Quantifying Protein-mRNA Interactions in Single Live Cells.

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ß-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ß-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.

Cell-surface-receptor pathways amplify weak, rare and local stimuli to induce cellular responses. This task is accomplished despite signaling components that segregate into nanometer-scale membrane domains. Here we describe a 'catch-and-release' mechanism that amplified and dispersed stimuli by releasing activated kinases from receptors lacking intrinsic catalytic activity. Specifically, we discovered a cycle of recruitment, activation and release for Zap70 kinases at phosphorylated T cell antigen receptors (TCRs). This turned the TCR into a 'catalytic unit' that amplified antigenic stimuli. Zap70 released from the TCR remained at the membrane, translocated, and phosphorylated spatially distinct substrates. The mechanisms described here are based on widely used protein domains and post-translational modifications; therefore, many membrane-associated pathways might employ similar mechanisms for signal amplification and dispersion.

Tagged actin mRNA dysregulation in IGF2BP1[Formula: see text] mice.

Gene expression is tightly regulated by RNA-binding proteins (RBPs) to facilitate cell survival, differentiation, and migration. Previous reports have shown the importance of the Insulin-like Growth Factor II mRNA-Binding Protein (IGF2BP1/IMP1/ZBP1) in regulating RNA fate, including localization, transport, and translation. Here, we generated and characterized a knockout mouse to study RBP regulation. We report that IGF2BP1 is essential for proper brain development and neonatal survival. Specifically, these mice display disorganization in the developing neocortex, and further investigation revealed a loss of cortical marginal cell density at E17.5. We also investigated migratory cell populations in the IGF2BP1[Formula: see text] mice, using BrdU labeling, and detected fewer mitotically active cells in the cortical plate. Since RNA localization is important for cellular migration and directionality, we investigated the regulation of ß-actin messenger RNA (mRNA), a well-characterized target with established roles in cell motility and development. To aid in our understanding of RBP and target mRNA regulation, we generated mice with endogenously labeled ß-actin mRNA (IGF2BP1[Formula: see text]; ß-actin-MS2[Formula: see text]). Using endogenously labeled ß-actin transcripts, we report IGF2BP1[Formula: see text] neurons have increased transcription rates and total ß-actin protein content. In addition, we found decreased transport and anchoring in knockout neurons. Overall, we present an important model for understanding RBP regulation of target mRNA.

International perspectives on the development, application, and evaluation of a multicancer early detection strategy.

The development and implementation of a multicancer early detection (MCED) test that is effective and affordable has the potential to change cancer care systems around the world. However, careful consideration is needed within the context of different health care settings (both low- and middle-income countries and high-income countries) to roll out an MCED test and promote equity in access.

Sustainable HIV treatment in Africa through viral-load-informed differentiated care.

There are inefficiencies in current approaches to monitoring patients on antiretroviral therapy in sub-Saharan Africa. Patients typically attend clinics every 1 to 3 months for clinical assessment. The clinic costs are comparable with the costs of the drugs themselves and CD4 counts are measured every 6 months, but patients are rarely switched to second-line therapies. To ensure sustainability of treatment programmes, a transition to more cost-effective delivery of antiretroviral therapy is needed. In contrast to the CD4 count, measurement of the level of HIV RNA in plasma (the viral load) provides a direct measure of the current treatment effect. Viral-load-informed differentiated care is a means of tailoring care so that those with suppressed viral load visit the clinic less frequently and attention is focussed on those with unsuppressed viral load to promote adherence and timely switching to a second-line regimen. The most feasible approach to measuring viral load in many countries is to collect dried blood spot samples for testing in regional laboratories; however, there have been concerns over the sensitivity and specificity of this approach to define treatment failure and the delay in returning results to the clinic. We use modelling to synthesize evidence and evaluate the cost-effectiveness of viral-load-informed differentiated care, accounting for limitations of dried blood sample testing. We find that viral-load-informed differentiated care using dried blood sample testing is cost-effective and is a recommended strategy for patient monitoring, although further empirical evidence as the approach is rolled out would be of value. We also explore the potential benefits of point-of-care viral load tests that may become available in the future.

Keep the quality high: the benefits of lot testing for the quality control of malaria rapid diagnostic tests.

BACKGROUND: The production and use of malaria rapid diagnostic tests (RDTs) has risen dramatically over the past 20 years. In view of weak or non-existing in vitro diagnostics (IVD) regulations and post-marketing surveillance (PMS) systems in malaria endemic countries, the World Health Organization, later joined by the Foundation for Innovative New Diagnostics, established an independent, centralized performance evaluation and Lot Testing (LT) programme to safeguard against poor quality of RDTs being distributed through the public health sector of malaria endemic countries. RDT performances and manufacturer quality management systems have evolved over the past decade raising questions about the future need for a centralized LT programme. RESULTS: Between 2007 and 2017, 6056 lots have been evaluated, representing approximately 1.6 Billion RDTs. A total of 69 lots (1.1%) failed the quality control. Of these failures, 26 were detected at receipt of the RDT lot in the LT laboratory, representing an estimated 7.9 million poor quality RDTs, and LT requesters were advised that RDTs were not of sufficient quality for use in patient management. Forty-three were detected after long-term storage in the laboratory, of which 24 (56%) were found to be due to a major issue with insufficient buffer volume in single use buffer vials, others predominantly showing loss of sensitivity. The annual cost of running the programme, based on expenses recorded in years 2014-2016, an estimated volume of 700 lots per year and including replenishment of quality control samples, was estimated at US$ 178,500 ($US 255 per lot tested). CONCLUSIONS: Despite the clear benefits of the centralized LT programme and its low cost compared with the potential costs of each country establishing its own PMS system for RDTs, funding concerns have made its future beyond 2020 uncertain. In order to manage the risks of misdiagnosis due to low quality RDTs, and to ensure the continued safety and reliability of malaria case management, there is a need to ensure that an effective and implementable approach to RDT quality control continues to be available to programmes in endemic countries.

ß-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration.

Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell's cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports ß-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of ß-actin mRNA and protein localization by tethering the mRNA to a specific location-in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes.

Economic considerations support C-reactive protein testing alongside malaria rapid diagnostic tests to guide antimicrobial therapy for patients with febrile illness in settings with low malaria endemicity.

Malaria is no longer a common cause of febrile illness in many regions of the tropics. In part, this success is a result of improved access to accurate diagnosis and effective anti-malarial treatment, including in many hard-to-reach rural areas. However, in these settings, management of other causes of febrile illness remains challenging. Health systems are often weak and other than malaria rapid tests no other diagnostics are available. With millions of deaths occurring annually due to treatable bacterial infections and the ever increasing spread of antimicrobial resistance, improvement in the management of febrile illness is a global public health priority. Whilst numerous promising point-of-care diagnostics are in the pipeline, substantial progress can be made in the interim with existing tools: C-reactive protein (CRP) is a highly sensitive and moderately specific biomarker of bacterial infection and has been in clinical use for these purposes for decades, with dozens of low-cost devices commercially available. This paper takes a health-economics approach to consider the possible advantages of CRP point-of-care tests alongside rapid diagnostic tests for malaria, potentially in a single multiplex device, to guide antimicrobial therapy for patients with febrile illness. Three rudimentary assessments of the costs and benefits of this approach all indicate that this is likely to be cost-effective when considering the incremental costs of the CRP tests as compared with either (i) the improved health outcomes for patients with bacterial illnesses; (ii) the costs of antimicrobial resistance averted; or (iii) the economic benefits of better management of remaining malaria cases and shorter malaria elimination campaigns in areas of low transmission. While CRP-guided antibiotic therapy alone cannot resolve all challenges associated with management of febrile illness in remote tropical settings, in the short-term a multiplexed CRP and malaria RDT could be highly cost-effective and utilize the well-established funding and distribution systems already in place for malaria RDTs. These findings should spark further interest amongst industry, academics and policy-makers in the development and deployment of such diagnostics, and discussion on their geographically appropriate use.

Inferring transient particle transport dynamics in live cells.

Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.

Specific interaction of KIF11 with ZBP1 regulates the transport of ß-actin mRNA and cell motility.

ZBP1-modulated localization of ß-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of ß-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of ß-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the ß-actin mRNA, and the ability of KIF11 to transport ß-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes ß-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein.

mRNA on the move: the road to its biological destiny.

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1.

Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, ß-actin, α-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.

Functional availability of medical oxygen for the management of hypoxaemia in Cameroon: A nationwide facility-based cross-sectional survey.

Background: Medical oxygen is essential for managing hypoxaemia, which has a multifactorial origin, including acute and chronic lung diseases such as pneumonia, asthma, and severe malaria. The coronavirus disease 2019 (COVID-19) revealed substantial gaps in the availability and accessibility of safe medical oxygen, especially in low- and middle-income countries (LMICs). This study aimed to assess the availability and sources, as well as the barriers to the availability of functional medical oxygen in hospitals in Cameroon. Methods: This was a nationwide cross-sectional descriptive study conducted from 26 March to 1 June 2021. Using a convenient sampling technique, we sampled accredited public and private COVID-19 treatment centres in all ten regions in Cameroon. Representatives from the selected hospitals were provided with a pre-designed questionnaire assessing the availability, type, and state of medical oxygen in their facilities. All analyses were performed using R. Results: In total, 114 hospitals were included in this study, with functional medical oxygen available in 65% (74/114) of the hospitals. About 85% (23/27) of the reference hospitals and only 59% (51/87) of the district hospitals had available functional medical oxygen. Compared to district hospitals, reference hospitals were more likely to have central oxygen units (reference vs. district: 10 vs. 0%), oxygen cylinders (74 vs. 42%), and oxygen concentrators (79 vs. 51%). The most common barriers to the availability of medical oxygen were inadequate oxygen supply to meet needs (district vs. reference hospitals: 55 vs. 30%), long delays in oxygen bottle refills (51 vs. 49%), and long distances from oxygen suppliers (57 vs. 49%). Conclusions: The availability of medical oxygen in hospitals in Cameroon is suboptimal and more limited in districts compared to reference hospitals. The cost of medical oxygen, delays related to refills and supplies, and long distances from medical sources were the most common barriers to availability in Cameroon.

i-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists.

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.

Optimizing diagnostic networks to increase patient access to TB diagnostic services: Development of the diagnostic network optimization (DNO) approach and learnings from its application in Kenya, India and the Philippines.

Diagnostic network optimization (DNO) is an analytical approach that enables use of available country data to inform evidence-based decision-making to optimize access to diagnostic services. A DNO methodology was developed using available data sources and a commercial supply chain optimization software. In collaboration with Ministries of Health and partners, the approach was applied in Kenya, India and the Philippines to map TB diagnostic networks, identify misalignments, and determine optimal network design to increase patient access to TB diagnostic services and improve device utilization. The DNO analysis was successfully applied to evaluate and inform TB diagnostic services in Kenya, India and the Philippines as part of national strategic planning for TB. The analysis was tailored to each country's specific objectives and allowed evaluation of factors such as the number and placement of different TB diagnostics, design of sample referral networks and integration of early infant diagnosis for HIV at national and sub-national levels and across public and private sectors. Our work demonstrates the value of DNO as an innovative approach to analysing and modelling diagnostic networks, particularly suited for use in low-resource settings, as an open-access approach that can be applied to optimize networks for any disease.

Implementation of a customised antimicrobial resistance laboratory scorecard in Cameroon, Ethiopia and Kenya.

Background: In low-resource settings, antimicrobial resistance (AMR) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. The AMR Scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures. Objective: To evaluate the performance of the AMR Scorecard in 11 pilot laboratory evaluations in three countries also assessed with the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist. Methods: Pilot laboratory evaluations were conducted in Cameroon, Ethiopia and Kenya between February 2019 and March 2019. Assessors with previous SLIPTA and microbiology experience were trained. Assessors performed the laboratory assessments using the SLIPTA and AMR Scorecard tools. Results: Weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. Safety had the highest mean performance score (SLIPTA: 68%; AMR Scorecard: 73%) while management review had the lowest (SLIPTA: 32%; AMR Scorecard: 8%) across all laboratories. The AMR Scorecard scores were generally consistent with SLIPTA scores. The AMR Scorecard identified technical weaknesses in AMR testing, and SLIPTA identified weaknesses in the quality management systems in the laboratories. Conclusion: Since the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA, it is recommended that microbiology laboratories use SLIPTA and the AMR Scorecard in parallel when preparing for accreditation. Expanding the use of the AMR Scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and AMR surveillance.

Target Product Profile for a mobile app to read rapid diagnostic tests to strengthen infectious disease surveillance.

The essential role of rapid diagnostic tests (RDTs) in disease control is compromised every time a test is not performed correctly or its result is not reported accurately and promptly. A mobile app that utilizes the camera and connectivity of a common smartphone can fill this role of supporting the test's proper execution and the automatic transmission of results. In a consensus process with 51 expert participants representing the needs of clinical users, healthcare programs, health information systems, surveillance systems, and global public health stakeholders, we developed a Target Product Profile describing the minimal and optimal characteristics of such an app. We collected feedback over two rounds and refined the characteristics to arrive at a preferred agreement level of greater than 75%, with an average of 92% agreement (range: 79-100%). As per this feedback, such an app should be compatible with many RDTs and mobile devices without needing accessories. The app should assist the user with RDT-specific instructions, include checks to facilitate quality control of the testing process and suggest results with ≥ 95% accuracy across common lighting conditions while allowing the user to determine the final result. Data from the app must be under the control of the health program that operates it, and the app should support at least one of the common data exchange formats HL7, FHIR, ASTM or JSON. The Target Product Profile also lays out the minimum data security and privacy requirements for the app.

Designing an optimized diagnostic network to improve access to TB diagnosis and treatment in Lesotho.

BACKGROUND: To reach WHO End tuberculosis (TB) targets, countries need a quality-assured laboratory network equipped with rapid diagnostics for tuberculosis diagnosis and drug susceptibility testing. Diagnostic network analysis aims to inform instrument placement, sample referral, staffing, geographical prioritization, integration of testing enabling targeted investments and programming to meet priority needs. METHODS: Supply chain modelling and optimization software was used to map Lesotho's TB diagnostic network using available data sources, including laboratory and programme reports and health and demographic surveys. Various scenarios were analysed, including current network configuration and inclusion of additional GeneXpert and/or point of care instruments. Different levels of estimated demand for testing services were modelled (current [30,000 tests/year], intermediate [41,000 tests/year] and total demand needed to find all TB cases [88,000 tests/year]). RESULTS: Lesotho's GeneXpert capacity is largely well-located but under-utilized (19/24 sites use under 50% capacity). The network has sufficient capacity to meet current and near-future demand and 70% of estimated total demand. Relocation of 13 existing instruments would deliver equivalent access to services, maintain turnaround time and reduce costs compared with planned procurement of 7 more instruments. Gaps exist in linking people with positive symptom screens to testing; closing this gap would require extra 11,000 tests per year and result in 1000 additional TB patients being treated. Closing the gap in linking diagnosed patients to treatment would result in a further 629 patients being treated. Scale up of capacity to meet total demand will be best achieved using a point-of-care platform in addition to the existing GeneXpert footprint. CONCLUSIONS: Analysis of TB diagnostic networks highlighted key gaps and opportunities to optimize services. Network mapping and optimization should be considered an integral part of strategic planning. By building efficient and patient-centred diagnostic networks, countries will be better equipped to meet End TB targets.

Electronic clinical decision support algorithms incorporating point-of-care diagnostic tests in low-resource settings: a target product profile.

Health workers in low-resource settings often lack the support and tools to follow evidence-based clinical recommendations for diagnosing, treating and managing sick patients. Digital technologies, by combining patient health information and point-of-care diagnostics with evidence-based clinical protocols, can help improve the quality of care and the rational use of resources, and save patient lives. A growing number of electronic clinical decision support algorithms (CDSAs) on mobile devices are being developed and piloted without evidence of safety or impact. Here, we present a target product profile (TPP) for CDSAs aimed at guiding preventive or curative consultations in low-resource settings. This document will help align developer and implementer processes and product specifications with the needs of end users, in terms of quality, safety, performance and operational functionality. To identify the characteristics of CDSAs, a multidisciplinary group of experts (academia, industry and policy makers) with expertise in diagnostic and CDSA development and implementation in low-income and middle-income countries were convened to discuss a draft TPP. The TPP was finalised through a Delphi process to facilitate consensus building. An agreement greater than 75% was reached for all 40 TPP characteristics. In general, experts were in overwhelming agreement that, given that CDSAs provide patient management recommendations, the underlying clinical algorithms should be human-interpretable and evidence-based. Whenever possible, the algorithm's patient management output should take into account pretest disease probabilities and likelihood ratios of clinical and diagnostic predictors. In addition, validation processes should at a minimum show that CDSAs are implementing faithfully the evidence they are based on, and ideally the impact on patient health outcomes. In terms of operational needs, CDSAs should be designed to fit within clinic workflows and function in connectivity-challenged and high-volume settings. Data collected through the tool should conform to local patient privacy regulations and international data standards.

Septins organize endoplasmic reticulum-plasma membrane junctions for STIM1-ORAI1 calcium signalling.

ORAI1 Ca2+ channels in the plasma membrane (PM) are gated by STIM1 at endoplasmic reticulum (ER)-PM junctions to effect store-dependent Ca2+ entry into cells, but little is known about how local STIM-ORAI signalling at junctions is coordinated with overall cellular architecture. Filamentous septins can specify cytoskeletal rearrangements and have been found recently to modulate STIM-ORAI signalling. Here we show by super-resolution imaging of ORAI1, STIM1, and septin 4 in living cells that septins facilitate Ca2+ signalling indirectly. Septin 4 does not colocalize preferentially with ORAI1 in resting or stimulated cells, assemble stably at ER-PM junctions, or specify a boundary that directs or confines ORAI1 to junctions. Rather, ORAI1 is recruited to junctions solely through interaction with STIM proteins, while septins regulate the number of ER-PM junctions and enhance STIM1-ORAI1 interactions within junctions. Thus septins communicate with STIM1 and ORAI1 through protein or lipid intermediaries, and are favorably positioned to coordinate Ca2+ signalling with rearrangements in cellular architecture.