Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells.

Two rare cancers of the exocrine pancreas: to treat or not to treat like ductal adenocarcinoma?

Pancreatic cancer is an aggressive malignancy with increasing incidence. Pancreatic ductal adenocarcinoma (PDAC) accounts for > 90% of pancreatic cancer diagnoses, while other exocrine tumors are much rarer. In this review, we have focused on two rare cancers of the exocrine pancreas: adenosquamous carcinoma of the pancreas (ASCP) and pancreatic acinar cell carcinoma (PACC).

First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and advances of new therapeutics.

The neurofibromatoses, which include neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis, are a group of syndromes characterized by tumor growth in the nervous system. The RASopathies are a group of syndromes caused by germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. The RASopathies include NF1, Noonan syndrome, Noonan syndrome with multiple lentigines, Costello syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, capillary malformation arterio-venous malformation syndrome, and SYNGAP1 autism.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit.

Inactivation of non-enveloped virus by 1,5 iodonaphthyl azide.

Background: A photoactive hydrophobic agent 1,5-iodonaphthyl-azide (INA), has been previously shown to completely inactivate the enveloped viruses. INA sequesters into the lipid bilayer of the virus envelope and upon UV-irradiation bind to the hydrophobic domains of the envelope glycoproteins. In our earlier study, we have shown that the Venezuelan equine encephalitis virus (VEEV) genomic RNA was also inactivated during the inactivation of the virus with INA.

Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma.

The primary objective of the study was to evaluate the feasibility and safety of a process which would utilize genome-wide expression data from tumor biopsies to support individualized treatment decisions. Current treatment options for recurrent neuroblastoma are limited and ineffective, with a survival rate of <10%. Molecular profiling may provide data which will enable the practitioner to select the most appropriate therapeutic option for individual patients, thus improving outcomes.

Direct competition assay for transcription fidelity.

Accurate transcription is essential for faithful information flow from DNA to RNA and to the protein. Mechanisms of cognate substrate selection by RNA polymerases are currently elucidated by structural, genetic, and biochemical approaches. Here, we describe a fast and reliable approach to quantitative analyses of transcription fidelity, applicable to analyses of RNA polymerase selectivity against misincorporation, incorporation of dNMPs, and chemically modified rNMP analogues.

A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.

We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible.

Nucleosomal elements that control the topography of the barrier to transcription.

The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component-the histone tails, the specific histone-DNA contacts, and the DNA sequence-contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones.

Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage.

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair.

RNA folding in transcription elongation complex: implication for transcription termination.

Intrinsic transcription termination signal in DNA consists of a short inverted repeat followed by a T-rich stretch. Transcription of this sequence by RNA polymerase (RNAP) results in formation of a "termination hairpin" (TH) in the nascent RNA and in rapid dissociation of the transcription elongation complex (EC) at termination points located 7-8 nt downstream of the base of TH stem. RNAP envelops 15 nt of the RNA following RNA growing 3'-end, suggesting that folding of the TH is impeded by a tight protein environment when RNAP reaches the termination points.

Data mining of functional RNA structures in genomic sequences.

The normal functions of genomes depend on the precise expression of messenger RNAs and noncoding RNAs (ncRNAs) such as transfer RNAs and microRNAs in eukaryotes. These ncRNAs and functional RNA structures (FRSs) act as regulators or response elements for cellular factors and participate in transcription, posttranscriptional processing, and translation. Knowledge discovery of these FRSs in huge DNA/RNA sequence databases is a very important step to reach our goal of going from genomic sequence data to biological knowledge for understanding RNA-based regulation.

Modeling opportunities in comparative oncology for drug development.

Successful development of novel cancer drugs depends on well-reasoned scientific drug discovery, rigorous preclinical development, and carefully conceived clinical trials. Failure in any of these steps contributes to poor rates of approval for new drugs to treat cancer. As technological and scientific advances have opened the door to a variety of novel approaches to cancer drug discovery and development, preclinical models that can answer questions about the activity and safety of novel therapies are increasingly necessary.

Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro.

Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro.

Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications.

Multicolor fluorescence-based approaches for imaging cytokine-induced alterations in the neovascularization, growth, metastasis, and apoptosis of murine neuroblastoma tumors.

Neuroblastoma is one of the most common solid tumors in children. The prognosis of patients with advanced neuroblastoma is poor overall despite standard therapeutic modalities and has stimulated substantial interest in the potential role for biologics such as immunotherapeutic and/or antiangiogenic agents for the treatment of neuroblastoma. To facilitate preclinical investigation of the efficacy and mechanisms of action of new biologic agents for the treatment of neuroblastoma, a comprehensive panel of disease-specific fluorescence-based model systems has been developed by our group to image the growth, neovascularization, metastasis, and apoptosis of neuroblastoma tumors.

Statistical inference for well-ordered structures in nucleotide sequences.

Distinct, local structures are frequently correlated with functional RNA elements involved in post-transcriptional regulation of gene expression. Discovery of microRNAs (miRNAs) suggests that there are a large class of small non-coding RNAs in eukaryotic genomes. These miRNAs have the potential to form distinct fold-back stem-loop structures.

An algorithm for detecting homologues of known structured RNAs in genomes.

Distinct RNA structures are frequently involved in a wide-range of functions in various biological mechanisms. The three dimensional RNA structures solved by X-ray crystallography and various well-established RNA phylogenetic structures indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop region. Discovery of well-ordered RNA structures and their homologues in genome-wide searches will enhance our ability to detect the RNA structural motifs and help us to highlight their association with functional and regulatory RNA elements.

Silencing in yeast: identification of clr4 targets.

Efficient handling of multiple reactions is a crucial prerequisite for productive RNA differential display (DD) analysis. To identify transcriptional targets of the histone H3 Lys9-specific methyltransferase Clr4, we applied a multiformat modification of DD to compare between clr4+ and clr4- transcriptomes of Schizosaccaromyces pombe. As a result, 14 differentially expressed bands were identified among 720 polymerase chain reaction (PCR) studied.

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita.

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule.