Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay

Item does not contain fulltex

Gene expression in sight: Single-molecule studies of gene expression control

Humans are multicellulair organisms, which means that the human body consists out of many individual cells. On average, the human body comprises an astounding number of 20 to 30 trillion cells. These cells are not all the same: for example there are skin cells, muscle cells, and nerve cells. Nowadays, it is estimated that over 400 different cell types can be found in the human body. The differences between our cells is not explained by the DNA that they contain. Instead, the differences arise because cells use or ‘read’ the DNA in distinct ways. The DNA consists of an enormous amount of nucleotides that together contain the information for approximately 30.000 genes. By controlling which genes are expressed, a cell can control if it becomes a skin cell or a muscle cell. Taken together this process is referred to as gene expression regulation. In this thesis we have studied three distinct processes that are involved in gene regulation. To study these processes we have used methods such as single-molecule imaging and single cell RNA sequencing. Using these methods we have studied how Argonaute2 finds and cleaves its RNA targets in a living cell. We have investigated how mRNA degradation is involved in remodelling the transcriptome during and after cell division. And, finally, we have set-up a method to assess how promoter-enhancer interactions result in active transcription

Quantification of mRNA translation in live cells using single-molecule imaging

mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3ʹ-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2–4 d to complete (after cell lines expressing all required transgenes have been generated)

Quantification of mRNA translation in live cells using single-molecule imaging

mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3ʹ-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2–4 d to complete (after cell lines expressing all required transgenes have been generated)

Buffers Strongly Modulate Fibrin Self-Assembly into Fibrous Networks

Fibrin is a plasma protein with a central role in blood clotting and wound repair. Upon vascular injury, fibrin forms resilient fibrillar networks (clots) via a multistep self-assembly process, from monomers, to double-stranded protofibrils, to a branched network of thick fibers. In vitro, fibrin self-assembly is sensitive to physicochemical conditions like the solution pH and ionic strength, which tune the strength of the noncovalent driving forces. Here we report a surprising finding that the bufferwhich is necessary to control the pH and is typically considered to be inertalso significantly influences fibrin self-assembly. We show by confocal microscopy and quantitative light scattering that various common buffering agents have no effect on the initial assembly of fibrin monomers into protofibrils but strongly hamper the subsequent lateral association of protofibrils into thicker fibers. We further find that the structural changes are independent of the molecular structure of the buffering agents as well as of the activation mechanism and even occur in fibrin networks formed from platelet-poor plasma. This buffer-mediated decrease in protofibril bundling results in a marked reduction in the permeability of fibrin networks but only weakly influences the elastic modulus of fibrin networks, providing a useful tuning parameter to independently control the elastic properties and the permeability of fibrin networks. Our work raises the possibility that fibrin assembly in vivo may be regulated by variations in the acute-phase levels of bicarbonate and phosphate, which act as physiological buffering agents of blood pH. Moreover, our findings add a new example of buffer-induced effects on biomolecular self-assembly to recent findings for a range of proteins and lipids

Incidence of hepatic decompensation after nucleos(t)ide analogue withdrawal : results from a large, international, multi-ethnic cohort of patients with chronic hepatitis B (RETRACT-B study)

INTRODUCTION: Despite improvements in the management of chronic hepatitis B (CHB), risk of cirrhosis and hepatocellular carcinoma remains. While hepatitis B surface antigen loss is the optimal end point, safe discontinuation of nucleos(t)ide analog (NA) therapy is controversial because of the possibility of severe or fatal reactivation flares.METHODS: This is a multicenter cohort study of virally suppressed, end-of-therapy (EOT) hepatitis B e antigen (HBeAg)-negative CHB patients who stopped NA therapy (n = 1,557). Survival analysis techniques were used to analyze off-therapy rates of hepatic decompensation and differences by patient characteristics. We also examined a subgroup of noncirrhotic patients with consolidation therapy of ≥12 months before cessation (n = 1,289). Hepatic decompensation was considered related to therapy cessation if diagnosed off therapy or within 6 months of starting retreatment. RESULTS: Among the total cohort (11.8% diagnosed with cirrhosis, 84.2% start-of-therapy HBeAg-negative), 20 developed hepatic decompensation after NA cessation; 10 events were among the subgroup. The cumulative incidence of hepatic decompensation at 60 months off therapy among the total cohort and subgroup was 1.8% and 1.1%, respectively. The hepatic decompensation rate was higher among patients with cirrhosis (hazard ratio [HR] 5.08, P &lt; 0.001) and start-of-therapy HBeAg-positive patients (HR 5.23, P &lt; 0.001). This association between start-of-therapy HBeAg status and hepatic decompensation remained significant even among the subgroup (HR 10.5, P &lt; 0.001). DISCUSSION: Patients with cirrhosis and start-of-therapy HBeAg-positive patients should be carefully assessed before stopping NAs to prevent hepatic decompensation. Frequent monitoring of viral and host kinetics after cessation is crucial to determine patient outcome.</p

Differential relapse patterns after discontinuation of entecavir vs tenofovir disoproxil fumarate in chronic hepatitis B

Background and Aims: Whether entecavir (ETV) and tenofovir disoproxil fumarate (TDF) differentially affect relapse and outcomes following treatment discontinuation across different patient subpopulations remains unclear. We aimed to compare rates of off-therapy hepatitis B surface antigen (HBsAg) loss, virological and clinical relapse, and retreatment between chronic hepatitis B (CHB) patients who discontinued TDF or ETV therapy. Methods: This study included 1402 virally suppressed CHB patients who stopped either ETV (n = 981) or TDF (n = 421) therapy between 2001 and 2020 from 13 participating centers across North America, Europe, and Asia. All patients were hepatitis B e antigen–negative at treatment discontinuation. Inverse probability of treatment weighting was used to balance the treatment groups. Outcomes were analyzed using survival methods. Results: During a median off-treatment follow-up of 18 months, HBsAg loss occurred in 96 (6.8%) patients overall. Compared with ETV, TDF was associated with a higher rate of HBsAg loss (P = .03); however, the association was no longer significant after statistical adjustment (P = .61). Virological relapse occurred earlier among TDF-treated patients (P < .01); nonetheless, rates became comparable after the first year off therapy (P = .49). TDF was significantly associated with a higher clinical relapse rate than ETV throughout follow-up (P < .01). The development of a virological or clinical relapse did not affect the rate of HBsAg loss. Retreatment rates were not significantly different between the treatment groups. Conclusions: TDF and ETV have differential relapse patterns but are associated with similar rates of HBsAg loss and retreatment following discontinuation. Finite therapy can be considered for CHB patients on either TDF or ETV therapy

Differential Relapse Patterns After Discontinuation of Entecavir vs Tenofovir Disoproxil Fumarate in Chronic Hepatitis B

Background and Aims: Whether entecavir (ETV) and tenofovir disoproxil fumarate (TDF) differentially affect relapse and outcomes following treatment discontinuation across different patient subpopulations remains unclear. We aimed to compare rates of off-therapy hepatitis B surface antigen (HBsAg) loss, virological and clinical relapse, and retreatment between chronic hepatitis B (CHB) patients who discontinued TDF or ETV therapy. Methods: This study included 1402 virally suppressed CHB patients who stopped either ETV (n = 981) or TDF (n = 421) therapy between 2001 and 2020 from 13 participating centers across North America, Europe, and Asia. All patients were hepatitis B e antigen–negative at treatment discontinuation. Inverse probability of treatment weighting was used to balance the treatment groups. Outcomes were analyzed using survival methods. Results: During a median off-treatment follow-up of 18 months, HBsAg loss occurred in 96 (6.8%) patients overall. Compared with ETV, TDF was associated with a higher rate of HBsAg loss (P = .03); however, the association was no longer significant after statistical adjustment (P = .61). Virological relapse occurred earlier among TDF-treated patients (P < .01); nonetheless, rates became comparable after the first year off therapy (P = .49). TDF was significantly associated with a higher clinical relapse rate than ETV throughout follow-up (P < .01). The development of a virological or clinical relapse did not affect the rate of HBsAg loss. Retreatment rates were not significantly different between the treatment groups. Conclusions: TDF and ETV have differential relapse patterns but are associated with similar rates of HBsAg loss and retreatment following discontinuation. Finite therapy can be considered for CHB patients on either TDF or ETV therapy