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Subramaniam, A.

Publications and source records attributed to Subramaniam, A..

9 recordsLinked to original sources

A DNM1L GTPase Effector Domain Deletion Causes Intermediate Defects in Peroxisomal and Mitochondrial Dynamics and Reveals a Targetable Metabolic Vulnerability

DNM1L encodes dynamin-related protein 1 (DRP1), a key GTPase regulating mitochondrial and peroxisomal dynamics. Pathogenic DNM1L variants are associated with a broad spectrum of neurodevelopmental and neurodegenerative disorders, with phenotypic severity varying by affected protein domain. While variants in the GTPase domain are typically associated with milder phenotypes and middle-domain variants with severe developmental and epileptic encephalopathy, the functional impact of GTPase effector domain (GED) variants remains less defined. Here, we describe four individuals harboring a recurrent heterozygous GED deletion (L700del) presenting with movement disorders and lacking severe infantile encephalopathy, consistent with an intermediate clinical phenotype. Patient-derived fibroblasts exhibited tubular peroxisomes and enlarged mitochondria, while Drosophila models demonstrated that DNM1LL700del has intermediate functional effects that were more pronounced than those of a GTPase-domain variant A192E but less severe than those of the middle-domain variant G350R. Comparison with another GED variant, Y691C, further demonstrated variant-specific differences in severity within the GED. Metabolic stress assays showed that L700del fibroblasts exhibited impaired growth under galactose conditions, consistent with increased vulnerability under conditions dependent on mitochondrial oxidative metabolism. Bezafibrate, a PPAR agonist that promotes mitochondrial biogenesis, partially rescued this growth defect. Together, these findings support a functional allelic spectrum in DNM1L-associated disease in which protein domain contributes to, but does not completely determine, functional severity. L700del represents an intermediate-strength allele associated with a movement-disorder phenotype and a pharmacologically modifiable metabolic vulnerability.

genetics↗

Cancer-associated KBTBD4 mutations induce differentiation defects and confer a unique therapeutic vulnerability

Epigenetic regulation governs stem cell fate and perturbations in these mechanisms often lead to tumor development. The CoREST complex, a critical regulator of neural and hematopoietic stem cell differentiation, is recurrently targeted by gain of function mutations in the ubiquitin ligase KBTBD4 in high-risk embryonal brain tumors. However, the tumorigenic potential of these mutations remains unresolved partly due to the challenges in modelling tumors that arise during early brain development. We and others recently demonstrated that small molecule UM171 mimics KBTBD4 mutations by promoting robust CoREST degradation and expansion of hematopoietic stem cells. Leveraging this mechanistic similarity, we modeled KBTBD4 mutations in hematopoietic stem and progenitor cells (HSPCs) and found that mutants induced expansion of immature stem and progenitor populations and impaired lineage differentiation. High-throughput screening identified HDAC inhibitors as specific agents that disrupt mutant KBTBD4 activity, by preventing interaction with the CoREST complex. Using our HSPC model, we demonstrated that the class I HDAC inhibitor mocetinostat alleviated differentiation defects caused by KBTBD4 mutations. Together, these findings reveal the tumorigenic mechanism of KBTBD4 mutations and uncover therapeutic vulnerabilities that may be exploited for clinical applications.

cancer biology↗

The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions

Bacterial non-homologous end joining (NHEJ) is a DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminus has been highlighted for its role in LigD recruitment to DNA DSBs and stimulation of ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences other LigD activities. Here, we combine NMR spectroscopy, structural modelling and mutational analysis to define the interaction interface between the Ku C-terminus and LigD. We identify critical residues in Ku (E246, V248, S258, K260, and N266) and the LigD polymerase (D162, V194, R198) and ligase (D522, K579, L580) domains that mediate this interaction. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both polymerase and ligase domains, while Ku attenuates template-dependent polymerase activity, contrasting previous studies with Pseudomonas aeruginosa homologs. Disrupting the Ku-LigD interface, either through Ku or LigD mutations, abolishes ligase stimulation and restores polymerase activity, highlighting a dual regulatory mechanism. Our data supports a model where Kus C-terminal region forms a bipartite interface with LigD to balance repair activity. These findings provide mechanistic insight into the Ku-LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/693941v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@802e3org.highwire.dtl.DTLVardef@1f5ac14org.highwire.dtl.DTLVardef@1f46677org.highwire.dtl.DTLVardef@7312b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Antibody Responses to Salmonella Typhi Antigens among Typhoid Recovered Individuals in an Endemic Region

BackgroundTyphoid fever remains a major public health concern in endemic regions, yet the nature of antibody responses to individual Salmonella Typhi antigens and their potential role in modulating cellular immune functions are not well understood. This gap also applies to the long-term immune response after the vaccination against the disease. ObjectivesTo evaluate the humoral and cellular immune response against the Salmonella Typhi antigens among the Typhoid recovered individuals. MethodsELISA was used to measure antibody responses against five S. Typhi antigens in plasma samples from typhoid-recovered individuals and healthy participants. Vi polysaccharide-specific plasma antibodies were tested for their ability to activate NK cells isolated from healthy donors. Flow cytometry-based assays were performed to analyze T cell and B cell profiles in the peripheral circulation of these individuals. ResultsPolysaccharide antigens mounted better IgA responses among typhoid-recovered individuals compared with healthy participants. Anti-S. Typhi IgM was significantly elevated in the typhoid group for all five antigens (p<0.05), while no significant differences were observed in IgG responses. NK cell activation assays showed comparable levels of activation markers of the NK cells isolated from healthy individuals and treated with the plasma of Typhoid recovered individuals. Analysis of B cell subsets revealed largely comparable distributions between the two groups; however, transitional B cells were elevated among the Typhoid recovered individuals. HlyE-specific memory B cells were detectable in both the groups. CD4 T cell subsets were similar between typhoid-recovered and healthy individuals, whereas differences were observed in CD8 T cell populations, with higher TEMRA cells among recovered individuals and relatively higher central memory CD8 T cells in healthy participants. ConclusionPolysaccharide antigens elicited significantly better IgA response in recovered typhoid participants, possibly reflecting mucosal immune priming from the gut. Persistent IgM antibodies after recovery suggest durable IgM responses following typhoid infection. Similar IgG levels between groups and the presence of antigen-specific memory cells in healthy individuals highlight the influence of repeated exposure in endemic settings. Elevated transitional B cells suggest the reinforcement of the B cells to the peripheral circulation during the disease which remained in the circulation of these individuals. However, elevated TEMRA population of the CD8 T cells has been observed in several other infectious diseases including this study. These findings demonstrate the complexity of humoral and cellular immune responses to S. Typhi in endemic regions.

immunology↗

SIRPα controls CD47-dependent platelet clearance in mice and humans

Over the last decade, more data has revealed that increased surface expression of the "dont eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRP can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRP signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRP axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRP axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy. Key pointsO_LIPlatelet homeostasis is regulated through the CD47-SIRP axis and therapeutic blockade to disrupt this interaction impacts platelet levels C_LIO_LICommon genetic variants at SIRPA locus associate with platelet levels C_LI

immunology↗

Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECM), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knockout VEGF and RUNX2 and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road towards the compositional eECMs editing and their exploitation in broad regenerative contexts.

bioengineering↗

Inducing synthetic lethality for selective targeting of acute myeloid leukemia cells harboring STAG2 mutations

Targeted therapies exploiting selective vulnerabilities of malignant cells are highly desired for clinical applications. The cohesin protein complex comprises of RAD21, SMC3, SMC1A as well as a fourth subunit that consists of either STAG1 or STAG2 and is essential for proper chromosomal segregation during mitosis. STAG2 loss-of-function mutations are recurrent driver events in acute myeloid leukemia (AML) and appear relatively early during leukemogenesis. Studies in cell lines have shown that STAG2 deficient cells are uniquely vulnerable to STAG1 perturbation, and this vulnerability could thus be exploited to selectively eliminate STAG2 null AML cells. Here we show that partial perturbation of STAG1 is well tolerated by normal human hematopoietic stem cells and does not affect their functionality. By contrast, STAG1 knockdown is lethal to STAG2 null human HSCs by inducing major mitotic defects. Moreover, STAG1 knockdown induced synthetic lethality in primary human AML cells harboring a STAG2 mutation and completely abrogated leukemia progression in xenograft models. Overall, our study provides proof-of-concept demonstration of a synthetic lethal approach to selectively target primary human cancer cells with STAG2 mutations

cancer biology↗

Skill acquisition and gaze behavior during laparoscopic surgical simulation

BackgroundExperts consistently exhibit more efficient gaze behaviors than non-experts during motor tasks. In surgery, experts have been shown to gaze more at surgical targets than surgical tools during simple simulations and when watching surgical recordings, suggesting a proactive control strategy with greater use of feedforward visual sampling. To investigate such expert gaze behaviors in a more dynamic and complex laparoscopic surgery simulation, the current study measured and compared gaze patterns between surgeons and novices who practiced extensively with laparoscopic simulation. MethodsThree surgeons were assessed in a testing visit and five novices were trained and assessed (at pre-, mid-, and post-training points) in a 5-visit protocol on the Fundamentals of Laparoscopic Surgery peg transfer task. The task was adjusted to have a fixed action sequence to allow recordings of dwell durations based on pre-defined areas of interest (AOIs). Novices individualized learning curves were analyzed using an inverse function model, and group-level differences were tested using analysis of variance on both behavioral performance and dwell duration measures. ResultsTrained novices were shown to reach more than 98% (M = 98.62%, SD = 1.06%) of their behavioral learning plateaus, leading to equivalent behavioral performance to that of surgeons. Despite this equivalence in behavioral performance, surgeons continued to show significantly shorter dwell durations at visual targets of current actions and longer dwell durations at future steps in the action sequence than trained novices (ps [&le;] .03, Cohens ds > 2). ConclusionThis study demonstrates that, whereas novices can train to match surgeons on behavioral performance, their gaze pattern is still less efficient than that of surgeons, suggesting that eye-tracking metrics might be more sensitive than behavioral performance in detecting surgical expertise. Such insight can be applied to develop training protocols so non-experts can internalize experts "gaze templates" to accelerate learning. Article SummaryGaze pattern differences persist between laparoscopic surgery experts and novices who have been trained to reach over 98% of individualized behavioral learning plateaus in the Fundamentals of Laparoscopic Surgery (FLS) peg transfer task. The importance of this finding lies in motivating the decision and method of including gaze behaviors via eyetracking technology in the present surgical training programs.

neuroscience↗

Detoxification of endogenous serine prevents cell lysis upon glucose depletion in bacteria

The amino acid serine, despite its diverse metabolic roles, can become toxic when present in excess. Indeed, many bacteria rapidly deaminate exogenously supplied serine into pyruvate and ammonia, even at the expense of biomass production. Here we report a surprising case in which endogenously produced serine must be detoxified in order for the bacterium Escherichia coli to survive. Specifically, we show that E. coli cells lacking the sdaCB operon, which encodes a serine transporter and a serine deaminase, lyse upon glucose depletion when serine is absent from the growth medium. Lysis can be prevented by omission of glycine or by inhibition of the glycine cleavage system, suggesting that activation of glycine catabolism upon glucose depletion causes a transient increase in intracellular serine levels. Heterologous expression of the serine transporter SdaC is sufficient to prevent lysis, indicating a dominant role for serine export, rather than deamination, in mitigating serine toxicity. Since lysis can be modulated by altering alanine availability, we further propose that mis-incorporation of serine instead of alanine into peptidoglycan crosslinks is the cause of lysis. Together, our results reveal that SdaC-mediated detoxification of intracellularly produced serine plays a protective role during sudden shifts in nutrient availability in bacteria.\n\nAuthor summaryThe amino acid serine is a building block used to make many types of macromolecules, yet bacteria actively degrade serine that is provided in growth media. Serine degradation is thought to prevent toxic serine accumulation, but the biological role of this process is not fully understood. We observed that cells lacking the sdaCB operon, which encodes a serine transporter and an enzyme that converts serine to pyruvate, suddenly lyse upon depletion of glucose from the growth medium. This surprising phenotype occurs only in media lacking serine, suggesting that sdaCB is required to detoxify intracellularly produced serine. Expression of the serine transporter SdaC is sufficient to prevent lysis, providing the first evidence that serine export can be an essential function of this protein. Our results reveal that sudden shifts in nutrient availability can increase the intracellular concentration of useful metabolites to toxic levels and suggest that increasing intracellular serine levels by manipulating SdaC activity may be a possible antimicrobial strategy.

microbiology↗