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.

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.

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.

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.

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.

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.

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.

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.

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.

Phage-Inducible Chromosomal Islands as a Diagnostic Platform to Capture and Detect Bacterial Pathogens.

Phage-inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user-friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL-1 . In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell-free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.

Gene therapy ameliorates spontaneous seizures associated with cortical neuron loss in a Cln2R207X mouse model.

Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9-mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.

A mouse mutant deficient in both neuronal ceroid lipofuscinosis-associated proteins CLN3 and TPP1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3, respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2, and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1.

Sequence terminus dependent PCR for site-specific mutation and modification detection.

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).