Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Fundamental physical cellular constraints drive self-organization of tissues.

Morphogenesis is driven by small cell shape changes that modulate tissue organization. Apical surfaces of proliferating epithelial sheets have been particularly well studied. Currently, it is accepted that a stereotyped distribution of cellular polygons is conserved in proliferating tissues among metazoans. In this work, we challenge these previous findings showing that diverse natural packed tissues have very different polygon distributions. We use Voronoi tessellations as a mathematical framework that predicts this diversity. We demonstrate that Voronoi tessellations and the very different tissues analysed share an overriding restriction: the frequency of polygon types correlates with the distribution of cell areas. By altering the balance of tensions and pressures within the packed tissues using disease, genetic or computer model perturbations, we show that as long as packed cells present a balance of forces within tissue, they will be under a physical constraint that limits its organization. Our discoveries establish a new framework to understand tissue architecture in development and disease.

Data-analysis strategies for image-based cell profiling.

Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences.

Protein quality control at the inner nuclear membrane.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

TimerQuant: a modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos.

Studies on signalling dynamics in living embryos have been limited by a scarcity of in vivo reporters. Tandem fluorescent protein timers provide a generic method for detecting changes in protein population age and thus provide readouts for signalling events that lead to changes in protein stability or location. When imaged with quantitative dual-colour fluorescence microscopy, tandem timers offer detailed 'snapshot' readouts of signalling activity from subcellular to organismal scales, and therefore have the potential to revolutionise studies in developing embryos. Here we use computer modelling and embryo experiments to explore the behaviour of tandem timers in developing systems. We present a mathematical model of timer kinetics and provide software tools that will allow experimentalists to select the most appropriate timer designs for their biological question, and guide interpretation of the obtained readouts. Through the generation of a series of novel zebrafish reporter lines, we confirm experimentally that our quantitative model can accurately predict different timer responses in developing embryos and explain some less expected findings. For example, increasing the FRET efficiency of a tandem timer actually increases the ability of the timer to detect differences in protein half-life. Finally, while previous studies have used timers to monitor changes in protein turnover, our model shows that timers can also be used to facilitate the monitoring of gene expression kinetics in vivo.

Directional tissue migration through a self-generated chemokine gradient.

The directed migration of cell collectives is a driving force of embryogenesis. The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients. One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them, a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion.

Upregulation of SPS100 gene expression by an antisense RNA via a switch of mRNA isoforms with different stabilities.

Pervasive transcription of genomes generates multiple classes of non-coding RNAs. One of these classes are stable long non-coding RNAs which overlap coding genes in antisense direction (asRNAs). The function of such asRNAs is not fully understood but several cases of antisense-dependent gene expression regulation affecting the overlapping genes have been demonstrated. Using high-throughput yeast genetics and a limited set of four growth conditions we previously reported a regulatory function for ∼25% of asRNAs, most of which repress the expression of the sense gene. To further explore the roles of asRNAs we tested more conditions and identified 15 conditionally antisense-regulated genes, 6 of which exhibited antisense-dependent enhancement of gene expression. We focused on the sporulation-specific gene SPS100, which becomes upregulated upon entry into starvation or sporulation as a function of the antisense transcript SUT169. We demonstrate that the antisense effect is mediated by its 3' intergenic region (3'-IGR) and that this regulation can be transferred to other genes. Genetic analysis revealed that SUT169 functions by changing the relative expression of SPS100 mRNA isoforms from a short and unstable transcript to a long and stable species. These results suggest a novel mechanism of antisense-dependent gene regulation via mRNA isoform switching.

Hospital discharges and patient activity associated with chronic pancreatitis in Ireland 2009-2013.

OBJECTIVE: To investigate trends in acute public hospital patient discharges in Ireland, to analyse hospital discharge activity for geographical variations, aetiological differences, and to estimate a national prevalence for chronic pancreatitis. METHOD: We performed a nationwide retrospective study of all in-patient discharges from acute public hospitals in Ireland, participating in the Hospital In-Patient Enquiry (HIPE) reporting system. We searched for International Classification of Disease, Tenth Revision, Australian Modification (ICD-10-AM) codes K86.0 alcohol-induced chronic pancreatitis, and K86.1 other chronic pancreatitis, and data were extracted for the years 2009-2013. RESULTS: There were 4098 emergency admissions for any aetiology chronic pancreatitis during the 5 year study period. Total discharges ranged from 753 in 2009 to 999 in 2013. Total patients ranged from 530 in 2009 to 601 in 2013. Prevalence of chronic pancreatitis is estimated at 11.6 per 100,000 to 13.0 per 100,000 over the five years. 'Other aetiology chronic pancreatitis' discharges were almost double that of 'alcohol chronic pancreatitis'. We found notable geographical variation in hospital discharge activity for chronic pancreatitis. CONCLUSIONS: We report a prevalence which is similar to those worldwide studies who adopted a similar methodology utilising exact counts of patients. Our data are an underestimated as they are based on in-patient discharges only, excluding those attending primary care, outpatient or emergency room visits without admission. Despite studying this disease in a population with high per capita alcohol consumption, we report almost twice as many discharges for non-alcohol aetiology chronic pancreatitis.

Control of tissue morphology by Fasciclin III-mediated intercellular adhesion.

Morphogenesis is dependent on the orchestration of multiple developmental processes to generate mature functional organs. However, the signalling pathways that coordinate morphogenesis and the mechanisms that translate these signals into tissue shape changes are not well understood. Here, we demonstrate that changes in intercellular adhesion mediated by the transmembrane protein Fasciclin III (FasIII) represent a key mediator of morphogenesis. Using the embryonic Drosophila hindgut as an in vivo model for organogenesis, we show that the tightening of hindgut curvature that normally occurs between embryonic stage 12 and 15 to generate the characteristic shepherd's crook shape is dependent on localised JAK/STAT pathway activation. This localised pathway activity drives the expression of FasIII leading to its subcellular lateralisation at a stage before formation of septate junctions. Additionally, we show that JAK/STAT- and FasIII-dependent morphogenesis also regulates folds within the third instar wing imaginal disc. We show that FasIII forms homophilic intercellular interactions that promote intercellular adhesion in vivo and in cultured cells. To explore these findings, we have developed a mathematical model of the developing hindgut, based on the differential interfacial tension hypothesis (DITH) linking intercellular adhesion and localised surface tension. Our model suggests that increased intercellular adhesion provided by FasIII can be sufficient to drive the tightening of tube curvature observed. Taken together, these results identify a conserved molecular mechanism that directly links JAK/STAT pathway signalling to intercellular adhesion and that sculpts both tubular and planar epithelial shape.

Screening and Brief Interventions for Illicit Drug Use and Alcohol Use in Methadone Maintained Opiate-Dependent Patients: Results of a Pilot Cluster Randomized Controlled Trial Feasibility Study.

BACKGROUND AND OBJECTIVES: The present study evaluated the effectiveness of a single clinician delivered brief intervention (BI) to reduce problem alcohol use and illicit substance use in an opiate-dependent methadone maintained cohort of patients attending for treatment. METHODS: Four addiction treatment centers were randomly assigned to either treatment as usual (TAU; control group) or BI (intervention group). Clinicians screened patients using the alcohol, smoking, and substance involvement screening test (ASSIST) screening tool at baseline and again at three-month follow up. Fidelity checks were performed to ensure that training was delivered effectively and uniformly across all study sites. Feasibility of administering a BI within daily practice was assessed through intervention fidelity checks, patient satisfaction questionnaires and process evaluation. RESULTS: A total of 465 patients were screened (66% of the overall eligible population) with a total of 433 (93%) ASSIST positive cases. Randomization was effective, with no differences in the control versus the intervention arms at baseline for key demographic or clinical indicators including substance us. There was a statistically significant difference between global risk score for the intervention (x = 39.36, sd = 25.91) group and the control group (x = 45.27, SD = 27.52) at 3-month follow-up (t(341) = -2.07, p < .05). CONCLUSIONS: This trial provides the first evidence that a single clinician delivered BI can result in a reduction in substance use within a methadone maintained opiate-dependent cohort, and this effect is sustained at three month follow up.

Biggest challenges in bioinformatics.

The third Heidelberg Unseminars in Bioinformatics (HUB) was held on 18th October 2012, at Heidelberg University, Germany. HUB brought together around 40 bioinformaticians from academia and industry to discuss the 'Biggest Challenges in Bioinformatics' in a 'World Café' style event.

Problem alcohol use among problem drug users in primary care: a qualitative study of what patients think about screening and treatment.

BACKGROUND: Problem alcohol use is common and associated with considerable adverse outcomes among patients who attend primary care in Ireland and other European countries for opiate substitution treatment. This paper aims to describe patients' experience of, and attitude towards, screening and therapeutic interventions for problem alcohol use in primary care. METHODS: This qualitative study recruited problem drug users (N = 28) from primary care based methadone programmes in the Ireland's Eastern region, using a stratified sampling matrix to include size of general practice and geographical area. Semi-structured interviews were conducted and analysed using thematic analysis, and audited by a third reviewer. RESULTS: We identified three overarching themes relevant to the purpose of this paper: (1) patients' experience of, and (2) attitude towards, screening and treatment for problem alcohol use in primary care, as well as their (3) views on service improvement. While most patients reported being screened for problem alcohol use at initial assessment, few recalled routine screening or treatment. Among the barriers and enablers to screening and treatment, patients highlighted the importance of the practitioner-patient relationship in helping them address the issue. Nevertheless, patients felt that healthcare professionals should be more proactive in the management of problem alcohol use at a primary care level and that primary care can play an important role in their treatment. CONCLUSIONS: Problem alcohol use is an important challenge in the care of problem drug users. While primary care is well placed to address this issue, little data has reported on this topic. The development of interventions which promote screening and brief interventions in practice are likely to benefit this at-risk group and further research and education, that help achieve this goal, are a priority. Strategies such as dissemination of clinical guidelines, educational videos, academic detailing and practice visits, should be explored.

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes.

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

A national study of the retention of Irish opiate users in methadone substitution treatment.

BACKGROUND: Retention in treatment is a key indicator of methadone treatment success. The study aims to identify factors that are associated with retention. OBJECTIVES: To determine retention in treatment at 12 months for Irish opiate users in methadone substitution treatment and to indicate factors that increase the likelihood of retention. METHODS: National cohort study of randomly selected opiate users commencing methadone treatment in 1999, 2001, and 2003 (n = 1269). RESULTS: Sixty-one percent of patients attending methadone treatment remained in continuous treatment for more than 1 year. Retention in treatment at 12 months was associated with age, gender, facility type, and methadone dose. Age and gender were no longer significant when adjusted for other variables in the model. Those who attended a specialist site were twice as likely to leave methadone treatment within 12 months compared with those who attended a primary care physician. The most important predictor of retention in treatment was methadone dose. Those who received <60 mg of methadone were three times more likely to leave treatment. CONCLUSION: Retention in methadone treatment is high in Ireland in a variety of settings. The main factors influencing retention in methadone treatment was an adequate methadone dose and access to a range of treatment settings including from primary care physicians. SCIENTIFIC SIGNIFICANCE: Providing an adequate dose of methadone during treatment will increase the likelihood of treatment retention. Methadone treatment by the primary care physician is a successful method of retaining opioid users in treatment.

Nα-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae.

N-terminal (Nt) acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt acetyltransferases (Nats) with differing specificities. Nt acetylation has been implicated in protein quality control, but its broad biological significance remains elusive. We investigate the roles of the two major Nats of S. cerevisiae, NatA and NatB, by performing transcriptome, translatome, and proteome profiling of natAΔ and natBΔ mutants. Our results reveal a range of NatA- and NatB-specific phenotypes. NatA is implicated in systemic adaptation control, because natAΔ mutants display altered expression of transposons, sub-telomeric genes, pheromone response genes, and nuclear genes encoding mitochondrial ribosomal proteins. NatB predominantly affects protein folding, because natBΔ mutants, to a greater extent than natA mutants, accumulate protein aggregates, induce stress responses, and display reduced fitness in the absence of the ribosome-associated chaperone Ssb. These phenotypic differences indicate that controlling Nat activities may serve to elicit distinct cellular responses.

Factors associated with early and later dropout from methadone maintenance treatment in specialist addiction clinics: a six-year cohort study using proportional hazards frailty models for recurrent treatment episodes.

BACKGROUND: Retention in methadone maintenance treatment (MMT) is associated with reduced illicit drug use, criminal activity, and mortality; however, many clients move in and out of MMT. This study aims to identify determinants of time to dropout of MMT across multiple treatment episodes in specialist addiction services in Ireland. METHODS: Cohort study of persons attending specialist addiction clinics between 2010 and 2015. MMT episodes were periods of continuous treatment if there were no interruptions to treatment lasting > 7days. Proportional hazards frailty models were used to assess factors associated with time to dropout from recurrent MMT episodes at 3 (90 days) and 12 months (91-365 days). MMT episodes were right- censored at time of death, transfer to prison or primary care, and study end. RESULTS: A total of 2,035 individuals experienced 4,969 MMT episodes, with 2,724 dropout events during the six-year follow-up. Factors associated with dropout at 3 months included low dose methadone (<60 mg/day) (HR = 1.49, 95% CI 1.29-1.73) and previous dropout (HR = 1.65, 95% CI 1.41-1.92). Adherence was protective (HR = 0.91, 95% CI 0.90-0.92). Dropout at 12 months was associated with low dose methadone (HR = 1.44, 95% CI 1.23-1.68), previous dropout (HR = 1.37, 95% CI 1.16-1.61), males (HR 1.26, 95% CI 1.06-1.50), benzodiazepines (HR = 1.22, 95% CI 1.03-1.45) and number of comorbidities (HR = 1.12, 95% CI 1.05-1.20); adherence was protective (HR = 0.86, 95% CI 0.84-0.87). CONCLUSIONS: Clients with a previous history of treatment dropout and those on low dose methadone should be identified as high risk for both early and later dropout. Inversely, adherence to treatment, not missing methadone doses, is protective.

Media Representations of Science during the First Wave of the COVID-19 Pandemic: A Qualitative Analysis of News and Social Media on the Island of Ireland.

COVID-19 is arguably the most critical science communication challenge of a generation, yet comes in the wake of a purported populist turn against scientific expertise in western societies. This study advances understanding of science-society relations during the COVID-19 pandemic by analysing how science was represented in news and social media coverage of COVID-19 on the island of Ireland. Thematic analysis was performed on a dataset comprising 952 news articles and 603 tweets published between 1 January and 31 May 2020. Three themes characterised the range of meanings attached to science: 'Defining science: Its subjects, practice and process', 'Relating to science: Between veneration and suspicion' and 'Using science: As solution, policy and rhetoric'. The analysis suggested that the COVID-19 pandemic represented a platform to highlight the value, philosophy, process and day-to-day activity of scientific research. However, the study also identified risks the pandemic might pose to science communication, including feeding public alienation by disparaging lay understandings, reinforcing stereotypical images of scientists, and amplifying the politicisation of scientific statements.

Bordering on crisis: A qualitative analysis of focus group, social media, and news media perspectives on the Republic of Ireland-Northern Ireland border during the 'first wave' of the COVID-19 pandemic.

RATIONALE: International border controls were among the earliest and most effective of measures to constrain transmission of COVID-19. However, such measures are complex when established borders are open yet politically contested, as for the border that divides the Republic of Ireland (ROI) from Northern Ireland (NI). Understanding how this border affected the everyday lives of both populations during the pandemic is important for informing the continued development of effective responses to COVID-19 and future health crises. OBJECTIVE: This multi-methods study aimed to explore public perspectives on how the ROI-NI border affected experiences of and responses to the 'first wave' of the pandemic. METHOD: The study collated data from focus groups (n = 8), news articles (n = 967), and Twitter posts (n = 356) on the island of Ireland, which mentioned the ROI-NI border in relation to COVID-19. Thematic analysis was used to explore the range of perspectives on the role played by the border during the early months of the pandemic. RESULTS: Analysis identified three themes: Cross-Border Interdependencies illustrated the complexity and challenges of living near the border; Interpretations of Cross-Border Policy Disparities showed that lay publics perceived NI and ROI policy approaches as discordant and politicised; and Responses to Cross-Border Policy Disparities revealed alternating calls to either strengthen border controls, or pursue a unified all-island approach. CONCLUSIONS: Results reveal clear public appetite for greater synchronisation of cross-border pandemic responses, emphasise the specific vulnerability of communities living near the border, and highlight the risk of long-term socio-political repercussions of border management decisions taken during the pandemic. Findings will inform implementation of pandemic responses and public health policies in jurisdictions that share a porous land border.