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.

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.

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.

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.

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.

Do interruptions to the continuity of methadone maintenance treatment in specialist addiction settings increase the risk of drug-related poisoning deaths? A retrospective cohort study.

AIMS: To examine the risk of mortality associated with interruptions to the continuity of methadone maintenance treatment (MMT), including transfers between services, in opioid-dependent individuals attending specialist addiction services. DESIGN: Retrospective cohort study using addiction services and primary care dispensing records, the National Methadone Register and National Drug-Related Death Index (NDRDI). SETTING: Geographically defined population in Dublin, Ireland. PARTICIPANTS: A total of 2899 people prescribed and dispensed methadone in specialist addiction services between January 2010 and December 2015. There were five exposure groups: weeks 1-4 following transfer between treatment providers; weeks 1-4 out of treatment; weeks 5-52 out of treatment; weeks 1-4 of treatment initiation; and weeks 5+ of continuous treatment (reference category). MEASUREMENTS: Primary outcome: drug-related poisoning (DRP) deaths. Secondary outcome: all-cause mortality (ACM). Mortality rates calculated by dividing number of deaths (DRP; ACM) in exposure groups by person-years exposure. Unadjusted and adjusted Poisson regression (covariates age, sex, incarceration, methadone dose and comorbidities) estimated differences in mortality rates. FINDINGS: There were 154 ACM deaths, 55 (35.7%) identified as DRP deaths. No deaths were observed in the first month following transfer between treatment providers. The risk of DRP mortality was highest in weeks 1-4 out of treatment [adjusted relative risk (aRR = 4.04, 95% confidence interval (CI) = 1.43-11.43, P = 0.009] and weeks 1-4 of treatment initiation (ARR = 3.4, 95% CI = 1.2-9.64, P = 0.02). Similarly, risk of ACM was highest in weeks 1-4 out of treatment (ARR = 11.78, 95% CI = 7.73-17.94, P < 0.001), weeks 1-4 of treatment initiation (aRR = 5.11, 95% CI = 2.95-8.83, P < 0.001) and weeks 5-52 off treatment (aRR = 2.04, 95% CI = 1.2-3.47, P = 0.009). CONCLUSIONS: Interruptions to the continuity of methadone maintenance treatment by treatment provider do not appear to be periods of risk for drug-related poisoning or all-cause mortality deaths. Risk of drug related poisoning and all-cause mortality deaths appears to be greatest during the first 4 weeks of treatment initiation/re-initiation and after treatment cessation.

Study protocol for the COvid-19 Toolbox for All IslaNd (CONTAIN) project: A cross-border analysis in Ireland to disentangle psychological, behavioural, media and governmental responses to COVID-19.

COVID-19 represents a serious challenge to governments and healthcare systems. In addition to testing/contact tracing, behavioural and social responses such as handwashing and social distancing or cocooning are effective tools for mitigating the spread of the disease. Psychological (e.g., risk perceptions, self-efficacy) and contextual factors (government, public health messaging, etc.) are likely to drive these behaviours. Collated real-time information of these indicators strengthens local, national and international public health advice and messaging. Further, understanding how well public health and government messages and measures are understood, communicated via (social) media and adhered to is vital. There are two governments and public health jurisdictions on the island of Ireland, the Republic of Ireland (ROI) and Northern Ireland (NI). This represents an opportunity to explore implications of differing measures and messaging across these two jurisdictions as they relate to COVID-19 on two similar populations. The expert research team are drawn from a range of disciplines in the two countries. This project has four nested studies: Assessment of key behavioural, social and psychological factors through a large, prospective representative telephone survey of individuals aged over-18 on a weekly basis over eight weeks (n=3072); and conduct qualitative focus groups over the same period.Interrogation of social media messaging and formal media responses in both jurisdictions to investigate the spread of (mis)information.Modelling data from Studies 1 and 2, plotting the psychosocial/behavioural and media messaging information with international, ROI and NI incidence and mortality data. Conducting an assessment of health policy transfer in an attempt to incorporate the most significant public health and political insights from each jurisdiction. The CONTAIN project will develop an evidence-based toolbox for targeting public health messaging and political leadership and will be created for use for the anticipated second wave of COVID-19, and subsequently for future epidemics/pandemics.

Histopathological Image QTL Discovery of Immune Infiltration Variants.

Genotype-to-phenotype association studies typically use macroscopic physiological measurements or molecular readouts as quantitative traits. There are comparatively few suitable quantitative traits available between cell and tissue length scales, a limitation that hinders our ability to identify variants affecting phenotype at many clinically informative levels. Here we show that quantitative image features, automatically extracted from histopathological imaging data, can be used for image quantitative trait loci (iQTLs) mapping and variant discovery. Using thyroid pathology images, clinical metadata, and genomics data from the Genotype-Tissue Expression (GTEx) project, we establish and validate a quantitative imaging biomarker for immune cell infiltration. A total of 100,215 variants were selected for iQTL profiling and tested for genotype-phenotype associations with our quantitative imaging biomarker. Significant associations were found in HDAC9 and TXNDC5. We validated the TXNDC5 association using GTEx cis-expression QTL data and an independent hypothyroidism dataset from the Electronic Medical Records and Genomics network.

Data Analysis Strategies in Medical Imaging.

Radiographic imaging continues to be one of the most effective and clinically useful tools within oncology. Sophistication of artificial intelligence has allowed for detailed quantification of radiographic characteristics of tissues using predefined engineered algorithms or deep learning methods. Precedents in radiology as well as a wealth of research studies hint at the clinical relevance of these characteristics. However, critical challenges are associated with the analysis of medical imaging data. Although some of these challenges are specific to the imaging field, many others like reproducibility and batch effects are generic and have already been addressed in other quantitative fields such as genomics. Here, we identify these pitfalls and provide recommendations for analysis strategies of medical imaging data, including data normalization, development of robust models, and rigorous statistical analyses. Adhering to these recommendations will not only improve analysis quality but also enhance precision medicine by allowing better integration of imaging data with other biomedical data sources. Clin Cancer Res; 24(15); 3492-9. ©2018 AACR.

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.

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.

J-shaped relationship between supervised methadone consumption and retention in methadone maintenance treatment (MMT) in primary care: National cohort study.

BACKGROUND: Supervised consumption ensures patients take methadone as prescribed and prevents diversion, however, the influence of supervised consumption on retention is unclear. We examined association between supervised consumption and retention across multiple treatment episodes. METHODS: Cohort study of persons experiencing ≥1 MMT episodes in primary care (2004-2010), excluding ongoing episodes at the start of follow-up. Length of treatment episodes based on methadone prescriptions, retention classified as no interruption in prescribed methadone lasting >7 days. When a patient did not receive a new prescription within seven days after the end of coverage of a prescription, they were considered to have ceased treatment. We evaluated the relationship between supervised consumption and time to discontinuation of treatment using proportional hazards gamma frailty models to account for recurrent MMT episodes. Age, gender, median daily methadone dose, and comorbidities included as potential confounders. RESULTS: 6393 patients experienced 19,715 treatment episodes over the six-year follow-up period. A J-shaped relationship was observed; having between 20 and 60% of methadone scripts supervised (compared to <20%) associated with reduced time to discontinuation (20-39% HR=0.88, 95% CI 0.81-0.95; 40-59%: HR=0.87, 95% CI 0.81-0.94). Beyond a threshold of 60%, retention reduced (60-79% of scripts: HR=1.28, 95% CI 1.20-1.36;>80% of scripts: HR=3.59, 95% CI 3.38-3.81). Median daily dose between 60 and 120mg/per day, and multiple treatment episodes also associated with longer time to discontinuation of treatment. CONCLUSION: A J-shaped relationship was observed between supervised consumption and retention in treatment. Additionally, patients experiencing multiple treatment episodes tend to stay in treatment for progressively longer periods of time.

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.

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.

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.

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.