Pandey, Akhilesh

Background: ALG13-CDG is an X-linked N-linked glycosylation disorder caused by pathogenic variants in the glycosyltransferase ALG13, leading to severe neurological manifestations. Despite the clear CNS involvement, the impact of ALG13 dysfunction on human brain glycosylation and neurodevelopment remains unknown. We hypothesize that ALG13-CDG causes brain-specific hypoglycosylation that disrupts neurodevelopmental pathways and contributes directly to cortical network dysfunction. Methods: We generated iPSC-derived human cortical organoids (hCOs) from individuals with ALG13-CDG to define the impact of hypoglycosylation on cortical development and function. Electrophysiological activity was assessed using MEA recordings and integrated with multiomic profiling, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was evaluated in both iPSCs and hCOs. Results: ALG13-CDG hCOs showed reduced glycosylation of proteins involved in ECM organization, neuronal migration, lipid metabolism, calcium homeostasis, and neuronal excitability. These pathway disruptions were supported by proteomic and scRNA-seq data and included altered intercellular communication. Trajectory analyses revealed mistimed neuronal maturation with early inhibitory and delayed excitatory development, indicating an E/I imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst structure, and shortened axonal projections, while transcriptomic and proteomic signatures suggested emerging hyperexcitability. Altered lipid and GlcNAc metabolism, along with skewed X-inactivation, were also observed. Conclusions: Our study reveals that ALG13-CDG is a disorder of brain-specific hypoglycosylation that disrupts key neurodevelopmental pathways and destabilizes cortical network function. Through integrated multiomic and functional analyses, we identify early network hypoactivity, mistimed neuronal maturation, and evolving E/I imbalance that progresses to compensatory hyperexcitability, providing a mechanistic basis for seizure vulnerability. These findings redefine ALG13-CDG as disorders of cortical network instability, offering a new framework for targeted therapeutic intervention.

Abstract Molecular profiling of human primary cell types is essential for understanding human biology. We present a transcriptome and proteome map of 28 primary human cell types. Three major clusters of epithelial, endothelial, and mesenchymal cell types were observed in both the transcriptome and proteome levels along with the discovery of cell type enriched molecules including GRAP and C1orf116. The epithelial cell specific protein C1orf116 was further validated using immunohistochemistry across various human tissues. An exhaustive protein database search considering 39 post-translational modifications (PTMs) revealed novel insights into the PTM landscape including identification of understudied PTMs such as serine O-acetylation and histidine methylation. This also enabled comprehensive characterization of proteins with diverse PTMs. Interestingly, an unexpectedly higher frequency of dioxidation on tryptophan compared to methionine led to the identification of oxidative mitochondria complex subunit proteins. Further, a search strategy accounting for alternative translational start sites, splice junctions and translational readthrough refined genome annotation using proteomic evidence. For example, peptides from translational readthrough including extended sequence of LDHB and MDH1 were detected representing the first peptide-level evidence of these protein readthrough isoforms. Our comprehensive transcriptome and proteome data revealed cell type-specific molecular cues and heterogeneity, offering new insights into disease mechanisms often overlooked by tissue proteomics.

Early postprandial glucagon concentrations are higher in type 1 diabetes (T1D) than in individuals with no diabetes (ND). To determine the cause, we infused stable [13C9, 15N1]glucagon before, during, and after a mixed meal in 16 ND and 16 T1D individuals to measure glucagon turnover. In a subcohort of 9 ND and 12 T1D individuals, we estimated [13C9, 15N1]glucagon kinetics during steady state. A linear, single-compartment model described [13C9, 15N1]glucagon kinetics and allowed precise estimation of the volume of distribution (VD) and clearance rate (CL). Model parameters were similar between groups, with the VD of [13C9, 15N1]glucagon at 42.1 ± 3.3 mL/kg, implying that [13C9, 15N1]glucagon distributes in a single compartment and with VD approximating the plasma volume and CL at 10.6 ± 0.9 mL/kg/min. Higher early (0–120 min after meal ingestion) postprandial glucagon concentrations (1,907.9 ± 373.4 vs. −93.6 ± 240.5 pg/mL · 120 min P < 0.001) observed in T1D was due to higher rates of glucagon appearance (3.39 ± 2.8 vs. −3.95 ± 2.0 ng/kg · 120 min, P < 0.04) and disappearance (2.13 ± 2.6 vs. −5.28 ± 2.1 ng/kg · 120 min, P < 0.04) compared with ND. We have determined postprandial glucagon turnover in humans and have demonstrated that changes in postprandial glucagon concentrations in T1D are due to increased rates of glucagon turnover during the early postprandial period. Article Highlights This study was conducted to determine postprandial glucagon metabolism in people with and without type 1 diabetes. We wanted to determine the cause for higher early postprandial glucagon concentrations in type 1 diabetes. We found that higher early postprandial glucagon turnover is the cause of higher early postprandial glucagon concentrations in type 1 diabetes Strategies that decrease early post prandial glucagon fluxes could improve postprandial glucose concentrations in type 1 diabetes.

Abstract BRCA2 mutations contribute to the pathogenesis and treatment sensitivity of a subset of ovarian, breast, prostate, and pancreatic cancers. When these cancers become therapy resistant, secondary mutations that restore the BRCA2 open reading frame are found in half the cases, but other causes of resistance remain incompletely understood. Here, we identified translational readthrough of a premature termination codon (PTC) as a cause of resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) and cisplatin in cells derived from the BRCA2-mutated ovarian cancer line PEO1 by PARPi selection. Despite persistence of the signature 4965C > G (p.Y1655X) BRCA2 mutation, low-level expression of full-length BRCA2 protein was detectable in these cells by immunoblotting and tandem mass spectrometry. Either BRCA2 knockdown or gene interruption 5′ or 3′ to the PTC restored treatment sensitivity, implicating BRCA2 in the resistance. Reporter assays demonstrated UAG-selective readthrough in the resistant clones but not parental cells. Moreover, custom searching of global proteomic data indicated readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. Finally, multi-omic analysis identified multiple changes in the nonsense-mediated decay and termination machineries that favor readthrough. Accordingly, the present results identify PTC readthrough as a potential mechanism of drug resistance in cells with BRCA2 nonsense mutations.

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer.

Key Points Increased protein expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase was observed in old adults compared with young adults. In old adults, overexpressed proteins were enriched in mitochondrial processes while under expressed proteins enriched in RNA metabolism. Validated differentially expressed proteins were not significantly correlated with kidney pathology after adjusting for age. Background Kidney aging is characterized by a loss of glomeruli, predominantly in the superficial cortex, with a resultant decline in GFR and an increased risk of various kidney-related diseases. The early molecular alterations in glomeruli associated with the aging process are not well studied. Methods We combined laser capture microdissection and mass spectrometry–based unbiased proteomic analysis of nonsclerosed, nonischemic glomeruli in the superficial cortex from young and old adults who underwent a radical nephrectomy for a tumor to understand the age-related molecular changes in glomeruli. Twenty-four young and 30 old adults were used for the discovery dataset, and the significant differentially expressed proteins were further validated using an independent set comprising six young and eight old adults. Results Kidneys from old adults had lower eGFR, less kidney parenchyma on computed tomography imaging, and more glomerulosclerosis and arteriosclerosis on histology. Quantitative proteomic analysis of nonsclerosed, nonischemic glomeruli identified increased expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase in old adults that were further validated in an independent set. Pathway analysis indicated that proteins with increased expression in old adults were enriched in mitochondrial translational processes, aerobic respiration, and TCA cycle, whereas proteins with decreased expression in old adults were enriched in mRNA splicing, mRNA processing, and nonsense mediated decay. Furthermore, validated differentially expressed proteins did not show any significant correlation with the kidney pathology independent of age group. Conclusions Overall, this study identified proteins that are specifically associated with the aging process in otherwise normal-appearing glomeruli.

Background and Aims: Cholangiocarcinoma (CCA) is one of the most lethal cancers, characterized by molecular heterogeneity and treatment resistance. To uncover new biological signals and therapeutic opportunities, we employed multiomic characterization combined with machine learning. Approach and Results: We profiled all anatomical CCA subtypes using whole exome sequencing, mRNA sequencing, and proteome/phosphoproteome analysis. Integrative dimensional reduction revealed RNA, protein, and phosphoprotein features driving tumor heterogeneity, enabling clustering. Machine learning algorithms identified molecular features for each cluster and mapped external datasets and patient-derived xenograft (PDX) models onto these clusters. Kinase enrichment analysis highlighted targetable kinases active in each cluster. In vivo validation was performed in cluster-specific PDX models using the selective TNK1 inhibitor, TP-5801. We identified 3 molecular clusters with distinct pathway characterization: immunomodulatory (cluster 1), metabolic (cluster 2), and gene regulation/cellular fate (cluster 3). Cluster assignment was independent of anatomic subtype but correlated with overall survival following curative-intent resection. We also identified multiomic features and pathways linked to overall survival and lymph node metastases, crucial for patient treatment selection. Kinase enrichment analysis pinpointed TNK1 as a highly active kinase in the metabolic cluster. Treatment with TP-5801 significantly reduced tumor growth in a metabolic PDX model, but not in models representing the other clusters. Combining internal data with publicly available datasets, we identified the immunomodulatory cluster as most responsive to gemcitabine/cisplatin therapy, confirmed in vivo using cluster-specific PDX models. Conclusions: Integrated multiomic characterization provides translational insights by defining unique molecular subtypes associated both with therapeutic response and overall clinical outcomes. This approach identified TNK1 as a previously unrecognized therapeutic target in a defined subset of CCA tumors.

ABSTRACT Periprosthetic joint infection (PJI) is the most common and difficult to treat form of arthroplasty failure. While treatment with debridement, antibiotics, and implant retention (DAIR) is preferable to one- or two-stage implant exchange based on morbidity and cost, outcomes are not successful in all cases selected for this management strategy. DAIR is currently recommended when infection is perceived to be in an “acute” phase, based on symptom duration; despite this selection strategy, DAIR failure rates are high. A more objective means of predicting DAIR success could improve patient outcomes by limiting DAIR failures. To explore this possibility, advanced proteomic analysis of sonicate fluid samples from 95 subjects with PJI was performed using proximity extension assay (PEA), with 77 samples additionally undergoing complementary proteomics analysis with liquid chromatography with tandem mass spectrometry (LC-MS/MS). Sparse partial least squares differentiation analysis (sPLS-DA) separated samples from patients who underwent DAIR with known outcomes ( n = 19) into successful or unsuccessful groups, predicting DAIR surgical outcome (area under the curve [AUC]: 1; mean “leave one out” AUC: 0.6). Proteins predictive of DAIR surgical outcome were significantly enriched for the “protein repair” Gene Ontology (GO) pathway ( P -adj = 0.02). Additionally, proteomic signatures associated with symptom duration in PJI categorized samples into short and long symptom duration PJI. In summary, an investigation of 7,261 unique human proteins separated PJI-associated samples based on symptom duration and identified a proteomic profile predictive of DAIR success. IMPORTANCE Chronic infections are generally understood to last months to years, with acute infections lasting days to weeks. The transition from acute to chronic infection is, however, poorly understood. Periprosthetic joint infection (PJI) has been considered more challenging to treat when “chronic” than when “acute.” A surgery preferred for its recovery time and cost—debridement, antibiotics, and implant retention (DAIR)—is recommended for PJI management when symptom duration is short; yet, even in this select patient group, DAIR is associated with a high treatment failure rate. A means to better identify those predicted to have a successful outcome if they undergo DAIR is needed. Here, over 7,000 proteins from human clinical PJI samples were measured and shown to be able to separate samples based on symptom duration. Importantly, a proteomic profile predictive of DAIR success was identified.

Abstract Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase ( cis- PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, Dhdds R37H+/- strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. Dhdds R37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used “off-label” to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in Dhdds R37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis- PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

ABSTRACT T‐cell receptor (TCR) signaling plays a crucial role in various biological processes and is usually studied using global mass spectrometry‐based phosphoproteomic studies. Despite advancements in targeted mass spectrometry‐based assays for protein quantification, their application in studying signaling processes, for example, reproducible measurements of post‐translational modifications (PTMs) such as phosphorylation, remains limited. Tyrosine phosphorylation is critical for many signaling pathways but presents challenges due to the low abundance of phosphotyrosine‐containing peptides. Conventional untargeted methods often encounter data gaps when analyzing large sample sets, particularly for low‐abundance peptides. To address this issue, a targeted proteomics method called “SureQuant” was employed, which relies on triggered data acquisition with heavy isotope‐labeled peptides. This method has been shown to provide sensitive and reproducible quantification of low‐abundance peptides. Here we describe the development of a SureQuant‐based method to quantify phosphotyrosine peptides that are involved in the TCR signaling pathway. To monitor the change in phosphotyrosine signals upon activation, the T‐cells were stimulated with anti‐CD3/CD28 antibodies. We successfully quantified changes in important phosphotyrosine peptides in primary T‐cells upon stimulation with anti‐CD3/CD28 antibodies. This study showcases the ability of the SureQuant approach to accurately quantify low‐abundance phosphotyrosine peptides, highlighting its broader potential to study a diverse set of PTMs in physiological or clinical settings. Summary T‐cell receptor (TCR) signaling plays a fundamental role in immune responses, regulating T‐cell activation, differentiation, and function. While tyrosine phosphorylation is a key regulatory mechanism in this pathway, the low abundance of phosphotyrosine peptides presents a major challenge for their detection and quantification in complex biological samples. By employing the SureQuant targeted mass spectrometry approach, we achieved highly sensitive and reproducible quantification of key phosphotyrosine sites involved in T‐cell activation. This study provides a systematic view of TCR signaling dynamics, revealing distinct phosphorylation patterns across different activation timepoints. Our findings demonstrate the effectiveness of SureQuant in quantifying low‐abundance, post‐translationally modified peptides, offering a valuable tool for studying signaling pathways with greater precision. Additionally, this methodological framework can be extended to investigate other signaling networks, immune cell functions, and disease‐associated phosphotyrosine modifications.