Status and future prospects for mobile phone-enabled diagnostics in Tanzania.

INTRODUCTION: Diagnosis is a key step towards the provision of medical intervention and saving lives. However, in low- and middle-income countries, diagnostic services are mainly centralized in large cities and are costly. Point of care (POC) diagnostic technologies have been developed to fill the diagnostic gap for remote areas. The linkage of POC testing onto smartphones has leveraged the ever-expanding coverage of mobile phones to enhance health services in low- and middle-income countries. Tanzania, like most other middle-income countries, is poised to adopt and deploy the use of mobile phone-enabled diagnostic devices. However, there is limited information on the situation on the ground with regard to readiness and capabilities of the veterinary and medical professionals to make use of this technology. METHODS: In this study we survey awareness, digital literacy and prevalent health condition to focus on in Tanzania to guide development and future implementation of mobile phoned-enable diagnostic tools by veterinary and medical professionals. Data was collected using semi-structured questionnaire with closed and open-ended questions, guided in-depth interviews and focus group discussion administered to the participants after informed consent was obtained. RESULTS: A total of 305 participants from six regions of Tanzania were recruited in the study. The distribution of participants across the six regions was as follows: Kilimanjaro (37), Arusha (31), Tabora (68), Dodoma (61), Mwanza (58), and Iringa (50). Our analysis reveals that only 48.2% (126/255) of participants demonstrated significant awareness of mobile phone-enabled diagnostics. This awareness varies significantly across age groups, professions and geographical locations. Interestingly, while 97.4% of participants own and can operate a smartphone, 62% have never utilized their smartphones for health services, including disease diagnosis. Regarding prevalent health condition to focus on when developing mobile phone -enabled diagnostics tools for Tanzania; there was disparity between medical and veterinary professionals. For medical professionals the top 4 priority diseases were Malaria, Urinary Tract Infections, HIV and Diabetes, while for veterinary professionals they were Brucellosis, Anthrax, Newcastle disease and Rabies. DISCUSSION: Despite the widespread ownership of smartphones among healthcare providers (both human and animal), only a small proportion have utilized these devices for healthcare practices, with none reported for diagnostic purposes. This limited utilization may be attributed to factors such as a lack of awareness, absence of policy guidelines, limited promotion, challenges related to mobile data connectivity, and adherence to cultural practices. CONCLUSION: The majority of medical and veterinary professionals in Tanzania possess the necessary digital literacy to utilize mobile phone-enabled diagnostics and demonstrate readiness to adopt digital technologies and innovations to enhance diagnosis. However, effective implementation will require targeted training and interventions to empower them to effectively apply such innovations for disease diagnosis and other healthcare applications.

Detection of foot-and-mouth disease virus RNA using a closed loop-mediated isothermal amplification system.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals responsible for economic losses that amount to >$20 billion annually. Rapid recognition of FMD cases provides vital information to guide control programmes. A range of point-of-need amplification technologies have been developed which allow sensitive detection of the causative virus (FMDV) in the field at locations remote from laboratories. Here we describe a novel system to detect FMDV RNA using loop-mediated isothermal amplification (LAMP). This test was evaluated using a panel of FMDV isolates (n = 79) and RNA standards demonstrating capability to amplify viral genome directly from clinical material in the absence of nucleic acid extraction. This extraction-free RT-LAMP assay was transferred to a bespoke closed-system lateral flow test (LFT) that was used in combination with a low-cost hand-held heater. Our results show that the RT-LAMP-LFT assay retains a high level of diagnostic and analytical sensitivity when using direct clinical material, with a limit of detection under 80 copies per reaction. Together, our data support the potential for the use of this assay at the point-of-need to facilitate rapid feedback on the status of suspect cases.

Test-retest reliability of a single isometric mid-thigh pull protocol to assess peak force and strength-endurance.

The purpose of this study was to examine the test-retest reliability of strength-endurance protocols using isometric mid-thigh pull (IMTP). Twenty-eight participants (23.2 ± 4.9 years) completed two protocols across four testing sessions. Protocol one consisted of 10 maximal IMTP tests lasting 5 seconds each with 10 seconds rest between. Protocol two consisted of a prolonged 60 second maximal IMTP. Data from protocol 1 was analysed in two ways; (a) use of the highest peak value from the first three IMTP efforts, and the lowest peak value from the final three IMTP efforts, and (b) use of the mean peak force from the first three IMTP efforts and mean peak force from the final three IMTP efforts. Data from protocol two used the highest and lowest peak values in the first- and final-15 seconds. Analyses revealed excellent reliability for peak force across all four testing sessions (ICC = 0.94), as well as good test-retest reliability for strength-endurance for protocol 1 (a; ICC = 0.81, b; ICC = 0.79). Test-retest reliability for protocol 2 was poor (ICC = 0.305). Bland-Altman bias values were smaller for protocol 1(a = -8.8 Nm, b = 21.7 Nm) compared to protocol 2 = (119.3 Nm). Our data suggest that 10 maximal IMTP tests performed as described herein is a reliable method for exercise professionals to assess both peak force and strength-endurance in a single, time-efficient protocol.

Cost-effectiveness of sentinel screening of endemic diseases alongside malaria diagnosis: A case study in schistosomiasis.

BACKGROUND: In countries where malaria is endemic, the use of rapid diagnostic tests(RDTs) has become routine, especially in rural settings. Such regions are characterised by often having other co-endemic infectious diseases, at high levels of prevalence. AIM: To illustrate the potential added-value of "sentinel" screening for patients presenting for a routine diagnostic test for malaria, at healthcare facilities in Uganda. METHODS: We developed an economic model by combining two decision trees, one for malaria and a second for the co-endemic disease schistosomiasis. The integrated model was designed to inform policy strategies for the co-endemic disease in addition to malaria (i.e., whether to test opportunistically for schistosomiasis or use mass drug administration(MDA) as per usual practice).We performed the analysis on three comparators varying testing accuracy and costs. RESULTS: Sentinel screening can provide added value to the testing of patients compared with the status quo: when schistosomiasis prevalence is high then MDA is preferential; if low prevalence, treating no one is preferred. If the disease has average levels of prevalence, then a strategy involving testing is preferred. Prevalence thresholds driving the dominant strategy are dependent upon the model parameters, which are highly context specific. At average levels of prevalence for schistosomiasis and malaria for Uganda, adding a sentinel screening was cost-effective when the accuracy of test was higher than current diagnostics and when economies of scope were generated(Expected value clinical Information = 0.65$ per DALY averted, 137.91$ per correct diagnoses).Protocols using diagnostics with current accuracy levels were preferred only for levels of MDA coverage below 75%. CONCLUSION: The importance of the epidemiological setting is crucial in determining the best cost-effective strategy for detecting endemic disease. Economies of scope can make sentinel screenings cost-effective strategies in specific contexts. Blanket thresholds recommended for MDA may not always be the preferred option for endemic diseases.

De Novo Design of Integrin α5ß1 Modulating Proteins for Regenerative Medicine.

Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.

Investigating Communication Dynamics in Neuronal Network using 3D Gold Microelectrode Arrays.

Although in vitro neuronal network models hold great potential for advancing neuroscience research, with the capacity to provide fundamental insights into mechanisms underlying neuronal functions, the dynamics of cell communication within such networks remain poorly understood. Here, we develop a customizable, polymer modified three-dimensional gold microelectrode array with sufficient stability for high signal-to-noise, long-term, neuronal recording of cultured networks. By using directed spatial and temporal patterns of electrical stimulation of cells to explore synaptic-based communication, we monitored cell network dynamics over 3 weeks, quantifying communication capability using correlation heatmaps and mutual information networks. Analysis of synaptic delay and signal speed between cells enabled us to establish a communication connectivity model. We anticipate that our discoveries of the dynamic changes in communication across the neuronal network will provide a valuable tool for future studies in understanding health and disease as well as in developing effective platforms for evaluating therapies.

Understanding patterns of fatigue in health and disease: protocol for an ecological momentary assessment study using digital technologies.

INTRODUCTION: Fatigue is prevalent across a wide range of medical conditions and can be debilitating and distressing. It is likely that fatigue is experienced differently according to the underlying aetiology, but this is poorly understood. Digital health technologies present a promising approach to give new insights into fatigue.The aim of this study is to use digital health technologies, real-time self-reports and qualitative interview data to investigate how fatigue is experienced over time in participants with myeloma, long COVID, heart failure and in controls without problematic fatigue. Objectives are to understand which sensed parameters add value to the characterisation of fatigue and to determine whether study processes are feasible, acceptable and scalable. METHODS AND ANALYSIS: An ecological momentary assessment study will be carried out over 2 or 4 weeks (participant defined). Individuals with fatigue relating to myeloma (n=10), heart failure (n=10), long COVID (n=10) and controls without problematic fatigue or a study condition (n=10) will be recruited. ECG patches will measure heart rate variability, respiratory rate, body temperature, activity and posture. A wearable bracelet accompanied by environment beacons will measure physical activity, sleep and room location within the home. Self-reports of mental and physical fatigue will be collected via smartphone app four times daily and on-demand. Validated fatigue and affect questionnaires will be completed at baseline and at 2 weeks. End-of-study interviews will investigate experiences of fatigue and study participation. A feedback session will be offered to participants to discuss their data.Data will be analysed using multilevel modelling and machine learning. Interviews and feedback sessions will be analysed using content or thematic analyses. ETHICS AND DISSEMINATION: This study was approved by the East of England-Cambridge East Research Ethics Committee (22/EE/0261). The results will be disseminated in peer-reviewed journals and at international conferences. TRIAL REGISTRATION NUMBER: NCT05622669.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

GABAergic interneurons contribute to the fatal seizure phenotype of CLN2 disease mice.

GABAergic interneuron deficits have been implicated in the epileptogenesis of multiple neurological diseases. While epileptic seizures are a key clinical hallmark of CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), the etiology of these seizures remains elusive. Given that Cln2 R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical interneuron populations, we hypothesized that those two events might be causally related. To address this hypothesis, we first generated an inducible transgenic mouse expressing lysosomal membrane-tethered TPP1 (TPP1LAMP1) on the Cln2 R207X/R207X genetic background to study the cell-autonomous effects of cell-type-specific TPP1 deficiency. We crossed the TPP1LAMP1 mice with Vgat-Cre mice to introduce interneuron-specific TPP1 deficiency. Vgat-Cre ; TPP1LAMP1 mice displayed storage material accumulation in several interneuron populations both in cortex and striatum, and increased susceptibility to die after PTZ-induced seizures. Secondly, to test the role of GABAergic interneuron activity in seizure progression, we selectively activated these cells in Cln2 R207X/R207X mice using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) in in Vgat-Cre : Cln2 R207X/R207X mice. EEG monitoring revealed that DREADD-mediated activation of interneurons via chronic deschloroclozapine administration accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre : Cln2 R207X/R207X mice, suggesting that modulating interneuron activity can exert influence over epileptiform abnormalities in CLN2 disease. Taken together, these results provide new mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

N-cadherin dynamically regulates pediatric glioma cell migration in complex environments.

Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and associated with shorter survival. We found that intercellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as leaders and followers change position, leading to altered N-cadherin levels and organization. Together, the results suggest that pediatric glioma cells adapt to different microenvironments by regulating N-cadherin dynamics and cell-cell contacts.

An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.

BACKGROUND: Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis. METHODS: A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis. RESULTS: Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively. CONCLUSION: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.

N-cadherin dynamically regulates pediatric glioma cell migration in complex environments.

Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and associated with shorter survival. We found that inter-cellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as leaders and followers change position, leading to altered N-cadherin levels and organization. Together, the results suggest that pediatric glioma cells adapt to different microenvironments by regulating N-cadherin dynamics and cell-cell contacts.

Single-Cell Sequencing Reveals Functional Alterations in Tuberculosis.

Despite its importance, the functional heterogeneity surrounding the dynamics of interactions between mycobacterium tuberculosis and human immune cells in determining host immune strength and tuberculosis (TB) outcomes, remains far from understood. This work now describes the development of a new technological platform to elucidate the immune function differences in individuals with TB, integrating single-cell RNA sequencing and cell surface antibody sequencing to provide both genomic and phenotypic information from the same samples. Single-cell analysis of 23 990 peripheral blood mononuclear cells from a new cohort of primary TB patients and healthy controls enables to not only show four distinct immune phenotypes (TB, myeloid, and natural killer (NK) cells), but also determine the dynamic changes in cell population abundance, gene expression, developmental trajectory, transcriptomic regulation, and cell-cell signaling. In doing so, TB-related changes in immune cell functions demonstrate that the immune response is mediated through host T cells, myeloid cells, and NK cells, with TB patients showing decreased naive, cytotoxicity, and memory functions of T cells, rather than their immunoregulatory function. The platform also has the potential to identify new targets for immunotherapeutic treatment strategies to restore T cells from dysfunctional or exhausted states.

Neuronal deletion of the circadian clock gene Bmal1 induces cell-autonomous dopaminergic neurodegeneration.

Circadian rhythm dysfunction is a hallmark of Parkinson disease (PD), and diminished expression of the core clock gene Bmal1 has been described in patients with PD. BMAL1 is required for core circadian clock function but also serves nonrhythmic functions. Germline Bmal1 deletion can cause brain oxidative stress and synapse loss in mice, and it can exacerbate dopaminergic neurodegeneration in response to the toxin MPTP. Here we examined the effect of cell type-specific Bmal1 deletion on dopaminergic neuron viability in vivo. We observed that global, postnatal deletion of Bmal1 caused spontaneous loss of tyrosine hydroxylase+ (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc). This was not replicated by light-induced disruption of behavioral circadian rhythms and was not induced by astrocyte- or microglia-specific Bmal1 deletion. However, either pan-neuronal or TH neuron-specific Bmal1 deletion caused cell-autonomous loss of TH+ neurons in the SNpc. Bmal1 deletion did not change the percentage of TH neuron loss after α-synuclein fibril injection, though Bmal1-KO mice had fewer TH neurons at baseline. Transcriptomics analysis revealed dysregulation of pathways involved in oxidative phosphorylation and Parkinson disease. These findings demonstrate a cell-autonomous role for BMAL1 in regulating dopaminergic neuronal survival and may have important implications for neuroprotection in PD.

A comprehensive multimodal dataset for contactless lip reading and acoustic analysis.

Small-scale motion detection using non-invasive remote sensing techniques has recently garnered significant interest in the field of speech recognition. Our dataset paper aims to facilitate the enhancement and restoration of speech information from diverse data sources for speakers. In this paper, we introduce a novel multimodal dataset based on Radio Frequency, visual, text, audio, laser and lip landmark information, also called RVTALL. Specifically, the dataset consists of 7.5 GHz Channel Impulse Response (CIR) data from ultra-wideband (UWB) radars, 77 GHz frequency modulated continuous wave (FMCW) data from millimeter wave (mmWave) radar, visual and audio information, lip landmarks and laser data, offering a unique multimodal approach to speech recognition research. Meanwhile, a depth camera is adopted to record the landmarks of the subject's lip and voice. Approximately 400 minutes of annotated speech profiles are provided, which are collected from 20 participants speaking 5 vowels, 15 words, and 16 sentences. The dataset has been validated and has potential for the investigation of lip reading and multimodal speech recognition.

Alignment-free construction of double emulsion droplet generation devices incorporating surface wettability contrast.

Although polydimethylsiloxane (PDMS) is a versatile and easy-to-use material for microfluidics, its inherent hydrophobicity often necessitates specific hydrophilic treatment to fabricate microchip architectures for generating double emulsions. These additional processing steps frequently lead to increased complexity, potentially creating barriers to the wider use of promising microfluidic techniques. Here we describe an alignment-free spatial hydrophilic PDMS patterning technique to produce devices for the creation of double emulsions using combinations of PDMS and PDMS/surfactant bilayers. The technique enables us to achieve selective patterning and alignment-free bonding, producing reliable and reproducible water-in-oil-in-water W/O/W droplet emulsions. Our method involves processing devices in a vertical orientation, with the wetting transition contrast being achieved simply by imaging whilst adjusting the PDMS pouring speed (using a mobile phone, for example). We successfully obtain hydrophilic surfaces without distinguishable hydrophobic recovery using a range of surfactant concentrations. Droplet emulsions were produced with low coefficients of variation aligned with those generated with other, more complex, techniques (e.g. 3.8% and 3.1% for the inner and outer diameters, respectively). As a further example, the methods were also demonstrated for liposome production. In future we anticipate that the technique may be applied to other fields, including e.g. reagent delivery, DNA amplification, and encapsulated cell studies.

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow µLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc µL-1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

Cas phosphorylation regulates focal adhesion assembly.

Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated and associated with its Crk/CrkL effectors in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin ß1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin ß1 is activated and core focal adhesion proteins including vinculin, talin, kindlin, and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs, and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin ß1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.

Computational analysis of peripheral blood smears detects disease-associated cytomorphologies.

Many hematological diseases are characterized by altered abundance and morphology of blood cells and their progenitors. Myelodysplastic syndromes (MDS), for example, are a group of blood cancers characterised by cytopenias, dysplasia of hematopoietic cells and blast expansion. Examination of peripheral blood slides (PBS) in MDS often reveals changes such as abnormal granulocyte lobulation or granularity and altered red blood cell (RBC) morphology; however, some of these features are shared with conditions such as haematinic deficiency anemias. Definitive diagnosis of MDS requires expert cytomorphology analysis of bone marrow smears and complementary information such as blood counts, karyotype and molecular genetics testing. Here, we present Haemorasis, a computational method that detects and characterizes white blood cells (WBC) and RBC in PBS. Applied to over 300 individuals with different conditions (SF3B1-mutant and SF3B1-wildtype MDS, megaloblastic anemia, and iron deficiency anemia), Haemorasis detected over half a million WBC and millions of RBC and characterized their morphology. These large sets of cell morphologies can be used in diagnosis and disease subtyping, while identifying novel associations between computational morphotypes and disease. We find that hypolobulated neutrophils and large RBC are characteristic of SF3B1-mutant MDS. Additionally, while prevalent in both iron deficiency and megaloblastic anemia, hyperlobulated neutrophils are larger in the latter. By integrating cytomorphological features using machine learning, Haemorasis was able to distinguish SF3B1-mutant MDS from other MDS using cytomorphology and blood counts alone, with high predictive performance. We validate our findings externally, showing that they generalize to other centers and scanners. Collectively, our work reveals the potential for the large-scale incorporation of automated cytomorphology into routine diagnostic workflows.