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We have studied the mechanism of A-769662,a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators,it directly activates native rat AMPK by mimicking both effects of AMP,i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated alpha subunit kinase domain,with or without the associated autoinhibitory domain,or on interaction of glycogen with the beta subunit glycogen-binding domain. Although it mimics actions of AMP,it has no effect on binding of AMP to the isolated Bateman domains of the gamma subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC),effects that are completely abolished in AMPK-alpha1(-/-)alpha2(-/-) cells but not in TAK1(-/-) mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1,a major upstream kinase for AMPK in this tissue. However,in HeLa cells,which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-beta,phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells,the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK. View Publication

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However,the role of PKCiota in lung tumor development has not been investigated. To address this question,we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene,Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore,the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus,Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore,aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. View Publication

Efficient allocation of deceased donor organs depends upon effective prediction of immunologic compatibility based on donor HLA genotype and recipient alloantibody profile,referred to as virtual crossmatching (VCXM). VCXM has demonstrated utility in predicting compatibility,though there is reduced efficacy for patients highly sensitized against allogeneic HLA antigens. The recently revised deceased donor kidney allocation system (KAS) has increased transplantation for this group,but with an increased burden for histocompatibility testing and organ sharing. Given the limitations of VCXM,we hypothesized that increased organ offers for highly-sensitized patients could result in a concomitant increase in offers rejected due to unexpectedly positive crossmatch. Review of 645 crossmatches performed for deceased donor kidney transplantation at our center did not reveal a significant increase in positive crossmatches following KAS implementation. Positive crossmatches not predicted by VCXM were concentrated among highly-sensitized patients. Root cause analysis of VCXM failures identified technical limitations of anti-HLA antibody testing as the most significant contributor to VCXM error. Contributions of technical limitations including additive/synergistic antibody effects,prozone phenomenon,and antigens not represented in standard testing panels,were evaluated by retrospective testing. These data provide insight into the limitations of VCXM,particularly those affecting allocation of kidneys to highly-sensitized patients. View Publication

Cardiomyocytes (CM) derived from human embryonic stem cells (hESC) are used for cardio-toxicity evaluation and tested in many preclinical trials for their potential use in regenerative therapeutics. As more efficient CM differentiation protocols are developed,reliable automated platforms for characterization and detection are needed. An automated time-resolved video analysis and management system (TVAMS) has been developed for the evaluation of hESC differentiation to CM. The system was used for monitoring the kinetics of embryoid bodies (EB) generation (numbers and size) and differentiation into beating EBs (percentage beating area and beating EB count) in two differentiation protocols. We show that the percentage beating areas of EBs (from total area of the EBs) is a more sensitive and better predictor of CM differentiation efficiency than percentage of beating EBs (from total EBs) as the percentage beating areas of EBs correlates with cardiac troponin-T and myosin heavy chain expression levels. TVAMS can also be used to evaluate the effect of drugs and inhibitors (e.g. isoproterenol and ZD7288) on CM beating frequency. TVAMS can reliably replace the commonly practiced,time consuming,manual counting of total and beating EBs during CM differentiation. TVAMS is a high-throughput non-invasive video imaging platform that can be applied for the development of new CM differentiation protocols,as well as a tool to conduct CM toxicology assays. View Publication

The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However,when ART is suspended,the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently,the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes,thus,restricting them from a broader level application. The novel TILDA,labelled as U-TILDA ('U' for universal),can detect all the major genetic subtypes of HIV-1 unbiasedly,and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies. Graphical abstract. View Publication

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens,T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However,the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe,and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC),a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm,which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the ? and ? subunits of a TCR,producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTC + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment,no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably,one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant. View Publication

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However,real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS),leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP),we created a novel,green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics,ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems,including human stem cell-derived neurons and cardiomyocytes,primary neurons and astrocytes,and mouse brain ex vivo and in vivo. These applications demonstrate oROS’s capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A?-putriscine-MAOB axis,highlighting the sensor’s relevance in validating neurodegenerative disease models. Lastly,we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool,offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology,with significant implications for understanding diseases associated with oxidative stress,such as cancer,neurodegenerative,and cardiovascular diseases. View Publication

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study,we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles,globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally,these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably,we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype,which reactivates fetal hemoglobin levels and rescues the disease phenotypes,thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether,we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling,drug screenings and cell and gene therapy-based applications. Sickle cell disease (SCD) and β-thalassemia (BT) are globally prevalent inherited blood disorders but,despite extensive research,no ex vivo system exists for SCD and BT. Here,the authors generate pathophysiologically relevant erythroid progenitor models of SCD and BT. View Publication

AbstractThere is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats),coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source,makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here,we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs,BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR,including indels,CRISPRa,and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing,achieving optogenetic gene editing in T lymphocytes in vivo. View Publication

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells,T cells,and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75{\%} of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7,the ocular T cell population increases to 50{\%} of CD45 + cells,leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible ({\textless} 1{\%} of CD45 + cells),the ocular B cell population increases to {\textgreater} 4{\%} by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models. View Publication