Synthetic biology approach towards engineering of Shewanella oneidensis MR-1 for microbial fuel cell technologies

In the past decade the emerging field of microbial electrochemical technologies (METs) has gained increased attention due to its potential for bioenergy production and bioremediation. By utilizing pollutants or waste as carbon sources electroactive bacteria (EAB) can convert chemical energy into electricity, thereby conceivably closing the waste disposal energy generation loop. These EABs can generate current anaerobically by forming an electroactive biofilm on conductive electrode materials via extracellular electron transfer (EET). The genetically tractable EAB model organism Shewanella oneidensis MR-1 (SOMR-1) already possesses several EET routes and a large respiration versatility. These traits make it feasible as a synthetic biology chassis to increase predictability, stability and novel functionalities of MET applications. However, as synthetic gene circuits become more elaborate in size and complexity and only relatively few well-characterized biological parts have been described for this organism, precise genetic engineering increasingly presents a bottleneck for this new technology. Here, the synthetic biology toolbox for SOMR-1 was expanded by establishing the Standardised European Vector Architecture (SEVA) plasmid platform providing characterisation of plasmid maintenance with a large range of replication origins, quantification of plasmid copy numbers and their compatibility as multi-plasmid bearing systems in SOMR-1. Further, establishment of transcriptional regulation using oxygen independent inducible promoters was realised. In this work the novel cyclohexanone inducible promoter PChnB/ChnR was introduced among others and characterised using oxygen independent reporter assays. A synthetic flavin gene operon under the control of PChnB/ChnR was used to show enhancement of SOMR-1 EET in small-scale MFCs using screen-printed electrode technology. Additional screening methods are presented which were aimed to identify novel EET capabilities in SOMR-1 using a colorimetric tungsten trioxide (WO3) assay

Diagnostics and monitoring tools for noncommunicable diseases: a missing component in the global response.

A key component of any health system is the capacity to accurately diagnose individuals. One of the six building blocks of a health system as defined by the World Health Organization (WHO) includes diagnostic tools. The WHO's Noncommunicable Disease Global Action Plan includes addressing the lack of diagnostics for noncommunicable diseases, through multi-stakeholder collaborations to develop new technologies that are affordable, safe, effective and quality controlled, and improving laboratory and diagnostic capacity and human resources. Many challenges exist beyond price and availability for the current tools included in the Package of Essential Noncommunicable Disease Interventions (PEN) for cardiovascular disease, diabetes and chronic respiratory diseases. These include temperature stability, adaptability to various settings (e.g. at high altitude), need for training in order to perform and interpret the test, the need for maintenance and calibration, and for Blood Glucose Meters non-compatible meters and test strips. To date the issues surrounding access to diagnostic and monitoring tools for noncommunicable diseases have not been addressed in much detail. The aim of this Commentary is to present the current landscape and challenges with regards to guidance from the WHO on diagnostic tools using the WHO REASSURED criteria, which define a set of key characteristics for diagnostic tests and tools. These criteria have been used for communicable diseases, but so far have not been used for noncommunicable diseases. Diagnostic tools have played an important role in addressing many communicable diseases, such as HIV, TB and neglected tropical diseases. Clearly more attention with regards to diagnostics for noncommunicable diseases as a key component of the health system is needed

Development of a target product profile for a point-of-care cardiometabolic device

INTRODUCTION: Multi-parameter diagnostic devices can simplify cardiometabolic disease diagnosis. However, existing devices may not be suitable for use in low-resource settings, where the burden of non-communicable diseases is high. Here we describe the development of a target product profile (TPP) for a point-of-care multi-parameter device for detection of biomarkers for cardiovascular disease and metabolic disorders, including diabetes, in primary care settings in low- and middle-income countries (LMICs). METHODS: A draft TPP developed by an expert group was reviewed through an online survey and semi-structured expert interviews to identify device characteristics requiring refinement. The draft TPP included 41 characteristics with minimal and optimal requirements; characteristics with an agreement level for either requirement of ≤ 85% in either the survey or among interviewees were further discussed by the expert group and amended as appropriate. RESULTS: Twenty people responded to the online survey and 18 experts participated in the interviews. Twenty-two characteristics had an agreement level of ≤ 85% in either the online survey or interviews. The final TPP defines the device as intended to be used for basic diagnosis and management of cardiometabolic disorders (lipids, glucose, HbA1c, and creatinine) as minimal requirement, and offering an expanded test menu for wider cardiometabolic disease management as optimal requirement. To be suitable, the device should be intended for level 1 healthcare settings or lower, used by minimally trained healthcare workers and allow testing using self-contained cartridges or strips without the need for additional reagents. Throughput should be one sample at a time in a single or multi-analyte cartridge, or optimally enable testing of several samples and analytes in parallel with random access. CONCLUSION: This TPP will inform developers of cardiometabolic multi-parameter devices for LMIC settings, and will support decision makers in the evaluation of existing and future devices

Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4(+/+)) or lack a complete Slc26a4 gene (Slc26a4(-/-)). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K(+ )concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4(-/- )mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4(-/- )mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K(+ )channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K(+ )concentrations were normal and membrane proteins necessary for K(+ )secretion were present, including the K(+ )channel KCNQ1 and KCNE1, Na(+)/2Cl(-)/K(+ )cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome

Development and validation of an LC-MS/MS method for the analysis of ivermectin in plasma, whole blood, and dried blood spots using a fully automatic extraction system.

Ivermectin is deployed in mass drug administration (MDA) campaigns to control parasitic diseases in the tropics, with billions of treatments having been administered in the last three decades. Simple blood sampling tools, like the dried blood spots (DBS) technique, are needed to monitor treatments in such challenging settings. Thus, we developed a fully automated method for the analysis of ivermectin in DBS microsamples, including a bioanalytical and clinical validation. Automated extraction was carried out using a DBS-MS 500 autosampler which was coupled to a LC-MS/MS system. DBS were extracted with 20 μL solvent and eluted on a C8 analytical column. Analysis was performed by multiple reaction monitoring in the positive mode. Automated DBS extraction resulted in consistent recoveries (62.8 ± 4.3%) and matrix effects (68.0 ± 8.1%) between different donors and concentration levels. Intra- and inter-day accuracy and precision deviations were ≤15%, while samples with hematocrits from 20 to 60% could be quantified reliably. The achieved sensitivity of 1 ng/mL in DBS samples is sufficient to analyze ivermectin at the dose given (single oral administration of 12 mg) over a period of at least 72 h post treatment. Importantly, DBS samples are stable after one-month storage at room temperature (accuracy: 88.8-96.2%), thus samples collected in the field must not be shipped on dry ice. Ivermectin concentrations in venous and capillary blood agreed strongly, with a mean difference of -4.8%. Moreover, the drying process of DBS did not alter the analysis and importantly plasma concentrations can be estimated from DBS data using the hematocrit and red blood cell partitioning as correction factor. Our method enables uncomplicated sample collection and shipment as well as automated analysis of large amounts of samples, which is key to surveying MDA campaigns in remote settings

Technologies for Diabetes Self-Monitoring: A Scoping Review and Assessment Using the REASSURED Criteria

Background: Self-management is an important pillar for diabetes control and to achieve it, glucose self-monitoring devices are needed. Currently, there exist several different devices in the market and many others are being developed. However, whether these devices are suitable to be used in resource constrained settings is yet to be evaluated.Aims: To assess existing glucose monitoring tools and also those in development against the REASSURED which have been previously used to evaluate diagnostic tools for communicable diseases.Methods: We conducted a scoping review by searching PubMed for peer-review articles published in either English, Spanish or Portuguese in the last 5 years. We selected papers including information about devices used for self-monitoring and tested on humans with diabetes; then, the REASSURED criteria were used to assess them.Results: We found a total of 7 continuous glucose monitoring device groups, 6 non-continuous, and 6 devices in development. Accuracy varied between devices and most of them were either invasive or minimally invasive. Little to no evidence is published around robustness, affordability and delivery to those in need. However, when reviewing publicly available prices, none of the devices would be affordable for people living in low- and middle-income countries.Conclusions: Available devices cannot be considered adapted for use in self-monitoring in resource constraints settings. Further studies should aim to develop less-invasive devices that do not require a large set of components. Additionally, we suggest some improvement in the REASSURED criteria such as the inclusion of patient-important outcomes to increase its appropriateness to assess non-communicable diseases devices.</p

Integration of point-of-care screening for type 2 diabetes mellitus and hypertension with COVID-19 rapid antigen screening in Johannesburg, South Africa

Aims: We sought to evaluate the yield and linkage-to-care for diabetes and hypertension screening alongside a study assessing the use of rapid antigen tests for COVID-19 in taxi ranks in Johannesburg, South Africa. Methods: Participants were recruited from Germiston taxi rank. We recorded results of blood glucose (BG), blood pressure (BP), waist circumference, smoking status, height, and weight. Participants who had elevated BG (fasting>7.0; random>11.1mmol/L) and/or BP (diastolic>90 and systolic>140mmHg) were referred to their clinic and phoned to confirm linkage. Results: 1169 participants were enrolled and screened for diabetes and hypertension. Combining participants with a previous diagnosis of diabetes (n=23, 2%; 95% CI:1.3-2.9%) and those that had an elevated BG measurement (n=60, 5.2%; 95%CI:4.1-6.6%) at study enrollment, we estimated an overall indicative prevalence of diabetes of 7.1% (95% CI:5.7-8.7%). When combining those with known hypertension at study enrollment (n=124, 10.6%; 95%CI:8.9-12.5%) and those with elevated BP (n=202; 17.3%; 95%CI:15.2-19.5%), we get an overall prevalence of hypertension of 27.9% (95% CI:25.4-30.1%). Only 31.7% of those with elevated BG and 16.0% of those with elevated BP linked-to-care. Conclusion: By opportunistically leveraging existing COVID-19 screening in South Africa to screen for diabetes and hypertension, 24% of participants received a potential new diagnosis. We had poor linkage-to-care following screening. Future research should evaluate options for improving linkage-to-care, and evaluate the large-scale feasibility of this simple screening tool.The Foundation for Innovative New Diagnostics (FIND) and the National Institute of Diabetes and Digestive and Kidney (NIDDK) grant 1K01DK116929-01A1

Integration of point-of-care screening for type 2 diabetes mellitus and hypertension into the COVID-19 vaccine programme in Johannesburg, South Africa

Abstract Background South Africa grapples with a substantial burden of non-communicable diseases (NCDs), particularly type 2 diabetes (diabetes) and hypertension. However, these conditions are often underdiagnosed and poorly managed, further exacerbated by the strained primary healthcare (PHC) system and the disruptive impact of the COVID-19 pandemic. Integrating NCD screening with large-scale healthcare initiatives, such as COVID-19 vaccination campaigns, offers a potential solution, especially in low- and middle-income countries (LMICs). We investigated the feasibility and effectiveness of this integration. Methods A prospective cohort study was conducted at four government health facilities in Johannesburg, South Africa. NCD screening was incorporated into the COVID-19 vaccination campaign. Participants underwent COVID-19 rapid tests, blood glucose checks, blood pressure assessments, and anthropometric measurements. Those with elevated blood glucose or blood pressure values received referrals for diagnostic confirmation at local PHC centers. Results Among 1,376 participants screened, the overall diabetes prevalence was 4.1%, combining previously diagnosed cases and newly identified elevated blood glucose levels. Similarly, the hypertension prevalence was 19.4%, comprising pre-existing diagnoses and newly detected elevated blood pressure cases. Notably, 46.1% of participants displayed waist circumferences indicative of metabolic syndrome, more prevalent among females. Impressively, 7.8% of all participants screened were potentially newly diagnosed with diabetes or hypertension. Approximately 50% of individuals with elevated blood glucose or blood pressure successfully linked to follow-up care within four weeks. Conclusion Our study underscores the value of utilizing even brief healthcare interactions as opportunities for screening additional health conditions, thereby aiding the identification of previously undiagnosed cases. Integrating NCD screenings into routine healthcare visits holds promise, especially in resource-constrained settings. Nonetheless, concerted efforts to strengthen care linkage are crucial for holistic NCD management and control. These findings provide actionable insights for addressing the NCD challenge and improving healthcare delivery in LMICs

Addressing the diagnostic gap in hypertension through possible interventions and scale-up: A microsimulation study

Background Cardiovascular diseases (CVDs) are the leading cause of mortality globally with almost a third of all annual deaths worldwide. Low- and middle-income countries (LMICs) are disproportionately highly affected covering 80% of these deaths. For CVD, hypertension (HTN) is the leading modifiable risk factor. The comparative impact of diagnostic interventions that improve either the accuracy, the reach, or the completion of HTN screening in comparison to the current standard of care has not been estimated. Methods and findings This microsimulation study estimated the impact on HTN-induced morbidity and mortality in LMICs for four different scenarios: (S1) lower HTN diagnostic accuracy; (S2) improved HTN diagnostic accuracy; (S3) better implementation strategies to reach more persons with existing tools; and, lastly, (S4) the wider use of easy-to-use tools, such as validated, automated digital blood pressure measurement devices to enhance screening completion, in comparison to the current standard of care (S0). Our hypothetical population was parametrized using nationally representative, individual-level HPACC data and the global burden of disease data. The prevalence of HTN in the population was 31% out of which 60% remained undiagnosed. We investigated how the alteration of a yearly blood pressure screening event impacts morbidity and mortality in the population over a period of 10 years. The study showed that while improving test accuracy avoids 0.6% of HTN-induced deaths over 10 years (13,856,507 [9,382,742; 17,395,833]), almost 40 million (39,650,363 [31,34,233, 49,298,921], i.e., 12.7% [9.9, 15.8]) of the HTN-induced deaths could be prevented by increasing coverage and completion of a screening event in the same time frame. Doubling the coverage only would still prevent 3,304,212 million ([2,274,664; 4,164,180], 12.1% [8.3, 15.2]) CVD events 10 years after the rollout of the program. Our study is limited by the scarce data available on HTN and CVD from LMICs. We had to pool some parameters across stratification groups, and additional information, such as dietary habits, lifestyle choice, or the blood pressure evolution, could not be considered. Nevertheless, the microsimulation enabled us to include substantial heterogeneity and stochasticity toward the different income groups and personal CVD risk scores in the model. Conclusions While it is important to consider investing in newer diagnostics for blood pressure testing to continuously improve ease of use and accuracy, more emphasis should be placed on screening completion. In a micro-simulation study, Lisa Koeppel and co-authors explore the comparative impact of interventions for addressing the diagnostic gap in hypertension screening in low- and middle-income countries. Author summary Why was this study done? Cardiovascular diseases (CVDs) are the leading cause of mortality globally, affecting low- and middle-income countries (LMICs) disproportionally highly. Hypertension (HTN) is the leading modifiable risk factor for CVDs. The diagnosis of HTN and thus the access to treatment is hampered by the necessity of at least one repeated measurement for a final diagnosis and the operator-dependent variability of blood pressure measurement. It is unclear which strategies would be the most impactful to close the diagnostic gap: more accurate, easy-to-use and/or more scalable tools or better implementation strategies to reach more persons with existing tools. What did the researchers do and find? We developed a stochastic microsimulation model that examines the impact of possible diagnostic interventions and implementation strategies on HTN-induced morbidity and mortality in LMICs. The different scenarios were applied over a period of 10 years and affected the individual risk of experiencing a CVD event. While improving test accuracy avoids only 0.6% of HTN-induced deaths over 10 years, scaling up test coverage and completion can avoid almost 40 million HTN-induced CVD events and 14 million (13.7%) related deaths. What do these findings mean? This simulation demonstrates the importance of increasing the coverage of testing for HTN and the improvement of screening completion over diagnostic accuracy of HTN testing. Strategies to narrow the diagnostic gap in HTN should put more emphasis on screening completion