Biology subjects

Guzman, G.

Publications and source records attributed to Guzman, G..

11 recordsLinked to original sources

bioRxiv2026

A critical signaling role for diacylglycerol in phagocytosis of M. tuberculosis

Mycobacterium tuberculosis (Mtb) establishes infection by entering host phagocytes through phagocytosis. While host lipids are known to influence this process, the specific contribution of the signaling lipid diacylglycerol (DAG) remains poorly defined. Here, we identify DAG as a critical regulator of phagocytosis. Disruption of DAG production, through inhibition or genetic deletion of adipose triglyceride lipase (ATGL) and phospholipase C gamma 2 (PLC{gamma}2), two major pathways that generate cellular DAG pools, markedly reduced uptake of both Mtb and zymosan-coated beads. Notably, loss of ATGL or PLC{gamma}2 did not impair receptor trafficking to the cell surface or cargo binding, indicating that DAG is not required for phagocytic recognition or initiation, but instead for a later step in phagosome formation. Mechanistically, cells lacking ATGL or PLC{gamma}2 displayed constitutive phosphoinositide 3-kinase (PI3K) phosphorylation, suggesting that dysregulated intracellular signaling prevents completion of phagocytosis. These findings uncover a previously unappreciated role for DAG biosynthesis in coordinating intracellular signaling required for phagocytosis and provide new insight into host pathways that govern Mtb entry.

microbiology↗

bioRxiv2026

An Integrated Method for Profiling Lipid-Protein Interactions Using Multifunctional Lipid Probes

Cellular lipids shape health and disease through specific protein interactions, yet lipid-protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid-protein interaction studies. We describe detailed methods for determining lipid interactomes from cells using multifunctionalized lipid derivatives. We provide protocols that detail 1) how to treat cells with lipid derivatives and perform photochemistry to obtain lipid-protein conjugates; 2) how to perform click chemistry with a fluorophore and observe lipid-protein conjugates by in-gel fluorescence; 3) how to perform click chemistry with azide beads and prepare lipid-protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables mapping lipid interactomes across diverse biological systems. The entire workflow from cell treatment to complete proteomic sample preparation requires [~]15 hours over four days, depending on the type of experimental readout (in-gel fluorescence or proteomics), and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major shift in the characterization of the biological roles of lipids in cellular systems.

biochemistry↗

bioRxiv2025

DINOcell: Learning Generalizable Perturbation Effects through Self-Distillation

AO_SCPLOWBSTRACTC_SCPLOWPredicting cellular responses to therapeutics is a promising approach for novel target discovery. However, state-of-the-art computational models designed to predict perturbation effects struggle to generalize and outperform simple baselines. We present DINOcell, a weakly supervised framework that adapts self-distillation to single-cell transcriptomics for predicting perturbation effects. We demonstrate that DINOcell outperforms baselines in predicting the effects of single gene perturbations. Furthermore, DINOcell accurately predicts non-additive effects of combination perturbations, indicating its capacity to model complex genetic interactions. Finally, we show that DINOcell learns representations that capture biological signals and is a promising, generalizable approach for in silico perturbation modeling, providing a valuable tool for accelerating therapeutic target discovery.

bioinformatics↗

bioRxiv2025

Functional interrogation uncovers a critical role for a high-plasticity cell state in lung adenocarcinoma

Plasticity--the ability of cells to undergo phenotypic transitions--drives cancer progression and therapy resistance1-3. To date, strategies targeting cancer plasticity have not advanced to the clinic due to a lack of fundamental understanding of the underlying mechanisms. Recent studies have suggested that plasticity in solid tumors is concentrated in a minority subset of cancer cells4-6, yet functional studies interrogating this high plasticity cell state (HPCS) in situ are lacking. Here, we developed mouse models enabling detection, longitudinal lineage tracing, and ablation of the HPCS in autochthonous lung tumors in vivo. Using lineage tracing, we uncover the HPCS cells are dedifferentiated but possess high capacity for cell state transitions, giving rise to both early neoplastic (differentiated) and advanced lung cancer cell states in situ. Longitudinal lineage tracing using secreted luciferases reveals HPCS-derived cells harbor high capacity for growth when compared to bulk cancer cells or another defined cancer cell state with features of differentiated lung epithelium. Suicide gene-mediated ablation of the HPCS in early neoplasias abrogates tumor progression. Ablating HPCS cells in established tumors by suicide gene or HPCS-directed CAR T cells robustly reduces tumor burden, whereas ablation of a differentiated lung cancer cell state had no effect. We further demonstrate that the HPCS gives rise to therapy-resistant cell states, whereas ablation of the HPCS abrogates resistance to chemotherapy and oncoprotein-targeted therapy. Interestingly, an HPCS-like state is ubiquitous in regenerating epithelia and in carcinomas of multiple other tissues, revealing a convergence of plasticity programs. Our work establishes the HPCS as a critical hub enabling reciprocal transitions between cancer cell states, including acquisition of states adapted to cancer therapies. Targeting the HPCS in lung cancer and in other carcinomas may suppress cancer progression and eradicate treatment resistance.

cancer biology↗

bioRxiv2025

CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer's Disease Susceptibility

The innate immune gene CD33, encoding a myeloid inhibitory sialic acid-binding receptor, is associated with Alzheimers disease (AD) susceptibility. The AD-associated rs3865444CC risk variant reduces splicing of the sialic acid-binding domain and increases expression of the full-length (sialic acid-binding) CD33 isoform seven-fold compared to the rs3865444AA protective genotype. Here we identify CD45 as an immune cell-specific sialic acid-dependent cis CD33 binding partner, whose phosphatase activity is inhibited by CD33. Overexpression of CD33 or loss of CD45 contributes to impaired microglial clearance of amyloid beta and amyloid beta-induced loss of dendritic spines in microglial-neuronal co-cultures, aligning with a detrimental effect of CD33-mediated inhibition of CD45. CD33-CD45 interaction frequency was increased in monocytes from individuals with the rs3865444CC risk variant compared to rs3865444AA, as well as in AD compared to controls, independent of genotype. Furthermore, an interaction between CD33 and PTPRC (encoding CD45) gene expression in human brain tissue was associated with a pathological diagnosis of AD and global burden of AD pathology. Our findings thus establish a functional interaction between CD33 and CD45 relevant to AD susceptibility and systemic myeloid dysfunction in this disease.

neuroscience↗

bioRxiv2025

In Vivo Production of a Protein via the Delivery of Plasmid DNA by Computationally Designed Polymer Nanoparticles (PNP)

While monoclonal antibodies (mAbs) are a prominent class of pharmaceutical products there remain significant issues of cost, complexity, and especially delivery: concepts that overcome the need to infuse antibodies frequently are a desirable objective. An attractive approach is to deliver non-integrating DNA directly to muscle tissue, allowing the patient to act as their own so-called "protein factory." Demonstrations of this concept have been made using lipid nanoparticles (LNPs) and viral vectors, but these delivery methods face significant challenges, including poor extrahepatic delivery, cargo compatibility, safety, redosability, and cost. Polymer nanoparticles (PNPs) offer a solution to these problems, but face their own challenges, such as the vast number of possible polymer structures, and polyplex formulation conditions. However, advances in machine learning, materials informatics, and high-throughput chemical synthesis techniques provide a foundation for addressing these challenges by efficiently exploring the polymer design space. Our SAYERTM platform utilizes a large computational dataset of plasmid DNA (pDNA) - polymer interactions to facilitate targeting agent discovery and refinement via deep learning, and to drive discovery of novel PNPs for a wide variety of target tissues. In this work, we demonstrate our ability to design PNPs that can deliver pDNA encoding for PGT121, a broadly neutralizing anti-HIV antibody that targets a V3 glycan-dependent epitope site on the HIV-1 envelope glycoprotein. SAYER-designed polymers formed small and stable PNPs with PGT121 plasmids. Intravenous delivery demonstrated strong serum PGT121 protein levels at 1-day post-transfection and higher levels of protein expression when compared to other state-of-the-art DNA-delivery vehicles. More importantly, intramuscular delivery of Nanite PNPs enabled greater than 1.0 {micro}g/mL peak protein expression levels, with meaningful, durable expression levels at > 56 days post-injection. Further, we showed that we can increase antibody levels and durability by redosing. A general trend of lower dose and lower N/P ratio providing higher antibody levels is seen when PNPs were delivered intramuscularly. These parameters, separate from polymer structure, provide different mechanisms to optimize PNP in vivo delivery performance using machine learning techniques. Extension of the concept to the continuous production of other antibodies, proteins or enzymes is possible, suggesting the broad applicability of pDNA depoting via PNPs as a therapeutic modality. Finally, we emphasize that this strategy of delivering a DNA-encoded secreted proteins via a safe and effective PNP in vivo could be applicable to a wide range of other disease modalities.

bioengineering↗

bioRxiv2024

Hepatic HKDC1 Deletion Alleviates Western Diet-Induced MASH in Mice

The global prevalence of Metabolic dysfunction-associated steatohepatitis (MASH) has been rising sharply, closely mirroring the increasing rates of obesity and metabolic syndrome. MASH exhibits a strong sexual dimorphism where females are affected with more severe forms after menopause. Hexokinase domain-containing protein 1 (HKDC1) has recently been recognized for its role in liver diseases, where its expression is minimal under normal conditions but significantly increases in response to metabolic stressors like obesity and liver injury. This selective upregulation suggests HKDC1s potential specialization in hepatic glucose and lipid dysregulation, linking it closely to the progression of MASLD and MASH. This study aims to clarify the role of HKDC1 in Western diet-induced MASH in female mice by examining its impact on hepatic glucose and lipid metabolism, offering insights into its potential as a therapeutic target and addressing the need for sex-specific research in liver disease. This study reveals that HKDC1 expression is elevated in obese women with MASH and correlates with liver pathology. In a mouse model, liver-specific HKDC1 knockout (HKDC1LKO) protected against Western diet-induced obesity, glucose intolerance, and MASH features, including steatosis, inflammation, and fibrosis. Transcriptomic analysis showed that HKDC1 deletion reduced pro-inflammatory and pro-fibrotic gene expression, while gut microbiome analysis indicated a shift toward MASH-protective bacteria. These findings suggest that HKDC1 may exacerbate MASH progression through its role in metabolic and inflammatory pathways, making it a potential therapeutic target.

biochemistry↗

bioRxiv2022

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice.

Background & AimsNon-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in humans. Although we previously showed that the expression of PPAR{gamma} in hepatocytes contributes to the development NASH in lean mice, the relevance of hepatocyte PPAR{gamma} in the development of NASH associated with obesity is still poorly understood. MethodsHepatocyte PPAR{gamma} was knocked out (Pparg{Delta}Hep) after the development of high-fat diet-induced obesity in male and female mice and before NASH was induced with a high fat, cholesterol and fructose (HFCF) diet. We assessed the effect of the diets and Pparg{Delta}Hep on body composition and glucose homeostasis, as well as on the liver pathology, gene expression, and metabolome. In addition, liver biopsies from a cohort of 102 bariatric surgery patients were assessed for liver histology and gene expression. ResultsPPAR{gamma} expression, specifically PPAR{gamma}2, is mostly derived from hepatocytes and increased by high fat diets. Pparg{Delta}Hep reduced HFCF-induced NASH progression without altering steatosis. Interestingly, Pparg{Delta}Hep reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and collagen- stained fibrotic area in the liver of HFCF-fed male mice. In addition, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPAR{gamma}-dependent manner. Specifically, Pparg{Delta}Hep increased betaine-homocysteine methyltransferase expression and reduced homocysteine levels in HFCF- fed male mice. In a cohort of 102 bariatric surgery patients, 16 cases of NASH were associated with increased insulin resistance and hepatic PPAR{gamma} expression. ConclusionsHepatocyte PPAR{gamma} expression associated with obesity could regulate methionine metabolism and the progression of fibrosis in NASH.

physiology↗

bioRxiv2021

The hexokinase ''HKDC1'' interaction with the mitochondria is essential for hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC.

cancer biology↗

bioRxiv2019

Sphingomyelin is critical in organizing phosphoinositide dynamics during phagocytic uptake of Mycobacterium tuberculosis.

Tuberculosis, the pathological result of infection by Mycobacterium tuberculosis (M.tb), represents a massive and persistent global health burden with vast overrepresentation in regions with poor access to healthcare. This pathogen survives intracellularly within lung macrophages, and has been shown to extensively reprogram the host lipid compartment to the effect of enhanced survival and immune evasion. Sphingolipids are a class of lipid with extensive functions in a wide variety of cellular contexts, including apoptosis, autophagy, stress response, and inflammation. There remains a significant knowledge gap surrounding the lipid-mediated interactions between M.tb and its host. Our investigation aims to explore the mechanisms through which M.tb harnesses host-derived sphingolipids to gain entry to the host and to survive intracellularly. To this end, we use chemical and genetic perturbations of the sphingolipid de novo synthesis pathway and fluorescence microscopy to show that sphingolipids are required for efficient phagocytosis of M.tb. We furthermore show that the dynamics of phosphoinositides at the phagosomal synapse are perturbed upon blockade of sphingolipid synthesis. Finally, we demonstrate that perturbing the homeostasis of a specific sphingolipid (sphingomyelin) through both enrichment and depletion at the cell surface reduces M.tb uptake. This work represents a first step in building comprehensive knowledge of the mechanisms of host-pathogen interactions in M.tb, and demonstrates a cohort of methodologies that may be applied to a variety of pathogenic contexts.

microbiology↗

bioRxiv2018

Opposing Roles of the Fork-head box genes FoxM1 and FoxA2 in Hepatocellular Carcinoma

The fork-head box transcription factor FoxMl is essential for hepatocellular carcinoma (HCC) development and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S/G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1, and inhibits FoxM1-induced tumorigenicity of HCC cells. Moreover, expression of FoxA2 in mouse liver expressing activated Ras inhibits FoxM1 expression and inhibits HCC progression. The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression.\n\nAUTHOR SUMMARYLiver cancer remains untreatable because it is diagnosed at a stage when the cancer is aggressive and resistant to therapeutics. The mechanism that drives aggressive liver cancer is poorly understood. These cancers are made up of poorly differentiated cancer cells. Interestingly, the FoxM1 gene is overexpressed in the aggressive liver cancers. Although FoxM1 is important for expression of the proliferation genes, it does not explain why it is overexpressed mainly in the undifferentiated cancers. The current study addresses this puzzle. Our previous studies demonstrated that FoxM1 increases expression of the pluripotency genes that are expressed mainly in the stem-like cells. In the current manuscript we show that, in addition to activating the pluripotency genes, FoxM1 inhibits expression of the liver differentiation gene FoxA2. Overexpression of FoxM1 is important for this inhibition function, as it involves the retinoblastoma family of proteins, which are often inactivated in cancer cells, and thus, are of low-abundance. Moreover, the inhibition of FoxA2 is significant because FoxA2 could inhibit expression of the pluripotency genes as well as FoxM1. The observations provide new insights into how FoxM1 drives progression of aggressive liver cancer.

cancer biology↗