Data Independent Acquisition Mass Spectrometry Enhanced Personalized Glycosylation Profiling of Haptoglobin in Hepatocellular Carcinoma.

Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC (p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.

Cerebrospinal Fluid Proteome Map Reveals Molecular Signatures of Reversible Cerebral Vasoconstriction Syndrome.

Reversible cerebral vasoconstriction syndrome (RCVS) is a complex neurovascular disorder characterized by repetitive thunderclap headaches and reversible cerebral vasoconstriction. The pathophysiological mechanism of this mysterious syndrome remains underexplored and there is no clinically available molecular biomarker. To provide insight into the pathogenesis of RCVS, this study reported the first landscape of dysregulated proteome of cerebrospinal fluid (CSF) in patients with RCVS (n = 21) compared to the age- and sex-matched controls (n  = 20) using data-independent acquisition mass spectrometry. Protein-protein interaction and functional enrichment analysis were employed to construct functional protein networks using the RCVS proteome. An RCVS-CSF proteome library resource of 1054 proteins was established, which illuminated large groups of upregulated proteins enriched in the brain and blood-brain barrier (BBB). Personalized RCVS-CSF proteomic profiles from 17 RCVS patients and 20 controls reveal proteomic changes involving the complement system, adhesion molecules, and extracellular matrix, which may contribute to the disruption of BBB and dysregulation of neurovascular units. Moreover, an additional validation cohort validated a panel of biomarker candidates and a two-protein signature predicted by machine learning model to discriminate RCVS patients from controls with an area under the curve of 0.997. This study reveals the first RCVS proteome and a potential pathogenetic mechanism of BBB and neurovascular unit dysfunction. It also nominates potential biomarker candidates that are mechanistically plausible for RCVS, which may offer potential diagnostic and therapeutic opportunities beyond the clinical manifestations.

Increasing DNA damage sensitivity through corylin-mediated inhibition of homologous recombination.

BACKGROUND: DNA repair allows the survival of cancer cells. Therefore, the development of DNA repair inhibitors is a critical need for sensitizing cancers to chemoradiation. Sae2CtIP has specific functions in initiating DNA end resection, as well as coordinating cell cycle checkpoints, and it also greatly interacts with the DDR at different levels. RESULTS: In this study, we demonstrated that corylin, a potential sensitizer, causes deficiencies in DNA repair and DNA damage checkpoints in yeast cells. More specifically, corylin increases DNA damage sensitivity through the Sae2-dependent pathway and impairs the activation of Mec1-Ddc2, Rad53-p and γ-H2A. In breast cancer cells, corylin increases apoptosis and reduces proliferation following Dox treatment by inhibiting CtIP. Xenograft assays showed that treatment with corylin combined with Dox significantly reduced tumor growth in vivo. CONCLUSIONS: Our findings herein delineate the mechanisms of action of corylin in regulating DNA repair and indicate that corylin has potential long-term clinical utility as a DDR inhibitor.

Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS.

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 µg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25-3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

Proteomic profiling of tumor microenvironment and prognosis risk prediction in stage I lung adenocarcinoma.

OBJECTIVES: With the increasing popularity of CT screening, more cases of early-stage lung cancer are being diagnosed. However, 24.5% of stage I non-small-cell lung cancer (NSCLC) patients still experience treatment failure post-surgery. Biomarkers to predict lung cancer patients at high risk of recurrence are needed. MATERIALS AND METHODS: We collected protein mass spectrometry data from the Taiwan Lung Cancer Moonshot Project and performed bioinformatics analysis on proteins with differential expressions between tumor and adjacent normal tissues in 74 stage I lung adenocarcinoma (LUAD) cases, aiming to explore the tumor microenvironment related prognostic biomarkers. Findings were further validated in 6 external cohorts. RESULTS: The analysis of differentially expressed proteins revealed that the most enriched categories of diseases and biological functions were cellular movement, immune cell trafficking, and cancer. Utilizing proteomic profiling of the tumor microenvironment, we identified five prognostic biomarkers (ADAM10, MIF, TEK, THBS2, MAOA). We then developed a risk score model, which independently predicted recurrence-free survival and overall survival in stage I LUAD. Patients with high risk scores experienced worse recurrence-free survival (adjusted hazard ratio = 8.28, p < 0.001) and overall survival (adjusted hazard ratio = 6.88, p = 0.013). Findings had been also validated in the external cohorts. CONCLUSION: The risk score model derived from proteomic profiling of tumor microenvironment can be used to predict recurrence risk and prognosis of stage I LUAD.

Lessons learned: establishing a CLIA-equivalent laboratory for targeted mass spectrometry assays - navigating the transition from research to clinical practice.

Mass spectrometry (MS) assays offer exceptional capabilities in high multiplexity, specificity, and throughput. As proteomics technologies continue advancements to identify new disease biomarkers, transition of these innovations from research settings to clinical applications becomes imperative. To meet the rigorous regulatory standards of clinical laboratories, development of a clinical protein MS assay necessitates adherence to stringent criteria. To illustrate the process, this project focused on using thyroglobulin (Tg) as a biomarker and an immuno-multiple reaction monitoring (iMRM) MS-based assay as a model for establishing a Clinical Laboratory Improvement Amendments (CLIA) compliant laboratory within the Centers of Genomic and Precision Medicine, National Taiwan University. The chosen example also illustrates the clinical utility of MS assays to complement conventional immunoassay-based methods, particularly in cases where the presence of autoantibodies in 10-30% of patients hinders accuracy. The laboratory design entails a comprehensive coordination in spatial layout, workflow organization, equipment selection, ventilation systems, plumbing, electrical infrastructure, documentation procedures, and communication protocols. Practical aspects of the transformation process, including preparing laboratory facilities, testing environments, instrument validation, assay development and validation, quality management, sample testing, and personnel competency, are discussed. Finally, concordant results in proficiency testing demonstrate the harmonization with the University of Washington Medical Center and the quality assurance of the CLIA-equivalent Tg-iMRM MS assay established in Taiwan. The realization of this model protein MS assay in Taiwan highlights the feasibility of international joint development and provides a detailed reference map to expedite the implementation of more MS-based protein assays in clinical laboratories for patient care.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

P25/CDK5-mediated Tau Hyperphosphorylation in Both Ipsilateral and Contralateral Cerebra Contributes to Cognitive Deficits in Post-stroke Mice.

OBJECTIVE: Post-stroke cognitive impairment (PSCI) develops in approximately one-third of stroke survivors and is associated with ingravescence. Nonetheless, the biochemical mechanisms underlying PSCI remain unclear. The study aimed to establish an ischemic mouse model by means of transient unilateral middle cerebral artery occlusions (MCAOs) and to explore the biochemical mechanisms of p25/cyclin-dependent kinase 5 (CDK5)-mediated tau hyperphosphorylation on the PSCI behavior. METHODS: Cognitive behavior was investigated, followed by the detection of tau hyperphosphorylation, mobilization, activation of kinases and/or inhibition of phosphatases in the lateral and contralateral cerebrum of mice following ischemia in MACO mice. Finally, we treated HEK293/tau cells with oxygen-glucose deprivation (OGD) and a CDK5 inhibitor (Roscovitine) or a GSK3ß inhibitor (LiCl) to the roles of CDK5 and GSK3ß in mediating ischemia-reperfusion-induced tau phosphorylation. RESULTS: Ischemia induced cognitive impairments within 2 months, as well as causing tau hyperphosphorylation and its localization to neuronal somata in both ipsilateral and contralateral cerebra. Furthermore, p25 that promotes CDK5 hyperactivation had significantly higher expression in the mice with MCAO than in the shamoperation (control) group, while the expression levels of protein phosphatase 2 (PP2A) and the phosphorylation level at Tyr307 were comparable between the two groups. In addition, the CDK5 inhibitor rescued tau from hyperphosphorylation induced by OGD. CONCLUSION: These findings demonstrate that upregulation of CDK5 mediates tau hyperphosphorylation and localization in both ipsilateral and contralateral cerebra, contributing to the pathogenesis of PSCI.

A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma.

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

Exosomes Derived from Hypoxia-Cultured Human Adipose Stem Cells Alleviate Articular Chondrocyte Inflammaging and Post-Traumatic Osteoarthritis Progression.

Osteoarthritis (OA) is the most common age-related degenerative joint disease. Inflammaging, linking inflammation and aging, is found in senescent cells with the secretions of matrix-degrading proteins and proinflammatory cytokines. The senescence-associated secretory phenotype (SASP) plays a very important role in OA progression. However, there remains no effective way to suppress OA progression, especially by suppressing inflammaging and/or the chondrocyte SASP. Recent studies have shown that exosomes derived from hypoxia-cultured BMSCs can regenerate cartilage in OA animal models. Some reports have further indicated that exosomes secreted from MSCs contribute to the efficacy of MSC therapy in OA. However, whether hypoxia-cultured ADSC-secreted exosomes (hypoxia-ADSC-Exos) can alleviate the chondrocyte SASP or OA progression remains unclear. Accordingly, we hypothesized that hypoxia-ADSC-Exos have a beneficial effect on the normal functions of human articular chondrocytes (HACs), can attenuate the SASP of OA-like HACs in vitro, and further suppress OA progression in rats. Hypoxia-ADSC-Exos were derived from ADSCs cultured in 1% O2 and 10% de-Exo-FBS for 48 h. The molecular and cell biological effects of hypoxia-ADSC-Exos were tested on IL1-ß-induced HACs as OA-like HACs in vitro, and the efficacy of OA treatment was tested in ACLT-induced OA rats. The results showed that hypoxia-ADSC-Exos had the best effect on GAG formation in normal HACs rather than those cultured in normoxia or hypoxia plus 2% de-Exo-FBS. We further found that hypoxia-ADSC-Exos alleviated the harmful effect in OA-like HACs by decreasing markers of normal cartilage (GAG and type II collagen) and increasing markers of fibrous or degenerative cartilage (type I or X collagen), matrix degradation enzymes (MMP13 and ADAMT5), and inflammatory cytokines (TNFα and IL-6). More importantly, intra-articular treatment with hypoxia-ADSC-Exos suppressed OA progression, as evidenced by the weight-bearing function test and cartilage GAG quantification in ACLT rats. Moreover, through NGS and bioinformatic analysis, seven potential miRNAs were found in hypoxia-ADSC-Exos, which may contribute to regulating cellular oxidative stress and attenuating cell senescence. In summary, we demonstrated that hypoxia-ADSC-Exos, carrying potent miRNAs, not only improve normal HAC function but also alleviate HAC inflammaging and OA progression. The results suggest that hypoxia-ADSC-Exo treatment may offer another strategy for future OA therapy.

Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection.

Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.

11th Asia Oceania Human Proteome Organization Congress Report.

As the first in-person Asia Oceania Human Proteomics Organization (AOHUPO) congress since 2018, the 11th AOHUPO congress was an opportune time for the research community to reconnect and to renew friendships after the long period of restricted travel due to the global pandemic. Moreover, this congress was a great opportunity for the many AO regional proteomics and mass spectrometry scientists to meet in Singapore to exchange ideas and to present their latest findings. Cohosted by the Singapore Society for Mass Spectrometry and the Malaysian Proteomics Society and held in conjunction with the seventh Asia Oceania Agricultural Proteomics Organization Congress and Singapore Society for Mass Spectrometry 2023, the meeting featured both human and agricultural proteomics. Over five hundred scientists from the AO region converged on the MAX Atria @ Singapore EXPO, Changi, Singapore from May 8 to 10 for the main congress. The diverse program was made up of 64 invited speakers and panellists for seven plenary lectures, 27 concurrent symposia, precongress and postcongress workshops, and 174 poster presentations. The AOHUPO society were able to celebrate not only their 20th anniversary but also the outstanding academic research from biological and agricultural proteomics and related 'omics fields being conducted across the Asia-Oceania region.

Dynamic association between phonemic awareness and disordered speech recognition moderated by transcription training.

BACKGROUND: Phonetic transcription of disordered speech samples is especially crucial for the assessment and treatment of functional or organic speech-sound disorders. Previous studies show that students who struggle with the identification and segmentation of speech sounds are more likely to encounter difficulties with clinical phonetic transcription. AIMS: To clarify the connection between phonemic awareness and phonetic transcription in Mandarin-speaking undergraduate students, as well as the improvement of these skills after phonetic transcription training. METHODS & PROCEDURES: A pretest-post-test design was adopted for this study. The experimental group (n = 38) consisted of students majoring in audiology and speech-language pathology who were given 12 weeks of transcription training. The control group (n = 33) consisted of audiology and speech-language pathology majors who completed the same module a year earlier without undergoing the transcription training. Two instruments were used to assess their skills: (1) the Phonemic Awareness Skill Test (PA test) and (2) the Phonetic Transcription Skill Test (PT test) designed for the purposes of the present study. OUTCOMES & RESULTS: Performance on most subtests of the PT and PA tests improved significantly at post-test for the experiment group, although only PT training was provided. After phonetic transcription training, the relationship between phonemic addition abilities and phonetic transcription strengthened. Phonemic identification was found to be a predictor of undergraduate students' PT skills of disordered speech samples. CONCLUSIONS & IMPLICATIONS: Results from this study provide insights into the role of phonetic transcription training in improving phonemic awareness and phonetic transcription skills. WHAT THIS PAPER ADDS: What is already known on the subject Phonetic transcription of disordered speech samples is especially crucial for the assessment and treatment of functional or organic speech-sound disorders. Phonemic awareness is not only an essential ability for reading alphabetic language but also associated with acquiring phonetic transcription skills. What this paper adds to existing knowledge A dynamically changing association between phonemic transcription and phonetic transcription during transcription training was reported. Phonetic transcription training of disordered speech samples acted as a driving force for deeper relationships between the two skills. What are the potential or actual clinical implications of this work? Training phonetic recognition and transcription equips future and practising speech-language pathologists with better skills in managing speech-sound disorders. Implications for phonemic awareness training also have the potential to extend to the literacy skills of alphabetic language speakers.

EGFR-T790M Mutation-Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Xenograft Model.

PURPOSE: Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. EXPERIMENTAL DESIGN: Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. RESULTS: We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. CONCLUSIONS: Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

A Pilot Study of Biliary Atresia Newborn Screening Using Dried Blood Spot Matrix Metalloproteinase-7.

OBJECTIVES: Timely diagnosis is a critical challenge and is associated with improved survival of biliary atresia (BA) patients. We aimed to measure matrix metalloproteinase-7 (MMP-7) levels in BA patients within 3 days of birth using the dried blood spot (DBS) method and evaluate its potential as a screening tool. METHODS: The study enrolled 132 patients, including 25 patients diagnosed with BA and 107 non-BA patients with other congenital or perinatal conditions from the National Taiwan University Children Hospital. The stored DBS samples collected from 48 to 72 hours of life were retrieved from newborn screening centers. MMP-7 on the DBS was quantified using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The MMP-7 levels of BA patients on the DBS were significantly higher than those of non-BA patients (19.2 ± 10.4 vs 5.6 ± 2.7 ng/mL, P value < 0.0001). MMP-7 levels in non-BA patients, including 5 patients with hepatobiliary structural anomaly, 9 patients with intrahepatic cholestasis, and 93 patients with other perinatal diseases, were 11.6 ± 4.2 ng/mL, 6.9 ± 3.0 ng/mL, and 5.2 ± 2.1 ng/mL, respectively. The DBS MMP-7 level showed good accuracy for identifying BA, with an area under the curve of 93.7% [95% confidence interval (CI): 87.7%-99.7%]. The MMP-7 cutoff at 8.0 ng/mL showed a sensitivity of 92.0% (95% CI: 75.0%-98.6%) and specificity of 92.5% (95% CI: 85.9%-96.1%) for detecting BA from other congenital or perinatal diseases. CONCLUSIONS: MMP-7 DBS analysis can be used to distinguish BA from other conditions as early as 3 days of age.

Recent advances in microfluidics for single-cell functional proteomics.

Single-cell proteomics (SCP) reveals phenotypic heterogeneity by profiling individual cells, their biological states and functional outcomes upon signaling activation that can hardly be probed via other omics characterizations. This has become appealing to researchers as it enables an overall more holistic view of biological details underlying cellular processes, disease onset and progression, as well as facilitates unique biomarker identification from individual cells. Microfluidic-based strategies have become methods of choice for single-cell analysis because they allow facile assay integrations, such as cell sorting, manipulation, and content analysis. Notably, they have been serving as an enabling technology to improve the sensitivity, robustness, and reproducibility of recently developed SCP methods. Critical roles of microfluidics technologies are expected to further expand rapidly in advancing the next phase of SCP analysis to reveal more biological and clinical insights. In this review, we will capture the excitement of the recent achievements of microfluidics methods for both targeted and global SCP, including efforts to enhance the proteomic coverage, minimize sample loss, and increase multiplexity and throughput. Furthermore, we will discuss the advantages, challenges, applications, and future prospects of SCP.

SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron.

The coronavirus nucleocapsid (N) protein is known to bind to nucleic acids and facilitate viral genome encapsulation. Here we report that the N protein can mediate RNA or DNA entering neighboring cells through ACE2-independent, receptor (STEAP2)-mediated endocytosis, and achieve gene expression. The effect is more pronounced for the N protein of wild-type SARS-CoV-2 than that of the Omicron variant and other human coronaviruses. This effect is enhanced by RANTES (CCL5), a chemokine induced by N protein, and lactate, a metabolite produced in hypoxia, to cause more damage. These findings might explain the clinical observations in SARS-CoV-2-infected cases. Moreover, the N protein-mediated function can be inhibited by N protein-specific monoclonal antibodies or p38 mitogen-activated protein kinase inhibitors. Since the N-protein-mediated nucleic acid endocytosis involves a receptor commonly expressed in many types of cells, our findings suggest that N protein may have an additional role in SARS-CoV-2 pathogenesis.