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person:Karim-Jean Armache (armack01) or Joel Belasco (belasj01) or bhabhg01 or burdes01 or cadwek01 or chaom01 or ekierd01 or froemr01 or gelmaj01 or jah12 or hubbas01 or knauth01 or lafaij01 or littmd01 or nancej01 or narask01 or neubet01 or novicr01 or ringsn01 or schwas13 or sfeira01 or skolne01 or smiths04 or stoked01 or torrej12 or treisj01 or turnbd01 or wangd01 or rifkid01 or ryooh01 or wilsoe01

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2559


Encoding the glucose identity by discrete hypothalamic neurons via the gut-brain axis

Kim, Jineun; Kim, Shinhye; Jung, Wongyo; Kim, Yujin; Lee, Seongju; Kim, Sehun; Park, Hae-Yong; Yoo, Dae Young; Hwang, In Koo; Froemke, Robert C; Lee, Seung-Hee; Park, Young-Gyun; Schwartz, Gary J; Suh, Greg S B
Animals need daily intakes of three macronutrients: sugar, protein, and fat. Under fasted conditions, however, animals prioritize sugar as a primary source of energy. They must detect ingested sugar-specifically D-glucose-and quickly report its presence to the brain. Hypothalamic neurons that can respond to the caloric content in the gut regardless of the identity of macronutrient have been identified, but until now, the existence of neurons that can encode the specific macronutrients remained unknown. We found that a subset of corticotropin-releasing factor (CRF)-expressing neurons in the hypothalamic paraventricular nucleus (CRFPVN) respond specifically to D-glucose in the gut, separately from other macronutrients or sugars. CRFPVN neuronal activity is essential for fasted mice to develop a preference for D-glucose. These responses of CRFPVN neurons to intestinal D-glucose require a specific spinal gut-brain pathway including the dorsal lateral parabrachial nuclei. These findings reveal the neural circuit that encodes the identity of D-glucose.
PMID: 40543511
ISSN: 1097-4199
CID: 5871472

Protocol for culture, purification, and target validation of a hybridoma-generated monoclonal antibody targeting Aβ truncated species

Valle, Maria Luisa; Getaneh, Bitseat; Loveland, James; Erdjument-Bromage, Hediye; William, Christopher; Neubert, Thomas A; Rostagno, Agueda; Ghiso, Jorge
Alzheimer's disease (AD) is characterized by the deposition of full-length and truncated amyloid beta (Aβ) species within brain parenchyma and cerebral vessels. However, Aβ truncated species remain understudied. Here, we present a protocol for culture and characterization of a mouse monoclonal antibody targeting N-terminally truncated proteoforms starting at position 4. We describe a detailed procedure for hybridoma culture, antibody collection, and isolation via affinity chromatography. We then describe steps for target validation via dot blot, as well as potential applications. For complete details on the use and execution of this protocol, please refer to Cabrera et al. and Rostagno et al.1
PMID: 40465455
ISSN: 2666-1667
CID: 5862422

Unravelling cysteine-deficiency-associated rapid weight loss

Varghese, Alan; Gusarov, Ivan; Gamallo-Lana, Begoña; Dolgonos, Daria; Mankan, Yatin; Shamovsky, Ilya; Phan, Mydia; Jones, Rebecca; Gomez-Jenkins, Maria; White, Eileen; Wang, Rui; Jones, Drew R; Papagiannakopoulos, Thales; Pacold, Michael E; Mar, Adam C; Littman, Dan R; Nudler, Evgeny
Around 40% of the US population and 1 in 6 individuals worldwide have obesity, with the incidence surging globally1,2. Various dietary interventions, including carbohydrate, fat and, more recently, amino acid restriction, have been explored to combat this epidemic3-6. Here we investigated the impact of removing individual amino acids on the weight profiles of mice. We show that conditional cysteine restriction resulted in the most substantial weight loss when compared to essential amino acid restriction, amounting to 30% within 1 week, which was readily reversed. We found that cysteine deficiency activated the integrated stress response and oxidative stress response, which amplify each other, leading to the induction of GDF15 and FGF21, partly explaining the phenotype7-9. Notably, we observed lower levels of tissue coenzyme A (CoA), which has been considered to be extremely stable10, resulting in reduced mitochondrial functionality and metabolic rewiring. This results in energetically inefficient anaerobic glycolysis and defective tricarboxylic acid cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen-rich compounds and amino acids. In summary, our investigation reveals that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism and stress signalling compared with other amino acid restrictions. These findings suggest strategies for addressing a range of metabolic diseases and the growing obesity crisis.
PMID: 40399674
ISSN: 1476-4687
CID: 5853222

Collagen type VI regulates TGF-β bioavailability in skeletal muscle in mice

Mohassel, Payam; Hearn, Hailey; Rooney, Jachinta; Zou, Yaqun; Johnson, Kory; Norato, Gina; Nalls, Matthew A; Yun, Pomi; Ogata, Tracy; Silverstein, Sarah; Sleboda, David A; Roberts, Thomas J; Rifkin, Daniel B; Bönnemann, Carsten G
Collagen VI-related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalization of collagen VI in the ECM underlies the noncell-autonomous dysfunction and dystrophic changes in skeletal muscle with a yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conducted a comprehensive natural history and outcome study in a mouse model of COL6-RDs (Col6a2-/- mice) using standardized (TREAT-NMD) functional, histological, and physiological parameters. Notably, we identify a conspicuous dysregulation of the TGF-β pathway early in the disease process and propose that the collagen VI-deficient matrix is not capable of regulating the dynamic TGF-β bioavailability both at baseline and in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGF-β with downstream skeletal muscle abnormalities, paving the way for the development and validation of therapeutics that target this pathway.
PMCID:12043086
PMID: 40309777
ISSN: 1558-8238
CID: 5834092

Transport of phenoxyacetic acid herbicides by PIN-FORMED auxin transporters

Schulz, Lukas; Ung, Kien Lam; Zuzic, Lorena; Koutnik-Abele, Sarah; Schiøtt, Birgit; Stokes, David L; Pedersen, Bjørn Panyella; Hammes, Ulrich Z
Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.
PMID: 40263580
ISSN: 2055-0278
CID: 5830192

Food sensing controls C. elegans reproductive behavior by neuromodulatory disinhibition

Chen, Yen-Chih; Zang, Kara E; Ahamed, Hassan; Ringstad, Niels
Like many organisms, the roundworm Caenorhabditis elegans incorporates an assessment of environmental quality into its reproductive strategy. C. elegans hermaphrodites release fertilized eggs into food-rich environments but retain them in the absence of food. Here, we report the discovery of a neural circuit required for the modulation of reproductive behavior by food sensing. A mutation that electrically silences the AVK interneurons uncouples egg laying from detection of environmental food cues. We find that AVK activity inhibits egg laying, and AVKs themselves are inhibited by dopamine released from food-sensing neurons. AVKs express a large number of structurally and functionally diverse neuropeptides. Coordination of food-sensing and reproductive behavior requires a subset of AVK neuropeptides that converge on a small ensemble of premotor neurons that coexpress their cognate receptors. Modulation of C. elegans reproductive behavior, therefore, requires a cascade of neuromodulatory signals that uses disinhibition and combinatorial neuropeptide signals to activate reproductive behavior when food is sensed.
PMCID:12002139
PMID: 40238881
ISSN: 2375-2548
CID: 5828242

Xenotransplantation: Current Understanding of the Mechanism of Immune Mediated Injury

Tatapudi, Vasishta S; Mattoo, Aprajita; Schiff, Tamar; Mehta, Sapna A; Skolnik, Edward Y; Montgomery, Robert A
The scarcity of transplantable organs represents a worldwide public health crisis, and as a result, thousands of people with end-stage kidney disease (ESKD) die waiting for a transplant each year. Xenotransplantation involves transplanting organs from an animal source into humans, offering a potential solution to this significant unmet need. Indeed, if there is a limitless supply of organs, many more patients who do not meet the current criteria for transplant eligibility could also be considered candidates. While there are examples of attempts to transplant animal tissues or organs into humans dating back over 300 years, none were successful due to cross-species immunologic incompatibility. Even so, significant advances in genetic engineering and the emergence of novel immunosuppressive agents have spurred impressive improvements in xenograft survival in preclinical studies involving nonhuman primates. Furthermore, recent reports of genetically modified pig kidney and heart xenotransplants in human decedents and living recipients on a compassionate use basis have provided impetus to advancing the field towards first-in-human trials. However, studies in nonhuman primates and humans thus far have described adaptive as well as innate immune-mediated xenograft injury. Understanding the mechanistic aspects of these responses at the cellular and molecular levels is critical to the development of targeted genetic modifications and innovative therapeutic strategies aimed at preventing rejection and inducing tolerance. Moreover, the physiological components of the bidirectional communication between the human host and pig xenograft must also be understood and manipulated. Here, we review the breakthroughs in renal xenotransplantation in the past few decades and highlight the immunologic hurdles that have yet to be overcome.
PMID: 40238253
ISSN: 1533-3450
CID: 5828222

Prdm16-dependent antigen-presenting cells induce tolerance to gut antigens

Fu, Liuhui; Upadhyay, Rabi; Pokrovskii, Maria; Chen, Francis M; Romero-Meza, Gabriela; Griesemer, Adam; Littman, Dan R
The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While RORγt+ antigen-presenting cells (RORγt-APCs) were shown to program gut microbiota-specific pTreg5-7, their definition remains incomplete, and the APC responsible for food tolerance has remained elusive. Here, we identify an APC subset required for differentiation of both food- and microbiota-specific pTreg cells and for establishment of oral tolerance. Development and function of these APCs require expression of the transcription factors Prdm16 and RORγt, as well as a unique Rorc(t) cis-regulatory element. Gene expression, chromatin accessibility, and surface marker analysis establish the pTreg-inducing APCs as myeloid in origin, distinct from ILC3, and sharing epigenetic profiles with classical dendritic cells (cDC), and designate them Prdm16+ RORγt+ tolerizing DC (tolDC). Upon genetic perturbation of tolDC, we observe a substantial increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTreg, leading to compromised tolerance in mouse models of asthma and food allergy. Single-cell analyses of freshly resected mesenteric lymph nodes from a human organ donor, as well as multiple specimens of human intestine and tonsil, reveal candidate tolDC with co-expression of PRDM16 and RORC and an extensive transcriptome shared with mice, highlighting an evolutionarily conserved role across species. Our findings suggest that a better understanding of how tolDC develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.
PMID: 40228524
ISSN: 1476-4687
CID: 5827502

scRNA-seq uncovers the transcriptional dynamics of Encephalitozoon intestinalis parasites in human macrophages

Jaroenlak, Pattana; McCarty, Kacie L; Xia, Bo; Lam, Cherry; Zwack, Erin E; Almasri, Nadia L; Sudar, Joseph; Aubry, Maelle; Yanai, Itai; Bhabha, Gira; Ekiert, Damian C
Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection, and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.
PMID: 40188181
ISSN: 2041-1723
CID: 5819552

Lateralized local circuit tuning in female mouse auditory cortex

Song, Soomin C; Froemke, Robert C
Most offspring are born helpless, requiring intense caregiving from parents especially during the first few days of neonatal life. For many species, infant cries are a primary signal used by parents to provide caregiving. Previously we and others documented how maternal left auditory cortex rapidly becomes sensitized to pup calls over hours of parental experience, enabled by oxytocin. The speed and robustness of this maternal plasticity suggests cortical pre-tuning or initial bias for pup call stimulus features. Here we examine the circuit basis of left-lateralized tuning to vocalization features with whole-cell recordings in brain slices. We found that layer 2/3 pyramidal cells of female left auditory cortex show selective suppression of inhibitory inputs with repeated stimulation at the fundamental pup call rate (inter-stimulus interval ∼150 msec) in pup-naïve females and expanded with maternal experience. However, optogenetic stimulation of cortical inhibitory cells showed that inputs from somatostatin-positive and oxytocin-receptor-expressing interneurons were less suppressed at these rates. This suggested that disynaptic inhibition rather than monosynaptic depression was a major mechanism underlying pre-tuning of cortical excitatory neurons, confirmed with simulations. Thus cortical interneuron specializations can augment neuroplasticity mechanisms to ensure fast appropriate caregiving in response to infant cries.
PMID: 40189152
ISSN: 1872-8111
CID: 5823522