The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Genomic Analyses from Non-invasive Prenatal Testing Reveal Genetic Associations, Patterns of Viral Infections, and Chinese Population History.

We analyze whole-genome sequencing data from 141,431 Chinese women generated for non-invasive prenatal testing (NIPT). We use these data to characterize the population genetic structure and to investigate genetic associations with maternal and infectious traits. We show that the present day distribution of alleles is a function of both ancient migration and very recent population movements. We reveal novel phenotype-genotype associations, including several replicated associations with height and BMI, an association between maternal age and EMB, and between twin pregnancy and NRG1. Finally, we identify a unique pattern of circulating viral DNA in plasma with high prevalence of hepatitis B and other clinically relevant maternal infections. A GWAS for viral infections identifies an exceptionally strong association between integrated herpesvirus 6 and MOV10L1, which affects piwi-interacting RNA (piRNA) processing and PIWI protein function. These findings demonstrate the great value and potential of accumulating NIPT data for worldwide medical and genetic analyses.

NIPT-PG: empowering non-invasive prenatal testing to learn from population genomics through an incremental pan-genomic approach.

Non-invasive prenatal testing (NIPT) is a quite popular approach for detecting fetal genomic aneuploidies. However, due to the limitations on sequencing read length and coverage, NIPT suffers a bottleneck on further improving performance and conducting earlier detection. The errors mainly come from reference biases and population polymorphism. To break this bottleneck, we proposed NIPT-PG, which enables the NIPT algorithm to learn from population data. A pan-genome model is introduced to incorporate variant and polymorphic loci information from tested population. Subsequently, we proposed a sequence-to-graph alignment method, which considers the read mis-match rates during the mapping process, and an indexing method using hash indexing and adjacency lists to accelerate the read alignment process. Finally, by integrating multi-source aligned read and polymorphic sites across the pan-genome, NIPT-PG obtains a more accurate z-score, thereby improving the accuracy of chromosomal aneuploidy detection. We tested NIPT-PG on two simulated datasets and 745 real-world cell-free DNA sequencing data sets from pregnant women. Results demonstrate that NIPT-PG outperforms the standard z-score test. Furthermore, combining experimental and theoretical analyses, we demonstrate the probably approximately correct learnability of NIPT-PG. In summary, NIPT-PG provides a new perspective for fetal chromosomal aneuploidies detection. NIPT-PG may have broad applications in clinical testing, and its detection results can serve as a reference for false positive samples approaching the critical threshold.

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei.

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level.

Glymphatic-lymphatic coupling: assessment of the evidence from magnetic resonance imaging of humans.

The discoveries that cerebrospinal fluid participates in metabolic perivascular exchange with the brain and further drains solutes to meningeal lymphatic vessels have sparked a tremendous interest in translating these seminal findings from animals to humans. A potential two-way coupling between the brain extra-vascular compartment and the peripheral immune system has implications that exceed those concerning neurodegenerative diseases, but also imply that the central nervous system has pushed its immunological borders toward the periphery, where cross-talk mediated by cerebrospinal fluid may play a role in a range of neoplastic and immunological diseases. Due to its non-invasive approach, magnetic resonance imaging has typically been the preferred methodology in attempts to image the glymphatic system and meningeal lymphatics in humans. Even if flourishing, the research field is still in its cradle, and interpretations of imaging findings that topographically associate with reports from animals have yet seemed to downplay the presence of previously described anatomical constituents, particularly in the dura. In this brief review, we illuminate these challenges and assess the evidence for a glymphatic-lymphatic coupling. Finally, we provide a new perspective on how human brain and meningeal clearance function may possibly be measured in future.

Utilizing non-coding RNA-mediated regulation of ATP binding cassette (ABC) transporters to overcome multidrug resistance to cancer chemotherapy.

Multidrug resistance (MDR) is one of the primary factors that produces treatment failure in patients receiving cancer chemotherapy. MDR is a complex multifactorial phenomenon, characterized by a decrease or abrogation of the efficacy of a wide spectrum of anticancer drugs that are structurally and mechanistically distinct. The overexpression of the ATP-binding cassette (ABC) transporters, notably ABCG2 and ABCB1, are one of the primary mediators of MDR in cancer cells, which promotes the efflux of certain chemotherapeutic drugs from cancer cells, thereby decreasing or abolishing their therapeutic efficacy. A number of studies have suggested that non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play a pivotal role in mediating the upregulation of ABC transporters in certain MDR cancer cells. This review will provide updated information about the induction of ABC transporters due to the aberrant regulation of ncRNAs in cancer cells. We will also discuss the measurement and biological profile of circulating ncRNAs in various body fluids as potential biomarkers for predicting the response of cancer patients to chemotherapy. Sequence variations, such as alternative polyadenylation of mRNA and single nucleotide polymorphism (SNPs) at miRNA target sites, which may indicate the interaction of miRNA-mediated gene regulation with genetic variations to modulate the MDR phenotype, will be reviewed. Finally, we will highlight novel strategies that could be used to modulate ncRNAs and circumvent ABC transporter-mediated MDR.

Controllable Assembly of a Quantum Dot-Based Aptasensor Guided by CRISPR/Cas12a for Direct Measurement of Circulating Tumor Cells in Human Blood.

Accurate and sensitive analysis of circulating tumor cells (CTCs) in human blood provides a non-invasive approach for the evaluation of cancer metastasis and early cancer diagnosis. Herein, we demonstrate the controllable assembly of a quantum dot (QD)-based aptasensor guided by CRISPR/Cas12a for direct measurement of CTCs in human blood. We introduce a magnetic bead@activator/recognizer duplex core-shell structure to construct a multifunctional platform for the capture and direct detection of CTCs in human blood, without the need for additional CTC release and re-identification steps. Notably, the introduction of magnetic separation ensures that only a target-induced free activator can initiate the downstream catalysis, efficiently avoiding the undesired catalysis triggered by inappropriate recognition of the activator/recognizer duplex structure by crRNAs. This aptasensor achieves high CTC-capture efficiency (82.72%) and sensitive detection of CTCs with a limit of detection of 2 cells mL-1 in human blood, holding great promise for the liquid biopsy of cancers.

EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD): Executive Summary.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously termed non-alcoholic fatty liver disease (NAFLD), is defined as steatotic liver disease (SLD) in the presence of one or more cardiometabolic risk factor(s) and the absence of harmful alcohol intake. The spectrum of MASLD includes steatosis, metabolic dysfunction-associated steatohepatitis (MASH, previously NASH), fibrosis, cirrhosis and MASH-related hepatocellular carcinoma (HCC). This joint EASL-EASD-EASO guideline provides an update on definitions, prevention, screening, diagnosis and treatment for MASLD. Case-finding strategies for MASLD with liver fibrosis, using non-invasive tests, should be applied in individuals with cardiometabolic risk factors, abnormal liver enzymes and/or radiological signs of hepatic steatosis, particularly in the presence of type 2 diabetes or obesity with additional metabolic risk factor(s). A stepwise approach using blood-based scores (such as the fibrosis-4 index [FIB-4]) and, sequentially, imaging techniques (such as transient elastography) is suitable to rule-out/in advanced fibrosis, which is predictive of liver-related outcomes. In adults with MASLD, lifestyle modification-including weight loss, dietary changes, physical exercise and discouraging alcohol consumption-as well as optimal management of comorbidities-including use of incretin-based therapies (e.g. semaglutide, tirzepatide) for type 2 diabetes or obesity, if indicated-is advised. Bariatric surgery is also an option in individuals with MASLD and obesity. If locally approved and dependent on the label, adults with non-cirrhotic MASH and significant liver fibrosis (stage ≥2) should be considered for a MASH-targeted treatment with resmetirom, which demonstrated histological effectiveness on steatohepatitis and fibrosis with an acceptable safety and tolerability profile. No MASH-targeted pharmacotherapy can currently be recommended for the cirrhotic stage. Management of MASH-related cirrhosis includes adaptations of metabolic drugs, nutritional counselling, surveillance for portal hypertension and HCC, as well as liver transplantation in decompensated cirrhosis.

Personalized depth-specific neuromodulation of the human primary motor cortex via ultrasound.

Non-invasive brain stimulation has the potential to boost neuronal plasticity in the primary motor cortex (M1), but it remains unclear whether the stimulation of both superficial and deep layers of the human motor cortex can effectively promote M1 plasticity. Here, we leveraged transcranial ultrasound stimulation (TUS) to precisely target M1 circuits at depths of approximately 5 mm and 16 mm from the cortical surface. Initially, we generated computed tomography images from each participant's individual anatomical magnetic resonance images (MRI), which allowed for the generation of accurate acoustic simulations. This process ensured that personalized TUS was administered exactly to the targeted depths within M1 for each participant. Using long-term depression and long-term potentiation (LTD/LTP) theta-burst stimulation paradigms, we examined whether TUS over distinct depths of M1 could induce LTD/LTP plasticity. Our findings indicated that continuous theta-burst TUS-induced LTD-like plasticity with both superficial and deep M1 stimulation, persisting for at least 30 min. In comparison, sham TUS did not significantly alter M1 excitability. Moreover, intermittent theta-burst TUS did not result in the induction of LTP- or LTD-like plasticity with either superficial or deep M1 stimulation. These findings suggest that the induction of M1 plasticity can be achieved with ultrasound stimulation targeting distinct depths of M1, which is contingent on the characteristics of TUS. KEY POINTS: The study integrated personalized transcranial ultrasound stimulation (TUS) with electrophysiology to determine whether TUS targeting superficial and deep layers of the human motor cortex (M1) could elicit long-term depression (LTD) or long-term potentiation (LTP) plastic changes. Utilizing acoustic simulations derived from individualized pseudo-computed tomography scans, we ensured the precision of TUS delivery to the intended M1 depths for each participant. Continuous theta-burst TUS targeting both the superficial and deep layers of M1 resulted in the emergence of LTD-like plasticity, lasting for at least 30 min. Administering intermittent theta-burst TUS to both the superficial and deep layers of M1 did not lead to the induction of LTP- or LTD-like plastic changes. We suggest that theta-burst TUS targeting distinct depths of M1 can induce plasticity, but this effect is dependent on specific TUS parameters.

Understanding genetic variability: exploring large-scale copy number variants through non-invasive prenatal testing in European populations.

Large-scale copy number variants (CNVs) are structural alterations in the genome that involve the duplication or deletion of DNA segments, contributing to genetic diversity and playing a crucial role in the evolution and development of various diseases and disorders, as they can lead to the dosage imbalance of one or more genes. Massively parallel sequencing (MPS) has revolutionized the field of genetic analysis and contributed significantly to routine clinical diagnosis and screening. It offers a precise method for detecting CNVs with exceptional accuracy. In this context, a non-invasive prenatal test (NIPT) based on the sequencing of cell-free DNA (cfDNA) from pregnant women's plasma using a low-coverage whole genome MPS (WGS) approach represents a valuable source for population studies. Here, we analyzed genomic data of 12,732 pregnant women from the Slovak (9,230), Czech (1,583), and Hungarian (1,919) populations. We identified 5,062 CNVs ranging from 200 kbp and described their basic characteristics and differences between the subject populations. Our results suggest that re-analysis of sequencing data from routine WGS assays has the potential to obtain large-scale CNV population frequencies, which are not well known and may provide valuable information to support the classification and interpretation of this type of genetic variation. Furthermore, this could contribute to expanding knowledge about the central European genome without investing in additional laboratory work, as NIPTs are a relatively widely used screening method.

An easy-to-implement, non-invasive head restraint method for monkey fMRI.

Functional magnetic resonance imaging (fMRI) in behaving monkeys has a strong potential to bridge the gap between human neuroimaging and primate neurophysiology. In monkey fMRI, to restrain head movements, researchers usually surgically implant a plastic head-post on the skull. Although time-proven to be effective, this technique could create burdens for animals, including a risk of infection and discomfort. Furthermore, the presence of extraneous objects on the skull, such as bone screws and dental cement, adversely affects signals near the cortical surface. These side effects are undesirable in terms of both the practical aspect of efficient data collection and the spirit of "refinement" from the 3R's. Here, we demonstrate that a completely non-invasive fMRI scan in awake monkeys is possible by using a plastic head mask made to fit the skull of individual animals. In all of the three monkeys tested, longitudinal, quantitative assessment of head movements showed that the plastic mask has effectively suppressed head movements, and we were able to obtain reliable retinotopic BOLD signals in a standard retinotopic mapping task. The present, easy-to-make plastic mask has a strong potential to simplify fMRI experiments in awake monkeys, while giving data that is as good as or even better quality than that obtained with the conventional head-post method.

Out-of-phase transcranial alternating current stimulation modulates the neurodynamics of inhibitory control.

Transcranial alternating current stimulation (tACS) is an efficient neuromodulation technique that enhances cognitive function in a non-invasive manner. Using functional magnetic resonance imaging, we investigated whether tACS with different phase lags (0° and 180°) between the dorsal anterior cingulate and left dorsolateral prefrontal cortices modulated inhibitory control performance during the Stroop task. We found out-of-phase tACS mediated improvements in task performance, which was neurodynamically reflected as putamen, dorsolateral prefrontal, and primary motor cortical activation as well as prefrontal-based top-down functional connectivity. Our observations uncover the neurophysiological bases of tACS-phase-dependent neuromodulation and provide a feasible non-invasive approach to effectively modulate inhibitory control.

Neural response during prefrontal theta burst stimulation: Interleaved TMS-fMRI of full iTBS protocols.

BACKGROUND: Left prefrontal intermittent theta-burst stimulation (iTBS) has emerged as a safe and effective transcranial magnetic stimulation (TMS) treatment protocol in depression. Though network effects after iTBS have been widely studied, the deeper mechanistic understanding of target engagement is still at its beginning. Here, we investigate the feasibility of a novel integrated TMS-fMRI setup and accelerated echo planar imaging protocol to directly observe the immediate effects of full iTBS treatment sessions. OBJECTIVE/HYPOTHESIS: In our effort to explore interleaved iTBS-fMRI feasibility, we hypothesize that TMS will induce acute BOLD signal changes in both the stimulated area and interconnected neural regions. METHODS: Concurrent TMS-fMRI with full sessions of neuronavigated iTBS (i.e. 600 pulses) of the left dorsolateral prefrontal cortex (DLPFC) was investigated in 18 healthy participants. In addition, we conducted four TMS-fMRI sessions in a single patient on long-term maintenance iTBS for bipolar depression to test the transfer to clinical cases. RESULTS: Concurrent TMS-fMRI was feasible for iTBS sequences with 600 pulses. During interleaved iTBS-fMRI, an increase of the BOLD signal was observed in a network including bilateral DLPFC regions. In the clinical case, a reduced BOLD response was found in the left DLPFC and the subgenual anterior cingulate cortex, with high variability across individual sessions. CONCLUSIONS: Full iTBS sessions as applied for the treatment of depressive disorders can be established in the interleaved iTBS-fMRI paradigm. In the future, this experimental approach could be valuable in clinical samples, for demonstrating target engagement by iTBS protocols and investigating their mechanisms of therapeutic action.

Non-invasive electrical brain stimulation modulates human conscious perception of mental representation.

Mental representation is a key concept in cognitive science; nevertheless, its neural foundations remain elusive. We employed non-invasive electrical brain stimulation and functional magnetic resonance imaging to address this. During this process, participants perceived flickering red and green visual stimuli, discerning them either as distinct, non-fused colours or as a mentally generated, fused colour (orange). The application of transcranial alternating current stimulation to the medial prefrontal region (a key node of the default-mode network) suppressed haemodynamic activation in higher-order subthalamic and central executive networks associated with the perception of fused colours. This implies that higher-order thalamocortical and default-mode networks are crucial in humans' conscious perception of mental representation.

Promoter profiles in plasma CfDNA exhibits a potential utility of predicting the efficacy of neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Gene expression profiles in breast tissue biopsies contain information related to chemotherapy efficacy. The promoter profiles in cell-free DNA (cfDNA) carrying gene expression information of the original tissues may be used to predict the response to neoadjuvant chemotherapy in breast cancer as a non-invasive biomarker. In this study, the feasibility of the promoter profiles in plasma cfDNA was evaluated as a novel clinical model for noninvasively predicting the efficacy of neoadjuvant chemotherapy in breast cancer. METHOD: First of all, global chromatin (5 Mb windows), sub-compartments and promoter profiles in plasma cfDNA samples from 94 patients with breast cancer before neoadjuvant chemotherapy (pCR = 31 vs. non-pCR = 63) were analyzed, and then classifiers were developed for predicting the efficacy of neoadjuvant chemotherapy in breast cancer. Further, the promoter profile changes in sequential cfDNA samples from 30 patients (pCR = 8 vs. non-pCR = 22) during neoadjuvant chemotherapy were analyzed to explore the potential benefits of cfDNA promoter profile changes as a novel potential biomarker for predicting the treatment efficacy. RESULTS: The results showed significantly distinct promoter profile in plasma cfDNA of pCR patients compared with non-pCR patients before neoadjuvant chemotherapy. The classifier based on promoter profiles in a Random Forest model produced the largest area under the curve of 0.980 (95% CI: 0.978-0.983). After neoadjuvant chemotherapy, 332 genes with significantly differential promoter profile changes in sequential cfDNA samples of pCR patients was observed, compared with non-pCR patients, and their functions were closely related to treatment response. CONCLUSION: These results suggest that promoter profiles in plasma cfDNA may be a powerful, non-invasive tool for predicting the efficacy of neoadjuvant chemotherapy breast cancer patients before treatment, and the on-treatment cfDNA promoter profiles have potential benefits for predicting the treatment efficacy.

Discrimination of pancreato-biliary cancer and pancreatitis patients by non-invasive liquid biopsy.

BACKGROUND: Current diagnostics for the detection of pancreato-biliary cancers (PBCs) need to be optimized. We therefore propose that methylated cell-free DNA (cfDNA) derived from non-invasive liquid biopsies serves as a novel biomarker with the ability to discriminate pancreato-biliary cancers from non-cancer pancreatitis patients. METHODS: Differentially methylated regions (DMRs) from plasma cfDNA between PBCs, pancreatitis and clinical control samples conditions were identified by next-generation sequencing after enrichment using methyl-binding domains and database searches to generate a discriminatory panel for a hybridization and capture assay with subsequent targeted high throughput sequencing. RESULTS: The hybridization and capture panel, covering around 74 kb in total, was applied to sequence a cohort of 25 PBCs, 25 pancreatitis patients, 25 clinical controls, and seven cases of Intraductal Papillary Mucinous Neoplasia (IPMN). An unbiased machine learning approach identified the 50 most discriminatory methylation markers for the discrimination of PBC from pancreatitis and controls resulting in an AUROC of 0.85 and 0.88 for a training (n = 45) and a validation (n = 37) data set, respectively. The panel was also able to distinguish high grade from low grade IPMN samples. CONCLUSIONS: We present a proof of concept for a methylation biomarker panel with better performance and improved discriminatory power than the current clinical marker CA19-9 for the discrimination of pancreato-biliary cancers from non-cancerous pancreatitis patients and clinical controls. This workflow might be used in future diagnostics for the detection of precancerous lesions, e.g. the identification of high grade IPMNs vs. low grade IPMNs.

Profiling of circulating glial cells for accurate blood-based diagnosis of glial malignancies.

Here, we describe a blood test for the detection of glial malignancies (GLI-M) based on the identification of circulating glial cells (CGCs). The test is highly specific for GLI-M and can detect multiple grades (II-IV) and subtypes including gliomas, astrocytomas, oligodendrogliomas, oligoastrocytomas and glioblastomas, irrespective of gender and age. Analytical validation of the test was performed as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Real-world performance characteristics of the test were evaluated in four clinical (observational) studies. The test has high analytical sensitivity (95%), specificity (100%) and precision (coefficient of variation [CV] = 13.7% for repeatability and CV = 23.5% for within laboratory precision, both at the detection threshold) and is not prone to interference from common drugs and serum factors. The ability of the test to detect and differentiate GLI-M from non-malignant brain tumours (NBT), brain metastases from primary epithelial malignancies (EPI-M) and healthy individual donors (HD) was evaluated in four clinical cohorts. Across these clinical studies, the test showed 99.35% sensitivity (95% confidence interval [CI]: 96.44%-99.98%) and 100% specificity (95% CI: 99.37%-100%). The performance characteristics of this test support its clinical utility for diagnostic triaging of individuals presenting with intracranial space-occupying lesions (ICSOL).