Abstract Accurate cell counting is essential in tissue engineering and cancer research. The ongoing transition towards advanced 3D in vitro tumour models raises a question about the validity of the standard cell counting protocols, particularly in the systems containing extracellular matrix-based scaffolds. Here, we provide a quantitative analysis of the performance of three popular plate reader-based cell counting/viability assays, such as the Alamar Blue, MTT, CellTiter Glo 3D assays, in 2D monolayer and 3D scaffold-based cultures of U251 human glioblastoma cells, including cell-laden Matrigel plugs, and original tissue engineering constructs based on the decellularised sheep brain scaffolds. We quantitatively characterized the assays’ linearity, precision, biological and technical reproducibility, proportionality, and inter-assay agreement. The study revealed that assays’ performance is highly platform-dependent, with 2D cultures allowing significantly more precise and reliable measurements than in 3D ECM scaffold-based cultures. The numerical results provided in this study can help researchers make informed decisions when working with 3D scaffold-based in vitro tumour models and for other tissue engineering purposes where precise cell counting is essential. ToC

Chronic liver disease (CLD) and hepatocellular carcinoma (HCC) account for approximately 2 million deaths annually, with HCC responsible for nearly 50% of these fatalities. The liver is the primary metabolic hub for the synthesis of NAD+, an essential cofactor for cellular metabolism and redox regulation that is produced through the kynurenine, Preiss–Handler, and salvage pathways. Emerging evidence suggests context-dependent dysregulation of NAD+ metabolism in chronic inflammation and tumour-associated settings, with links to immune suppression and tumour metabolic reprogramming. This review summarises reported dysregulation of NAD+ metabolism across CLD and HCC, highlighting disease-specific perturbations and relevance to HCC pathophysiology. Potential therapeutic implications of targeting NAD+ biosynthetic enzymes or modulating NAD+ availability in HCC are discussed, emphasising the need for biomarker-guided patient stratification.

BACKGROUND:Environmental biotoxins, such as mould, are linked to neurological and visual dysfunction, including impaired visual contrast sensitivity (VCS). Testing VCS alterations is recommended as a biomarker for biotoxicity. This study compared four VCS tests in detecting VCS deficits in individuals with clinical signs of biotoxicity. METHODS:VCS was measured in 28 biotoxin-exposed individuals and 30 controls using four VCS tests: Shoemaker handheld chart, Online Contrast Sensitivity Test (OCSTTM), Clinic CSF App, and an Experimental VCS test. Neurotoxicity symptoms were assessed with the modified Q16 questionnaire, and pupil size was measured under standard lighting. RESULTS:Biotoxin-exposed participants had smaller pupil diameters (mean difference: 0.703 mm, p < 0.0001) and higher neurotoxicity scores (45.50 ± 13.0 vs. 22.00 ± 7.7). Contrast sensitivity was significantly reduced in exposed participants on digital VCS tests only. The Experimental VCS test demonstrated the highest diagnostic performance (100% sensitivity; 60-80% specificity), followed by OCSTTM and the Clinic CSF App. The Shoemaker handheld chart did not distinguish between groups. CONCLUSION:These results highlight the effectiveness of digital VCS testing and symptom questionnaires as practical tools for detecting visual and neurological impairments in individuals with suspected biotoxin exposure.

Formalin-fixed, paraffin-embedded (FFPE) tumour archives represent a vast, clinically annotated resource for biomarker discovery. However, reliable detection and quantification of lower-abundance proteins, particularly cancer biomarkers, remain challenging. Here, we applied an optimised recombinant protein spectral library data-independent acquisition MS (rPSL-DIA-MS) workflow to FFPE colorectal cancer (CRC) tissues to demonstrate efficient protein extraction, compare library-based and library-free DIA analysis using FragPipe/DIA-NN and Spectronaut, and assess sensitive detection and quantification of candidate biomarkers. Proteins were extracted from 20 CRC FFPE tumours and analysed by DIA-MS. An independent cohort (n = 20) processed separately was used to evaluate reproducibility. Optimised extraction and deep DDA library generation enabled identification of > 7,000 proteins (> 5,500 quantified) from FFPE CRC tissues. Across both platforms, library-based DIA achieved greater proteome coverage than library-free analysis. Recombinant proteins representing 34 cancer-associated proteins were used to generate rPSL-only and merged biological-rPSL libraries. rPSL-based DIA improved detection of most cancer-associated proteins, enabling quantification of all 34 proteins with multiple high-stringency peptides and increased coverage compared with standard biological-library and library-free DIA-MS workflows. Independent batch analysis showed minimal variation. Overall, rPSL-DIA-MS enables deep, sensitive and reproducible proteomic profiling of CRC FFPE tissues, supporting retrospective biomarker discovery in archival cohorts.

This chapter presents a segmentation-free, cell ensemble-level workflow for detecting treatment-induced responses in confluent microglial cultures in vitro, using standard phase-contrast microscopy and Python-based imaging data analysis. Entire fields of view are analyzed after minimal preprocessing, avoiding fixation, staining, and single-cell segmentation. We implement multifractal, lacunarity, and texture analysis (MFTA) to characterize the microglial monolayers. In parallel, we introduce cell radiomics as the adaptation of radiomics principles to label-free microscopy, extracting a high-dimensional panel of intensity- and texture-based features directly from grayscale images of live cells. Together, MFTA and cell radiomics provide complementary, quantitative readouts that are sensitive to subtle changes in ensemble organization. Statistical comparisons employ false discovery rate control to identify robust discriminative features between conditions, with optional validation by conventional morphometry on a subset of segmented cells. The workflow is demonstrated on an experimental dataset by the successful reveal of the effect of a moderate static magnetic field on human C20 microglia cell line.

Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling.In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.

Microglia, Müller cells, and astrocytes play a crucial role in maintaining retinal structure, homeostasis, and neuronal function. In disease, they undergo reprogramming that drives chronic inflammation and neurodegeneration. Unique to the retina, these glial cells occupy specialized niches and interact closely with the blood-retinal barrier, creating distinct vulnerabilities. We summarized the glial activation mechanisms, shared triggers, including oxidative stress, metabolic dysfunction, aging, and systemic inflammation, as well as key pathways, such as nuclear factor kappa-B, mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription, the inflammasome, and the complement system. Disease-specific responses in glaucoma, age-related macular degeneration, diabetic retinopathy, and vascular occlusions were compared, highlighting the heterogeneity of gliosis and its impact on neuronal and vascular pathology. We also discussed emerging human-derived platforms alongside proteomics approaches, highlighting their utility for mechanistic insights and discovering biomarkers. Despite advances, critical gaps remain in understanding glial-glial interactions and in developing robust models focused on glia. Despite these advances, major gaps remain in our understanding of glial-glial communication, state transitions, and their temporal relationship to neurodegeneration. Moreover, the lack of experimental models explicitly designed to interrogate glial biology continues to limit translational progress. Addressing these challenges will be essential to reposition glial cells as central drivers of retinal disease rather than secondary responders. A strategic shift toward glia-centered models, integrative multi-omics analyses, and human-relevant systems holds promise for advancing biomarker discovery and developing targeted therapeutic strategies that aim to modulate glial dysfunction and preserve vision.

Alzheimer’s disease (AD) is characterized by the buildup of extracellular aggregated amyloid-β (Aβ) peptides, following sequential enzymatic cleavage of amyloid precursor protein, along with intraneuronal accumulation of hyperphosphorylated Tau proteins and subsequent neuronal loss. Despite extensive research, the precise mechanisms underlying Aβ and Tau-mediated neurodegeneration remain elusive. Inhibiting protein aggregation has been a primary focus for mitigating neuronal toxicity. Probiotics have emerged as a promising preventative measure against cognitive decline in AD, with several in vivo and clinical trials demonstrating the efficacy of select bacterial strains in slowing AD progression. However, these studies lack direct molecular evidence on the effects of probiotics on Aβ aggregation kinetic. Inhibiting protein aggregation is key to reducing neuronal toxicity. While probiotics have shown promise in preventing cognitive decline in Alzheimer’s disease, supported by in vivo and clinical studies, direct molecular evidence of their impact on Aβ aggregation kinetics remains lacking. In this study, we conducted bioinformatic and physicochemical assessments, including molecular docking of proteins derived from 13 probiotic strains against Aβ and Tau, identifying four strains predicted to efficiently inhibit Aβ aggregation. Kinetic studies confirmed that both the probiotic formulation and its derived supernatant significantly inhibited the conversion of monomeric Aβ and Tau into aggregated forms. To explore bioavailability, we administered the probiotic formulation to healthy individuals and detected its presence in stool samples, demonstrating survival through the gastrointestinal tract. These findings suggest that specific probiotic strains may serve as therapeutic candidates for targeting Aβ and/or Tau aggregation, with further studies warranted to assess their potential clinical utility in AD.

Glioblastoma (GB) is the most aggressive brain cancer with a poor survival rate. While molecular markers have been established to improve treatment response with modest outcomes, the cause of this cancer remains unknown. Viruses were proposed as potential contributing factors to glioblastoma, particularly given that approximately 20% of all human cancers are virus-induced. While previous studies have detected viral presence in glioblastoma tissues, these investigations were limited by small sample sizes and often focused exclusively on either viral DNA or proteins, restricting the scope of their findings. To address these shortcomings, we explored the role of viruses in glioblastoma by utilising advanced analytical chemistry and high-throughput technologies to study viral proteins and genes and their interaction with tumour signalling pathway(s). We analysed 233 publicly accessible mass spectrometry metaproteome datasets encompassing 196 glioblastoma, 21 adjacent, and 16 normal brain tissues (PMID: 31331834, 31154438, 36720864), using the Trans-Proteomic Pipeline (PMID: 36648445) which converts raw data files, identifies, validates and quantifies peptides, and protein inference. To expand our analysis, we incorporated metaproteomic data from 100 samples (52 glioblastoma, 48 normal) and metagenomic data from seven glioblastoma tumours sourced from Hunter Cancer Biobank. Our comprehensive analysis, integrating meta-proteomics and meta-genomics, revealed a higher prevalence of multiple herpesvirus species, including human Herpes Simplex Virus-1, Herpes Simplex Virus-2, and Herpesvirus 8 within tumour tissues compared to control and adjacent tissues. To further elucidate the role of viral infections in tumour progression, we are currently investigating meta-transcriptomic data from 15 tumours.We are also investigating human proteins within a cohort of 100 samples to identify potential associations with our detected viruses. We aim to explore whether viral infections may influence human proteins involved in critical biological processes in glioblastoma. In a preliminary analysis, we quantified 3, 991 human proteins, of which 1, 155 were found to be deregulated. Gene enrichment analysis revealed up-regulated gene ontology biological processes associated with interspecies interactions and defence responses to other organisms. This suggests that these genes may potentially play a role in the host's reaction to viral infections. Additionally, Ingenuity Pathway Analysis identified 182 deregulated pathways, including several that may be implicated in viral responses, warranting further investigation.Collectively, our findings indicate a potential role of viruses in tumour development, possibly having the capacity to redefine the stratification of tumour types in GB patients. Bavani Gunasegaran, Shamini Ayyadhury, Shivani Krishnamurthy, Seong Beom Ahn, Benjamin Heng. Exploring the presence and role of causative viruses in glioblastoma using a multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2205.

Tumour immune suppression has been identified as an emerging hallmark of cancer that facilitates cancer progression, including hepatocellular carcinoma (HCC). Studies suggest that cancer cells can remain undetected and proliferate by evading local immune surveillance. A major pathway in tryptophan metabolism, the kynurenine pathway (KP), has been suggested to be one of the key mechanisms mediating tumoral immune evasion. In a pro-inflammatory tumour microenvironment, the KP is highly activated which can suppress activation of tumour-targeting immune cells and enhance cancer cell growth. To elucidate the mechanisms underlying KP activation and its contribution to tumour progression in HCC, this study aims to characterize the KP profile and tumour secretome, including cytokines and proteins in HCC patients, to examine the relationship between changes in plasma KP activity and the inflammation-associated secretome. We have acquired plasma samples collected from patients with HCC (n=30), cirrhosis only (n=30), and healthy control (n=30). The KP metabolomic analysis was carried out using high pressure liquid chromatography (HPLC), Ultra-HPLC and Gas-chromatography Mass-spectrometry. The cytokine and proteomics analysis was performed using flow cytometry and data-independent acquisition mass-spectrometry respectively. Results/Discussion: Our findings demonstrate that the KP is highly activated in HCC, as inferred by the elevated enzymatic activities of indoleamine-2, 3-dioxygenase/tryptophan-2, 3-dioxygenase and kynurenine 3-monooxygenase in HCC patients. Activation of these KP enzymes implies that HCC patients have a suppressed immune profile to facilitate the progression of cancer. Cytokine profiling revealed a more inflamed systemic profile in HCC patients compared to controls and cirrhotic patients. Given that the KP is induced by inflammation, this provides a potential explanation for the elevated KP activity measured in HCC patients. Proteomics analysis further identified differentially expressed proteins linked to inflammation and immune system activation, highlighting that the immune system plays a major role in HCC pathophysiology, potentially driving inflammation and the observed KP profile in HCC patients. Collectively, these results suggest that KP activation may potentially be one of the mechanisms used by HCC tumour to create an immune-tolerant environment, facilitating tumour progression and development. Shivani Krishnamurthy, Bavani Gunasegaran, Sharron Chow, Vincent Lam, Ken Liu, Fiona Guan, Joo-Shik Shin, Avik Majumdar, Geoffrey McCaughan, Seong Beom Ahn, Benjamin Heng. Hiding in plain sight: Understanding the role of tryptophan metabolism in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4161.

Microglia are the primary resident immune cells of the central nervous system (CNS) that respond to injury and infections. Being critical to CNS homeostasis, microglia also have been shown to contribute to neurodegenerative diseases and brain cancer. Hence, microglia are regarded as a potential therapeutic target in CNS diseases, resulting in an increased demand for reliable in vitro models. Two human microglia cell lines (HMC3 and C20) are being used in multiple in vitro studies, however, the knowledge of their biological and immunological characteristics remains limited. Our aim was to identify and compare the biological changes in these immortalised immune cells under normal physiological and immunologically challenged conditions. Using high-resolution quantitative mass spectrometry, we have examined in-depth proteomic profiles of non-stimulated and LPS or IFN-γ challenged HMC3 and C20 cells. Our findings reveal that HMC3 cells responded to both treatments through upregulation of immune, metabolic, and antiviral pathways, while C20 cells showed a response associated with mitochondrial and immune activities. Additionally, the secretome analysis demonstrated that both cell lines release IL-6 in response to LPS, while IFN-γ treatment resulted in altered kynurenine pathway activity, highlighting distinct immune and metabolic adaptations.

Introduction:Peripheral immune dysfunction may be critically involved in the pathophysiology of migraine. Some evidence supports a role for peripheral T cells, monocytes, and humoral factors including kynurenine metabolites and cytokines, however a comprehensive picture has yet to emerge. Objective:This study sought to undertake a systematic assessment of the immune changes in episodic and chronic migraine across phases of the migraine cycle. Methods:Migraine patients in different phases of the migraine cycle with a confirmed diagnosis of episodic or chronic migraine and age- and sex-matched healthy controls were recruited. Peripheral blood was assessed for circulating immune cells, plasma proteins, and kynurenine pathway metabolites in a cross-sectional case-control design. Data were acquired using high-dimensional approaches including proteomics, single-cell mass cytometry, and imaging flow cytometry. Results:Plasma proteins related to increased cell-cell adhesion and altered enzymatic activity were increased in migraine. The migraine prodrome displayed a strong and distinct proinflammatory phenotype defined by increased platelet-neutrophil aggregation, quinolinic acid production, and matrix metalloproteinase-9 expression. Migraine patients in the attack phase instead expressed higher levels of cytokine receptors and phosphorylated transcription factors in Th17 cells, monocytes, natural killer cells, and B cells. T cells were shifted to a mobilised, recirculating phenotype across all migraine phases. Episodic and chronic migraine patients were only distinguished by subtle changes in T-cell phenotype. Conclusion:Distinct proinflammatory peripheral signatures were detected between migraine phases, while few alterations distinguished episodic and chronic status. These data provide a resource that may aid in the identification of peripheral immune cells and mediators contributing to migraine attack onset.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disorder with undefined mechanisms, no diagnostic tools and treatments. To investigate concurrent system dysfunctions, we recruited age- and sex-matched ME/CFS patients and healthy controls for a multimodal analysis of energy metabolism, immune profiles, and plasma proteomics. Immune cells from ME/CFS patients show elevated adenosine monophosphate (AMP) and adenosine diphosphate (ADP) with a reduced ATP/ADP ratio, indicating decreased ATP generation and cellular energy stress. Immune profiling reveals skewing toward less mature effector subsets of CD4+, CD8+, and γδ T cells, with reduced CD1c+CD141- conventional DC type 2 and CD56lowCD16+ terminal natural killer cells. Elevated levels of plasma proteins associated with thrombus formation and vascular reactivity may contribute to the endothelial dysfunction observed in ME/CFS patients. Classification and regression tree modeling identifies variables with strong predictive potential for ME/CFS. Together, this study provides insights into the somatic symptoms and underlying biology of ME/CFS.

BACKGROUND AND OBJECTIVES:Despite the absence of acute lesion activity in multiple sclerosis (MS), chronic neurodegeneration continues to progress, and a potential underlying mechanism could be the kynurenine pathway (KP). Prolonged activation of the KP from chronic inflammation is known to exacerbate the progression of neurodegenerative diseases through the production of neurotoxic metabolites. Among the 8 KP metabolites, six of them, namely kynurenine (KYN), 3-hydroxylkynurenine (3HK), anthranilic acid (AA), kynurenic acid (KYNA), and quinolinic acid (QUIN), have been associated with neurodegeneration. METHODS:To gain insights into the links between the KP and neurodegeneration in MS, we investigated the KP metabolomics profile of relapsing remitting MS (RRMS) patients and their correlation with parameters of neurodegeneration in brain and retinal. Outpatients with a clinical diagnosis of RRMS (n = 98) or age-matched and sex-matched healthy controls (n = 39) were included. MS participants undertook yearly evaluation of MRI and optical coherence tomography scan to evaluate neuroaxonal loss. Blood samples were collected at the baseline from all participants for the biochemical analysis of KP metabolites. RESULTS:We identified increased plasma levels of AA and 3HAA in the MS group, indicating an anti-inflammatory response alongside active neurodegeneration. By contrast, plasma levels of KYNA and 3HK were lower in the MS group than in healthy controls. Our analysis revealed a higher KYN:tryptophan (TRP) and QUIN:KYNA ratios in the MS cohort, suggesting activation of the pathway toward the production of neurotoxic QUIN. Another important finding was that KP metabolites were correlated with measures of axonal degeneration in patients with MS. Notably, central brain atrophy positively correlated with the TRP levels, but negatively correlated with KYN and level KYN:TRP ratio. Finally, the choroid plexus volume was inversely correlated with KYNA plasma levels. DISCUSSION:These findings highlight changes in the biosynthesis of KP during the progression of RRMS and its correlation with axonal loss. This study underscores the potential of targeting the KP in developing novel treatments for neuroaxonal damage in MS and warrants future research in greater depth.

Data-independent acquisition mass spectrometry (DIA-MS) is a powerful tool for quantitative proteomics, but a well-constructed reference spectral library is crucial to optimize DIA analysis, particularly for low-abundance proteins. In this study, we evaluate the efficacy of a recombinant protein spectral library (rPSL), generated from tryptic digestion of 42 human recombinant proteins, in enhancing the detection and quantification of lower-abundance cancer-associated proteins. Additionally, we generated a combined sample-specific biological-rPSL by integrating the rPSL with a spectral library derived from pooled biological samples. We compared the performance of these libraries for DIA data extraction with standard methods, including sample-specific biological spectral library and library-free DIA methods. Our specific focus was on quantifying cancer-associated proteins, including key enzymes involved in kynurenine pathway, across patient-derived tissues and cell lines. Both rPSL and biological-rPSL DIA approaches provided significantly improved coverage of lower-abundance proteins, enhancing sensitivity and more consistent protein quantification across matched tumour and adjacent noncancerous tissues from breast and colorectal cancer patients and in cancer cell lines. Overall, our study demonstrates that rPSL and biological-rPSL coupled with DIA-MS workflows, can address the limitations of both biological library-based and library-free DIA methods, offering a robust approach for quantifying low-abundance cancer-associated proteins in complex biological samples.

Alzheimer's disease (AD) pathogenesis is not restricted to amyloid-beta, Aβ, and tau pathologies but involves dysregulation in diverse cellular and molecular processes. Numerous metabolomic studies revealed plasma metabolite alterations in AD individuals compared to healthy controls. Nevertheless, plasma P-tau181, an established biomarker for AD diagnosis and prognosis, has been described to reflect initial multiple cortical region Aβ deposition in cognitively intact adults. The current study aims to identify plasma metabolites associated with plasma P-tau181 at the preclinical stage and better understand the associated biochemical mechanisms for AD pathogenesis. In the current study, 100 older adults with no objective cognitive impairment, MoCA and MMSE ≥ 26, from the Kerr Anglican Retirement Village Initiative in Ageing Health (KARVIAH) cohort, comprising 65 CI Aβ- (cognitively intact normal brain Aβ) and 35 CI Aβ+ (cognitively intact higher brain Aβ) individuals, were assessed for plasma P-tau181, via ultra-sensitive Quanterix Simoa technology, and plasma metabolites, via mass spectrometry-based BIOCRATES kit, and then investigated for associations, both before and after adjusting for confounding variables, in the study groups. Additionally, P-tau181-associated plasma metabolites were evaluated using the receiver operating characteristic (ROC) curves for the potential to classify brain Aβ status. In the entire cohort, significant positive associations of plasma metabolites, including acylcarnitines, amino acid citrulline, and three biogenic amines (creatinine, kynurenine and SDMA), were observed with P-tau181 and similar associations, except for kynurenine, were detected in CI Aβ-. In contrast, in CI Aβ+, only acylcarnitine, AC(10:3), was found to have a positive association with P-tau181 and further, upon including AC(10:3), the AUC for P-tau181 (AUC=70.9%) potentially outperformed (AUC=76.2%), which additionally topped to 83.9% when combined with a base model (Abstract Figure). These findings suggest that the higher the plasma P-tau181, the higher the medium chain acylcarnitine, AC(10:3), in plasma in cognitively intact older adults at risk for AD, indicating a link between early Aβ pathology and fatty acid oxidation mediated energy metabolism pathway. Additionally, associated metabolite strengthens the significance of P-tau181 in classifying brain Aβ status in cognitively intact older adults. Therefore, plasma P-tau181-associated plasma metabolite may serve as potential predictive marker for preclinical AD pathogenesis.

INTRODUCTION:Metastasis remains the major cause of death in breast cancer (BrCa) and lacks specific treatment strategies. The kynurenine pathway (KP) has been suggested as a key mechanism facilitating progression of BrCa. While KP activity has been explored in primary BrCa, its role in metastasis remains unclear. To better understand this, we examined changes in the KP of BrCa with no metastasis compared to BCa that produced local or distant metastases. Given that the cancer cell secretome plays a role in metastasis, we also investigated the relationship between changes in KP activity and serum proteins of patients with local or distant metastases. METHODS:To investigate changes in the KP in BrCa, with and without metastasis, we quantified KP metabolites in blood sera collected from patients with stage 1 BrCa (n = 34), BrCa with local metastases (n = 46), BrCa with distant metastases (n = 20) and healthy controls (n = 39). The serum protein profile of the BrCa patients with local or distant metastasis was determined before correlation analyses were carried out to examine the relationship between changes in the KP and cancer serum proteins using SPSS. RESULTS:We found that the KP was elevated in BrCa patients with local and distant metastasis compared to healthy controls and stage 1 BrCa patients. The activity of kynurenine monooxygenase (KMO) and kynureninase (KYNU) A was positively associated with disease stage and was higher compared to healthy controls. Proteome analysis in patients with local or distant metastasis revealed the dysregulation of 14 proteins, 9 of which were up-regulated and 5 down-regulated at the distant metastasis stage. Importantly, three of these proteins have not been previously linked to BrCa metastasis. In the correlation studies between the KP profile, cancer serum proteins and metastasis status, KYNU A had the greatest number of significant associations with cancer serum protein, followed by KMO. CONCLUSION:Our findings reveal that the KP was regulated differently at various stages of BrCa and was more dysregulated in patients with local or distant metastasis. These KP activity changes showed a significant association with cancer serum proteins in BrCa patients with local or distant metastasis, highlighting the potential role of KP in BrCa metastasis.

ABSTRACTLongitudinal studies that continuously generate data enable the capture of temporal variations in experimentally observed parameters, facilitating the interpretation of results in a time-aware manner. We propose IL-VIS (Incrementally Learned Visualizer), a new machine learning pipeline that incrementally learns and visualizes a progression trajectory representing the longitudinal changes in longitudinal studies. At each sampling time point in an experiment, IL-VIS generates a snapshot of the longitudinal process on the data observed thus far, a new feature that is beyond the reach of classical static models. We first verify the utility and correctness of IL-VIS using simulated data, for which the true progression trajectories are known. We find that it accurately captures and visualizes the trends and (dis)similarities between high-dimensional progression trajectories. We then apply IL-VIS to longitudinal Multi-Electrode Array data from brain cortical organoids when exposed to different levels of Quinolinic Acid, a metabolite contributing to many neuroinflammatory diseases including Alzheimer’s disease, and its blocking antibody. We uncover valuable insights into the organoids’ electrophysiological maturation and response patterns over time under these conditions.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic illness often triggered by an initiating acute event, mainly viral infections. The transition from acute to chronic disease remains unknown, but interest in this phenomenon has escalated since the COVID-19 pandemic and the post-COVID-19 illness, termed ‘long COVID’ (LC). Both ME/CFS and LC share many clinical similarities. Here, we present recent findings in ME/CFS research focussing on proposed disease pathologies shared with LC. Understanding these disease pathologies and how they influence each other is key to developing effective therapeutics and diagnostic tests. Given that ME/CFS typically has a longer disease duration compared with LC, with symptoms and pathologies evolving over time, ME/CFS may provide insights into the future progression of LC.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly contagious respiratory disease Corona Virus Disease 2019 (COVID-19) that may lead to various neurological and psychological disorders that can be acute, lasting days to weeks or months and possibly longer. The latter is known as long-COVID or more recently post-acute sequelae of COVID (PASC). During acute COVID-19 infection, a strong inflammatory response, known as the cytokine storm, occurs in some patients. The levels of interferon‐γ (IFN‐γ), interferon-β (IFN-β), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) are particularly increased. These cytokines are known to activate the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), catalysing the first step of tryptophan (Trp) catabolism through the kynurenine pathway (KP) leading to the production of several neurotoxic and immunosuppressive metabolites. There is already data showing elevation in KP metabolites both acutely and in PASC, especially regarding cognitive impairment. Thus, it is likely that KP involvement is significant in SARS-CoV-2 pathogenesis especially neurologically.