Selective Occupation by E2F and RB of Loci Expressed by RNA Polymerase III.

In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Utilizing the Un-Meeting model to advance innovative translational and team science.

Advances in translational science require innovative solutions, and engagement of productive transdisciplinary teams play a critical role. While various forms of scientific meetings have long provided venues for sharing scientific findings and generating new collaborations, many conferences lack opportunities for active discussions. We describe the use of an Un-Meeting to foster innovative translational science teams through engaged discussions across multidisciplinary groups addressing a shared theme. The Un-Meeting was delivered by the University of Rochester Center for Leading Innovation and Collaboration, the national coordinating center for the National Institutes of Health Clinical and Translational Science Awards (CTSA) program. This pilot CTSA program Un-Meeting focused on engaging translational scientists, policy-makers, community members, advocates, and public health professionals to address the opioid crisis. The participant-driven format leveraged lightning talks, attendee-led idea generation, and extensive breakout discussions to foster multidisciplinary networking. Results indicated participation by a broad set of attendees and a high level of networking during the meeting. These results, coupled with the growth of the Un-Meeting across the CTSA Consortium, provide practices and models to potentially advance team and translational science. While future work will further assess the impact of Un-Meetings, this format presents a promising approach to enhance translational science.

Selection of tRNA Genes in Human Breast Tumours Varies Substantially between Individuals.

Abnormally elevated expression of tRNA is a common feature of breast tumours. Rather than a uniform increase in all tRNAs, some are deregulated more strongly than others. Elevation of particular tRNAs has been associated with poor prognosis for patients, and experimental models have demonstrated the ability of some tRNAs to promote proliferation or metastasis. Each tRNA isoacceptor is encoded redundantly by multiple genes, which are commonly dispersed across several chromosomes. An unanswered question is whether the consistently high expression of a tRNA in a cancer type reflects the consistent activation of the same members of a gene family, or whether different family members are activated from one patient to the next. To address this question, we interrogated ChIP-seq data to determine which tRNA genes were active in individual breast tumours. This revealed that distinct sets of tRNA genes become activated in individual cancers, whereas there is much less variation in the expression patterns of families. Several pathways have been described that are likely to contribute to increases in tRNA gene transcription in breast tumours, but none of these can adequately explain the observed variation in the choice of genes between tumours. Current models may therefore lack at least one level of regulation.

Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology.

Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing.

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

TFIIIC-based chromatin insulators through eukaryotic evolution.

Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types.

Widespread association of ERα with RMRP and tRNA genes in MCF-7 cells and breast cancers.

tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at tRNALeu and 5S rRNA genes in this breast cancer cell line. While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer.

Impact on Arabidopsis growth and stress resistance of depleting the Maf1 repressor of RNA polymerase III.

Maf1 is a transcription factor that is conserved in sequence and structure between yeasts, animals and plants. Its principal molecular function is also well conserved, being to bind and repress RNA polymerase (pol) III, thereby inhibiting synthesis of tRNAs and other noncoding RNAs. Restrictions on tRNA production and hence protein synthesis can provide a mechanism to preserve resources under conditions that are suboptimal for growth. Accordingly, Maf1 is found in some organisms to influence growth and/or stress survival. Because of their sessile nature, plants are especially vulnerable to environmental changes and molecular adaptations that enhance growth under benign circumstances can increase sensitivity to external challenges. We tested if Maf1 depletion in the model plant Arabidopsis affects growth, pathogen resistance and tolerance of drought or soil salinity, a common physiological challenge that imposes both osmotic and ionic stress. We find that disruption of the Maf1 gene or RNAi-mediated depletion of its transcript is well-tolerated and confers a modest growth advantage without compromising resistance to common biotic and abiotic challenges.

Un-Meetings as tools for translational idea generation: A semantic content analysis of an Opioid Crisis Un-Meeting.

Background: Team development and idea generation are key intertwined steps in translational science that need a framework to accommodate unstructured, participatory interactions. To this end, we introduced Un-Meetings to the Clinical and Translational Science Awards (CTSA) Program, innovative events that facilitate cross-disciplinary idea generation and informal discussions between translational scientists, policy makers, community members, advocates, and public health professionals. Here we describe a mixed methods study to characterize the conceptual diversity and clusterization of ideas generated through an Opioid Crisis Un-Meeting. Methods: An Un-Meeting targeting translation science approaches to the opioid crisis were hosted at the University of Rochester Center for Leading Innovation and Collaboration (CLIC). We used semantic analysis and conceptual mapping of keywords to analyze how attendee-led idea generation sessions identified topics for breakout discussions. Results: One hundred and two individuals from 40 institutions proposed 150 unique ideas that were grouped into 23 breakout sessions. Network analysis showed that diverse pools of experts were bridged by topics addressing the complexities of the opioid crisis. Two clusters emerged: (1) systems, contexts, and community engagement, and (2) technologies, innovations, and treatment advancements. Conclusions: The cross-disciplinary nature of topic areas that bridge across thematic communities provide opportunities for CTSA programs to engage and support development of diverse translational teams. Potential opportunities for team building include technological advancements of opioid prevention, treatment, surveillance, systems approaches, and studies focusing on special populations and health disparities. The analysis method here may be useful in identifying naturally emerging teams of experts and community gaps when addressing large problems.

Association of Hospitalization with Long-Term Cognitive Trajectories in Older Adults.

IMPORTANCE: Hospitalizations are associated with cognitive decline in older adults. OBJECTIVE: To determine the association between hospitalization characteristics and the trajectory of cognitive function in older adults. DESIGN: Population-based longitudinal study of cognitive aging. SETTING: Olmsted Medical Center and Mayo Clinic, the only centers in Olmsted County, Minnesota, with hospitalization capacity. PARTICIPANTS: Individuals without dementia at baseline, with consecutive cognitive assessments from 2004 through 2017, and at least one visit after the age of 60. MEASUREMENTS: The primary outcome was longitudinal changes in global cognitive z-score. Secondary outcomes were changes in four cognitive domains: memory, attention/executive function, language, and visuospatial skills. Hospitalization characteristics analyzed included elective versus nonelective, medical versus surgical, critical care versus no critical care admission, and long versus short duration admissions. RESULTS: Of 4,587 participants, 1,622 had 1 and more hospital admission. Before hospitalization, the average slope of the global z-score was -0.031 units/year. After hospitalization, the rate of annual global z-score accelerated by -0.051 (95% CI = -0.057, -0.045) units, P < .001, resulting in an estimated annual slope after the first hospitalization of -0.082. The accelerated decline was found in all four cognitive domains (memory, visuospatial, language, and executive, all P < .001). The acceleration of the decline in global z-score following hospitalization was greater for medical compared to surgical hospitalizations (slope change following hospitalization = -0.064 vs -0.034 for medical vs surgical, P < .001), and nonelective compared to elective admissions (slope change following hospitalization = -0.075 vs -0.037 for nonelective vs elective, P < .001). The acceleration of cognitive decline was not different for hospitalization with intensive care unit admission versus not. CONCLUSIONS: Hospitalization of older adults is associated with accelerated decline of global and domain-specific cognitive domains, with the rate of decline dependent upon type of admission. The clinical impact of this accelerated decline will depend on the individual's baseline cognitive reserve and expected longevity.

Inhibition of tRNA Gene Transcription by the Immunosuppressant Mycophenolic Acid.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling.

Predicting hospital-onset Clostridium difficile using patient mobility data: A network approach.

OBJECTIVE: To examine the relationship between unit-wide Clostridium difficile infection (CDI) susceptibility and inpatient mobility and to create contagion centrality as a new predictive measure of CDI. DESIGN: Retrospective cohort study. METHODS: A mobility network was constructed using 2 years of patient electronic health record data for a 739-bed hospital (n = 72,636 admissions). Network centrality measures were calculated for each hospital unit (node) providing clinical context for each in terms of patient transfers between units (ie, edges). Daily unit-wide CDI susceptibility scores were calculated using logistic regression and were compared to network centrality measures to determine the relationship between unit CDI susceptibility and patient mobility. RESULTS: Closeness centrality was a statistically significant measure associated with unit susceptibility (P < .05), highlighting the importance of incoming patient mobility in CDI prevention at the unit level. Contagion centrality (CC) was calculated using inpatient transfer rates, unit-wide susceptibility of CDI, and current hospital CDI infections. The contagion centrality measure was statistically significant (P < .05) with our outcome of hospital-onset CDI cases, and it captured the additional opportunities for transmission associated with inpatient transfers. We have used this analysis to create easily interpretable clinical tools showing this relationship as well as the risk of hospital-onset CDI in real time, and these tools can be implemented in hospital EHR systems. CONCLUSIONS: Quantifying and visualizing the combination of inpatient transfers, unit-wide risk, and current infections help identify hospital units at risk of developing a CDI outbreak and, thus, provide clinicians and infection prevention staff with advanced warning and specific location data to inform prevention efforts.

Come from away: Best practices in mini-sabbaticals for the development of young investigators: a White Paper by the SEQUIN (mini-Sabbatical Evaluation and QUality ImprovemeNt) Group.

Mini-sabbaticals are formal short-term training and educational experiences away from an investigator's home research unit. These may include rotations with other research units and externships at government research or regulatory agencies, industry and non-profit programs, and training and/or intensive educational programs. The National Institutes of Health have been encouraging training institutions to consider offering mini-sabbaticals, but given the newness of the concept, limited data are available to guide the implementation of mini-sabbatical programs. In this paper, we review the history of sabbaticals and mini-sabbaticals, report the results of surveys we performed to ascertain the use of mini-sabbaticals at Clinical and Translational Science Award hubs, and consider best practice recommendations for institutions seeking to establish formal mini-sabbatical programs.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Visualizing nationwide variation in medicare Part D prescribing patterns.

BACKGROUND: To characterize the regional and national variation in prescribing patterns in the Medicare Part D program using dimensional reduction visualization methods. METHODS: Using publicly available Medicare Part D claims data, we identified and visualized regional and national provider prescribing profile variation with unsupervised clustering and t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction techniques. Additionally, we examined differences between regionally representative prescribing patterns for major metropolitan areas. RESULTS: Distributions of prescribing volume and medication diversity were highly skewed among over 800,000 Medicare Part D providers. Medical specialties had characteristic prescribing patterns. Although the number of Medicare providers in each state was highly correlated with the number of Medicare Part D enrollees, some states were enriched for providers with > 10,000 prescription claims annually. Dimension-reduction, hierarchical clustering and t-SNE visualization of drug- or drug-class prescribing patterns revealed that providers cluster strongly based on specialty and sub-specialty, with large regional variations in prescribing patterns. Major metropolitan areas had distinct prescribing patterns that tended to group by major geographical divisions. CONCLUSIONS: This work demonstrates that unsupervised clustering, dimension-reduction and t-SNE visualization can be used to analyze and visualize variation in provider prescribing patterns on a national level across thousands of medications, revealing substantial prescribing variation both between and within specialties, regionally, and between major metropolitan areas. These methods offer an alternative system-wide and pattern-centric view of such data for hypothesis generation, visualization, and pattern identification.

Association of Optic Nerve Head Drusen with Best Vitelliform Macular Dystrophy: A Case Series.

PURPOSE: To report the association of optic nerve head (ONH) drusen with Best vitelliform macular dystrophy (BVMD). METHODS: Chart review. PATIENTS: Five patients from 3 families. RESULTS: Multimodal imaging and ophthalmic examination demonstrated findings consistent with ONH drusen, in association with BVMD, in 5 patients. CONCLUSION: We report the association of BVMD with ONH drusen in 5 patients. This combination has previously been reported only once. We recommend that patients with a diagnosis of BVMD undergo autofluorescence and ultrasound imaging of the optic nerve to help facilitate this diagnosis, as some ONH drusen can be buried.

Knobloch syndrome associated with Polymicrogyria and early onset of retinal detachment: two case reports.

BACKGROUND: Knobloch Syndrome (KS) is a rare congenital syndrome characterized by occipital skull defects and vitreoretinal degeneration. Retinal detachment (RD) often occurs at the end of the first decade of life or later. Aside from occipital skull defects, central nervous system abnormalities are uncommon. CASE PRESENTATIONS: We report on two siblings with KS. The first, a seven month old male, presented with nystagmus and was found to have a serous RD and a tessellated retinal appearance. His sister had a history of multiple visual abnormalities and had a similar retinal appearance although no signs of RD, but retina staphylomas. Genetic testing performed on both siblings showed a mutation in COL18A1, diagnostic of KS. MRI of both siblings demonstrated polymicrogyria but did not show occipital defects. CONCLUSIONS: Although several families with KS have been described previously, our case is noteworthy for several reasons. The RD observed in our first patient occurred at an early age, and we find evidence of only one patient with KS who had an RD identified at an earlier age. The findings of polymicrogyria are not characteristic of KS, and we found only a few previous reports of this association. Additionally, we review potential treatment options for this condition.

Properties of healthcare teaming networks as a function of network construction algorithms.

Network models of healthcare systems can be used to examine how providers collaborate, communicate, refer patients to each other, and to map how patients traverse the network of providers. Most healthcare service network models have been constructed from patient claims data, using billing claims to link a patient with a specific provider in time. The data sets can be quite large (106-108 individual claims per year), making standard methods for network construction computationally challenging and thus requiring the use of alternate construction algorithms. While these alternate methods have seen increasing use in generating healthcare networks, there is little to no literature comparing the differences in the structural properties of the generated networks, which as we demonstrate, can be dramatically different. To address this issue, we compared the properties of healthcare networks constructed using different algorithms from 2013 Medicare Part B outpatient claims data. Three different algorithms were compared: binning, sliding frame, and trace-route. Unipartite networks linking either providers or healthcare organizations by shared patients were built using each method. We find that each algorithm produced networks with substantially different topological properties, as reflected by numbers of edges, network density, assortativity, clustering coefficients and other structural measures. Provider networks adhered to a power law, while organization networks were best fit by a power law with exponential cutoff. Censoring networks to exclude edges with less than 11 shared patients, a common de-identification practice for healthcare network data, markedly reduced edge numbers and network density, and greatly altered measures of vertex prominence such as the betweenness centrality. Data analysis identified patterns in the distance patients travel between network providers, and a striking set of teaming relationships between providers in the Northeast United States and Florida, likely due to seasonal residence patterns of Medicare beneficiaries. We conclude that the choice of network construction algorithm is critical for healthcare network analysis, and discuss the implications of our findings for selecting the algorithm best suited to the type of analysis to be performed.

Codon-Driven Translational Efficiency Is Stable across Diverse Mammalian Cell States.

Whether codon usage fine-tunes mRNA translation in mammals remains controversial, with recent papers suggesting that production of proteins in specific Gene Ontological (GO) pathways can be regulated by actively modifying the codon and anticodon pools in different cellular conditions. In this work, we compared the sequence content of genes in specific GO categories with the exonic genome background. Although a substantial fraction of variability in codon usage could be explained by random sampling, almost half of GO sets showed more variability in codon usage than expected by chance. Nevertheless, by quantifying translational efficiency in healthy and cancerous tissues in human and mouse, we demonstrated that a given tRNA pool can equally well translate many different sets of mRNAs, irrespective of their cell-type specificity. This disconnect between variations in codon usage and the stability of translational efficiency is best explained by differences in GC content between gene sets. GC variation across the mammalian genome is most likely a result of the interplay between genome repair and gene duplication mechanisms, rather than selective pressures caused by codon-driven translational rates. Consequently, codon usage differences in mammalian transcriptomes are most easily explained by well-understood mutational biases acting on the underlying genome.