUUCU) repeats, that causes spinocerebellar ataxia type 10 (SCA10) in patient-derived cells. Indeed, 2AU-2 (50 nM) ameliorates various aspects of SCA10 pathology including improvement of mitochondrial dysfunction, reduced activation of caspase 3, and reduction of nuclear foci. These studies provide a first-in-class chemical probe to study SCA10 RNA toxicity and potentially define broadly applicable compounds targeting RNA AU base pairs in cells.

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation.

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.