Biology subjects

Dutt, S.

Publications and source records attributed to Dutt, S..

11 recordsLinked to original sources

bioRxiv2026

A conserved vesicular circuit enables metabolic adaptation and maintains systemic heme homeostasis

Metabolic homeostasis depends on adaptive control of intracellular metabolite flux, yet how such control is reconfigured when canonical transport pathways fail is unknown. Here we define a conserved vesicular circuit that preserves systemic heme balance by rerouting intracellular heme flux. We show that loss of the intestinal heme exporter MRP-5 in Caenorhabditis elegans causes lethal heme sequestration within endolysosomal compartments. This defect is bypassed by disabling the vesicular adaptor AP-3, which stabilizes and reroutes the heme importer HRG-1, restoring heme export without increasing cytosolic heme. Unbiased genetics and transcriptomics identify two previously uncharacterized SLC49A family members, HRG-13 and HRG-14, as heme exporters with distinct affinities that engage in a vesicular importer-exporter handoff. Live imaging reveals heme-enhanced contacts between HRG-1 and SLC49A-containing vesicles, consistent with direct vesicular transfer. This circuitry extends to vertebrates as disruption of the SLC49A3 homolog impairs erythropoiesis in zebrafish and causes intracellular heme overload, premature hemoglobinization and apoptosis in differentiating human erythroid cells. Together, these findings establish SLC49A3 proteins as conserved heme exporters and uncover a general principle of metabolic adaptation in which reprogramming intracellular compartmentalization, rather than increasing nutrient supply, restores systemic homeostasis.

cell biology↗

bioRxiv2026

Detection of attomolar concentration of heart-type fatty acid binding protein using ion current rectification sensing with conical SiO2 nanopores

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

biochemistry↗

bioRxiv2026

Donor-derived CD8+CD122+ Tregs generated in mixed donor chimeric NOD mice delete autoreactive T cells

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes.

immunology↗

bioRxiv2026

A cell-nonautonomous heme acquisition pathway enables erythroid hemoglobinization under stress

Heme, an iron-containing cofactor, is synthesized in mitochondria by an eight-enzyme pathway. Although cells were thought to manage heme autonomously, over 1,000 proteins contribute to its production, transport, and regulation. During terminal erythroid differentiation, mitochondria are discarded yet hemoglobin production continues, implying a cell-nonautonomous heme supply. We show that, under stress, erythroblasts import heme through the permease Heme Responsive Gene 1 (HRG1), which localizes to the plasma membrane and accumulates during stress erythropoiesis, the emergency program that expands red cell output. HRG1 loss impaired heme uptake, inhibited terminal erythroid differentiation, and caused anemia. In {beta}-thalassemic mice, partial HRG1 loss reduces ineffective erythropoiesis, underscoring the importance of balanced heme import. These findings reveal intercellular heme sharing and identify HRG1 as a potential therapeutic target in hemoglobinopathies.

cell biology↗

bioRxiv2025

Solid-state nanopore sensing reveals conformational changes induced by a mutation in a neuron-specific tRNAArg

We demonstrate that solid-state nanopore sensing is a powerful single-molecule method for analyzing RNA conformational ensembles. As a model, we employed n-Tr20, a neuron-specific cytoplasmic tRNAArgUCU, whose C50U mutation is associated with neurodegeneration in C57BL/6J mice. Maturation of the Tr20C50U precursor is impaired as the mutation stabilizes a conformational ensemble different from the wild-type. To gain insights into how this mutation engenders structural differences, we used solid-state nanopore sensing for the real-time identification of metastable conformers that are not easily observable by ensemble methods. Ion-current traces recorded using an 8-nm nanopore revealed broad contours of the conformational landscape of n-Tr20/n-Tr20C50U {+/-} Mg2+. Additionally, cryo-EM analysis and small-angle X-ray scattering studies revealed structural plasticity even more than predicted from the nanopore-sensing data. Since dynamics undergird RNA (dys)function in cellular physiology and pathology, nanopore sensing to determine RNA conformational sampling is a valuable addition to the growing RNA structural analysis toolkit. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/647894v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@45bforg.highwire.dtl.DTLVardef@1e31110org.highwire.dtl.DTLVardef@75388forg.highwire.dtl.DTLVardef@1d45037_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Effectiveness of FLASH vs conventional dose rate radiotherapy in a model of orthotopic, murine breast cancer

PurposeRadiotherapy is an effective breast cancer treatment that enhances local tumor control and prolongs overall survival yet is associated with undesirable side effects which can impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has been shown to induce less normal tissue toxicity while producing comparable tumor growth delay in a variety of preclinical tumor models when compared with conventional dose rate radiotherapy (CONV). However, growth delay is not a surrogate for tumor eradication, which is a critical endpoint of cancer therapy, and studies using FLASH in breast cancer are limited. We sought to evaluate whether FLASH produced comparable tumor control to CONV in a breast cancer model with tumor eradication as the primary endpoint. Methods and Materials106 cells from the radiation sensitive mammary tumor cell line Py117 were used to create non-metastatic, syngeneic, orthotopic tumors in the left 4th mammary fat pad of C57BL/6J mice (n=67). Tumors were established for two distinct sequential irradiation studies (Rounds 1 and 2), utilizing either large (7.5 mm into the body) or small (5 mm) treatment tumor margins, respectively. For Round 1, mice were divided into groups with either small (20-40 mm3) or large (250-800 mm3) tumors, whereas only small tumors were included in Round 2. Tumors were irradiated with FLASH (93, 192 and 200 Gy/s) or CONV (0.08 Gy/s) using 16.6 MeV FLASH and 15.7 MeV CONV electron beams. Mice in the small tumor cohort were treated with single fractions of 20, 25, or 30 Gy. The larger tumors were treated with a single fraction of 30 Gy. Tumor eradication was determined by palpation and with histology as needed to clarify physical findings. ResultsSingle fractions of FLASH and CONV demonstrated comparable treatment responses within matched cohorts of small and large tumors. A portion of small tumors treated with single fractions of 20 or 25 Gy were eradicated though most regrew within 2 to 3 weeks. Eradication of small tumors was best seen treated with 30 Gy and a large treatment tumor margin. These mice had no tumor regrowth at 30 days with either FLASH or CONV: however, euthanasia criteria were met at the 30-day time point due to concerns over skin toxicity for both FLASH and CONV groups. Small tumors treated with 30 Gy and a smaller treatment tumor margin had less skin toxicity with 75% of mice remaining tumor free at 48 days. 30 Gy FLASH and CONV applied to larger tumors demonstrated growth delay equally with a partial reduction in size but without tumor eradication. ConclusionsFLASH and CONV produced comparable tumor control in this model of orthotopic, murine breast tumors. Single fractions of 30 Gy with both FLASH and CONV applied to small tumors achieved the highest rates of tumor eradication in particular when delivered with a wider treatment margin. Skin toxicity seen at this dose and in this location could be ameliorated with the use of multiple fractions or different tangents in future studies. Efforts at eradicating larger tumors would require testing higher single fraction doses, multiple fractions, and/or hypofractionated treatment regimens. The equivalent effectiveness between FLASH and CONV in this study of murine breast tumors supports ongoing evaluation of FLASH for use in treating human breast cancer. To this end future efforts at tumor eradication with single fraction FLASH doses with comprehensive evaluation of the toxicity of organs at risk as compared to CONV will be necessary. Additionally, studies of dose-response in a range of tumor volumes with additional breast cancer cell lines and tumors, including human xenografts, along with refined target margins, will guide future studies into the use of FLASH in the adjuvant therapy of primary human breast cancer.

cancer biology↗

bioRxiv2024

Rapid Sterilization of Clinical Apheresis Blood Products using Ultra-High Dose Rate Radiation

BACKGROUND AND OBJECTIVESApheresis platelets products and plasma are essential for medical interventions, but both still have inherent risks associated with contamination and viral transmission. Platelet products are vulnerable to bacterial contamination due to storage conditions, while plasma requires extensive screening to minimize virus transmission risks. Here we investigate rapid irradiation to sterilizing doses for bacteria and viruses as an innovative pathogen reduction technology. MATERIALS AND METHODSWe configured a clinical linear accelerator to deliver ultra-high dose rate (6 kGy/min) irradiation to platelet and plasma blood components. Platelet aliquots spiked with 105 CFU of E.coli were irradiated with 0.1-20 kGy, followed by E.coli growth and platelet count assays. COVID Convalescent Plasma (CCP) aliquots were irradiated at a virus-sterilizing dose of 25 kGy and subsequently, RBD- specific antibody binding was assessed. RESULTS1 kGy irradiation of bacteria-spiked platelets reduced E.coli growth by 2.7- log without significant change of platelet count, and 5 kGy or higher produced complete growth suppression. The estimated sterilization (6-log bacterial reduction) dose was 2.3 kGy, corresponding to 31% platelet count reduction. A 25 kGy virus sterilizing dose to CCP produced a 9.2% average drop of RBD-specific IgG binding. CONCLUSIONThis study shows proof-of-concept of a novel rapid blood sterilization technique using a clinical linear accelerator. Promising platelet counts and CCP antibody binding were maintained at bacteria and virus sterilizing doses, respectively. This represents a potential point-of-care blood product sterilization solution. If additional studies corroborate these findings, this may be a practical method for ensuring blood products safety. HIGHLIGHTSO_LI1 kGy irradiation of bacteria-spiked platelets reduced E.coli growth by 2.7-log without significant change of platelet count, and 5 kGy or higher produced complete growth suppression. C_LIO_LIThe estimated sterilization (6-log bacterial reduction) dose was 2.3 kGy, corresponding to 31% platelet count reduction. C_LIO_LIA 25 kGy virus sterilizing dose to CCP produced a 9.2% average drop of RBD- specific IgG binding. C_LI

microbiology↗

bioRxiv2024

A multi-institutional study to investigate the sparing effect after whole brain electron FLASH in mice: Reproducibility and temporal evolution of functional, electrophysiological, and neurogenic endpoints

PurposeUltra-high dose-rate radiotherapy (FLASH) has been shown to mitigate normal tissue toxicities associated with conventional dose rate radiotherapy (CONV) without compromising tumor killing in preclinical models. A prominent challenge in preclinical radiation research, including FLASH, is validating both the physical dosimetry and the biological effects across multiple institutions. MethodsWe previously demonstrated dosimetric reproducibility of two different electron FLASH devices at separate institutions using standardized phantoms and dosimeters. In this study, we compared the outcome of FLASH and CONV 10 Gy whole brain irradiation on female adult mice at both institutions to evaluate the reproducibility and temporal evolution of multiple endpoints. ResultsFLASH sparing of behavioral performance on novel object recognition (4 months post-irradiation) and electrophysiologic long-term potentiation (LTP, 5-months post-irradiation) was reproduced between institutions. Interestingly, differences between FLASH and CONV on the endpoints of hippocampal neurogenesis (Sox2, doublecortin), neuroinflammation (microglial activation), and electrophysiology (LTP) at late times were not observed at early times. ConclusionsIn summary, we demonstrated reproducible FLASH sparing effects between two beams and two institutions with validated dosimetry. FLASH sparing effects on the endpoints evaluated manifested at late but not early time points.

cancer biology↗

bioRxiv2022

The integrity of dopaminergic and noradrenergic brain regions is associated with different aspects of late-life memory performance

Researchers have identified changes in dopaminergic neuromodulation as playing a key role in adult memory decline. Facilitated by technical advancements, recent research has also implicated noradrenergic neuromodulation in shaping late-life memory performance. However, it is not yet clear whether these two neuromodulators have distinct roles in age-related cognitive changes. Combining longitudinal high-resolution magnetic resonance imaging of the dopaminergic substantia nigra-ventral tegmental area (SN-VTA) and the noradrenergic locus coeruleus (LC) in younger (n = 69) and older adults (n = 251), we found that dopaminergic and noradrenergic integrity are differentially associated with individual differences in memory performance. While LC integrity was related to better episodic memory across several memory tasks, SN-VTA integrity was linked to working memory. Moreover, consistent with their dense interconnection and a largely shared biosynthesis, dopaminergic and noradrenergic brain regions integrity were positively related, and correlated with medial temporal lobe volumes. Longitudinally, we found that older age was associated with more-negative change in SN- VTA and LC integrity (time point 1-time point 2; mean delay [~]1.9 years). Importantly, changes in LC integrity reliably predicted future episodic memory performance (at time point 3). These findings support the feasibility of in-vivo indices for catecholaminergic integrity with potential clinical utility, given the degeneration of both neuromodulatory systems in several age-associated diseases. Moreover, they highlight differential roles of dopaminergic and noradrenergic neuromodulatory nuclei in late-life cognitive decline.

neuroscience↗

bioRxiv2021

Redox state and cellular uptake of copper is regulated by the N-terminus of human Copper Transporter-1

Copper(I) is essential for all life forms. Though Cu(II) is most abundant state in environment, its reduction to Cu(I) is prerequisite for bio-utilization, by a mechanism that is uncharacterized. We show that in human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) and Cu(I) preceding its import. The endocytosis of hCTR1 from basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and Cu(I) coordination by methionines. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis that is correctable by reduced and non-reoxidizable Cu(I). Histidines clusters are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. We propose a mechanistic model where His-Met-Asp residues of amino-terminal hCTR1 coordinates copper and maintains its reduced state crucial for uptake.

cell biology↗

bioRxiv2018

ERBB2 and KRAS Alterations Mediate Response to EGFR Inhibitors in early stage Gallbladder Cancer

The uncommonness of gallbladder cancer has contributed to the generally poor understanding of the disease, with scant reports restricted to advance-stage tumors. Here, using an integrated analysis of whole exome and phospho-proteome, we show recurrent activating ERBB2 and KRAS somatic mutations are present in 6 and 3 of 44 early-stage rare gallbladder tumors, respectively. In vitro and in vivo cell-based and biochemical assays reveal an essential role of ErbB pathway activation for the survival of gallbladder cells. Interestingly, the genetic and pharmacological dependencies of gallbladder cells are dependent on the KRAS mutant allele status, reminiscent of the clinical algorithm commonly practiced to opt for anti-EGFR treatment in colorectal cancer. In overall, we present the first evidence that the presence of KRAS (G12V), but not KRAS (G13D) mutation, may preclude gallbladder cancer patients to respond to anti-EGFR treatment, leading to an early adoption of an approved treatment regimen for gallbladder cancer patients.

cancer biology↗