Biology subjects

Machado, N.

Publications and source records attributed to Machado, N..

7 recordsLinked to original sources

bioRxiv2026

A Hypothalamic Pathway for Energy Conservation

Many animals utilize a survival strategy of hypothermia, inactivity, and a dramatic decrease in their energy metabolism when faced with a shortfall in energy supplies. Mice, for example, show daily torpor that may last several hours to bridge the lack of energy availability in a cold environment. While our recent studies identified a population of neurons in the median preoptic nucleus (MnPO) necessary for fasting-induced torpor regulation, the specific brain circuits that trigger energy conservation in response to food restriction are unknown. We show here that the energy state of the animal is conveyed to preoptic neurons by inputs from the arcuate nucleus (ARC), a region known to be indispensable for driving metabolic and behavioral adaptations to the fasted state. We first found that the activity of ARC neurons expressing Agouti-Related Peptide (ARCAgRP) is required for the generation of fasting-induced torpor. Then, we showed that torpor-active ARCAgRP neurons innervate the preoptic area of the hypothalamus (POA), including the MnPO. Using optogenetic methods, we also found that activating the ARCAgRP-MnPO pathway induces key features of torpor: decreased body temperature, bradycardia, reduction of energy expenditure, and suppression of brown adipose tissue thermogenesis. Remarkably, we also show that inhibition of the ARCAgRP-MnPO brain circuit entirely prevents fasting-induced torpor. Furthermore, we identified that both NPY and GABA are neuromodulators used by ARCAgRP neurons to enable hypothermic responses during starvation. Our studies provide a detailed view of a hypothalamic pathway enabling energy conservation, a survival mechanism in conditions of low energy availability.

neuroscience↗

bioRxiv2026

Fentanyl + Xylazine Co Administration Leads to Sustained Depression of Breathing and Body Temperature Likely Driven by mu Opioid and alpha2a Adrenergic Pathway Interactions

Background and PurposeIts been reported that illicit drug supplies increasingly contain the 2-adrenergic agonist, xylazine, alongside fentanyl, yet the pharmacological basis for the greater lethality of this combination remains unclear. Prior research has shown that -opioid (Oprm1) receptors, on which fentanyl acts, and 2-adrenergic (Adra2a) receptors, on which xylazine acts, are both expressed within brainstem circuits that govern autonomic control, especially the parabrachial (PB) and Kolliker-Fuse (KF) nuclei that regulate respiration. Thus, we propose that co-activation of these inhibitory receptors and their respective pathways could potentiate or additively suppress respiratory and thermoregulatory function. Experimental ApproachFreely behaving C57BL/6J mice received intraperitoneal injections of either saline, fentanyl, xylazine, or fentanyl-xylazine (F+X) solutions. Continuous recordings of respiration using whole-body plethysmography, sleep/wake state using EEG/EMG and body temperature using both infrared thermography, and telemetry were collected for several hours following injection. RNAscope was used to identify Oprm1 and Adra2a expression within PB and KF nuclei. ResultsFentanyl alone produced dose-dependent respiratory depression that was not associated with body temperature changes, whereas the dose we used of xylazine alone had no effect on either respiration or body temperature. In contrast, F+X induced a markedly prolonged (>5 h) reduction in respiratory rate and profound hypothermia lasting 7-8 h, exceeding the effects of either drug alone. Mortality increased to 58.8% following F+X exposure. RNAscope revealed that both Oprm1 and Adra2a receptors are expressed in PB/KF FoxP2-positive neurons, identifying a plausible substrate for convergent inhibitory signaling. ImplicationsThis manuscript provides the first direct experimental evidence that fentanyl and xylazine may interact through convergent -opioid and 2-adrenergic receptor signaling to produce additive and sustained suppression of respiratory and thermoregulatory function. These findings address a critical mechanistic gap in understanding the disproportionate lethality of fentanyl-xylazine mixtures, an emerging public-health crisis. The work further identifies the PB/KF FoxP2 population as a plausible site of dual-receptor convergence and highlights a previously unrecognized pharmacodynamic interaction with immediate implications for overdose reversal strategies. Given the novelty, mechanistic insight, and translational urgency of these results, rapid dissemination will help accelerate scientific and clinical responses to this evolving threat. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/719036v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@5b54aeorg.highwire.dtl.DTLVardef@148b7dorg.highwire.dtl.DTLVardef@d1ebccorg.highwire.dtl.DTLVardef@1cfa9c8_HPS_FORMAT_FIGEXP M_FIG Possible convergent -opioid (Oprm1) and 2-adrenergic (Adra2a) signaling within parabrachial FoxP2-expressing neurons likely produces additive suppression of respiratory and thermoregulatory drive during fentanyl-xylazine co-exposure. Fentanyl and xylazine engage parallel inhibitory GPCR pathways in Parabrachial/ Kolliker Fuse nucleus (PB/KF) neurons that project to the pre-Botzinger complex (preBotC) to depress respiratory rhythm and to the dorsomedial hypothalamus (DMH) to blunt thermogenic output. Co-activation of these pathways results in sustained bradypnea, profound hypothermia, and reduced survival, providing a possible mechanistic basis for the increased lethality of fentanyl-xylazine mixtures. C_FIG

neuroscience↗

bioRxiv2025

SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy

DNA polymerase theta (Pol{theta}) plays a crucial role in the repair of DNA double-strand breaks (DSBs) by microhomology-mediated end joining (MMEJ). We previously demonstrated that Pol{theta} inhibition (Pol{theta}i) is an effective and well-tolerated approach to sensitise tumours to radiotherapy (RT). Here, we profiled 54 cancer cell lines and found that Pol{theta}i induces significant radiosensitisation in most models, though with marked variability not explained by indicators of Pol{theta} activity. To pinpoint molecular determinants of radiosensitisation by Pol{theta}i, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 (FAM35A) as a vulnerability to Pol{theta}i combined with RT. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, but also enhances the radiosensitising effect of Pol{theta}i, both in vitro and in vivo. Importantly, we found that SHLD2 is deleted in a subset of human prostate cancers, often co-occurring with PTEN loss, an adverse prognostic factor. Furthermore, we show that SHLD2-deficient cancer cells are more reliant on Pol{theta} to prevent DSB accumulation and chromosomal instability. In summary, we discovered SHLD2 loss as a novel collateral vulnerability that can be exploited through combined treatment with Pol{theta}i and RT.

cancer biology↗

bioRxiv2024

UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment by regulating tumour cell fate

Dysregulation of the C5a-C5a receptor 1 (C5aR1) signalling axis underlies inflammation and immune-driven pathology. C5aR1 was traditionally thought to be primarily expressed on the cell membrane, although recent reports indicate the importance of intracellular C5aR1 expression for the inflammatory effector functions of various cell types. However, the mechanisms regulating C5aR1 expression and localisation remain unclear. In tumours with an immunosuppressive microenvironment, we recently found robust C5aR1 expression on malignant epithelial cells, highlighting potential tumour cell-specific functions. Here, we show that hypoxia, a hallmark of immunosuppressive microenvironments, induces C5aR1 expression in an unfolded protein response (UPR)-dependent manner via enhanced endoplasmic reticulum stress. Furthermore, hypoxia drives endocytosis, relocating C5aR1 from the cell membrane to the intracellular compartment. By genetically and pharmacologically targeting the C5a/C5aR1 axis, we show that C5aR1 mediates cellular adaptation to hypoxia by regulating processes associated with cell fate, including autophagy and apoptosis. In line with hypoxia-induced intracellular C5aR1 pools, pharmacological inhibition of C5aR1, particularly with cell-permeable small molecule inhibitors, significantly reduces tumour cell survival. These results suggest that the dysregulated C5a/C5aR1 axis and the hypoxia-induced shift in C5aR1 localisation, support tumour cell survival and provide new insights into therapeutic strategies for targeting the C5a/C5aR1 axis.

cancer biology↗

bioRxiv2022

Small-molecule Polθ inhibitors provide safe and effective tumor radiosensitization in preclinical models

DNA polymerase theta (Pol{theta}) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Pol{theta} has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Pol{theta} DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, normal fibroblasts are not radiosensitized by Pol{theta} inhibition. Mechanistically, we show that the radiosensitization caused by Pol{theta} inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Pol{theta} inhibitor with improved metabolic stability. In vivo, the combination of Pol{theta} inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared to radiation alone. These results pave the way for future clinical trials of Pol{theta} inhibitors in combination with radiotherapy.

cancer biology↗

bioRxiv2022

Integrated genomic analysis of AgRP neurons reveals that IRF3 regulates leptin's hunger-suppressing effects

AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated AgRP neuron-specific transcriptomic and chromatin accessibility profiles during opposing states of fasting-induced hunger and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 28 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons, while live-cell imaging in vitro indicate that leptin can activate neuronal IRF3 in a cell autonomous manner. Finally, we employ CUT&RUN to uncover direct transcriptional targets of IRF3 in AgRP neurons in vivo. Thus, our findings identify AgRP neuron-expressed IRF3 as a key transcriptional effector of the hunger-suppressing effects of leptin.

neuroscience↗

bioRxiv2019

Essential gene networks in acute myeloid leukemia identified using a microRNA-knockout CRISPR library screen

MicroRNAs (miRNA) are small RNAs that function as key modulators of gene expression. Due to their promiscuity of binding, a single miRNA may regulate several genes and hence, multiple pathways simultaneously. In addition, the 3-UTR of mRNA can be recognized by several miRNA for suppression or degradation. We built a microRNA-only Knock-out (miRKo) CRISPR/Cas-9 library to identify essential miRNA in Acute Myeloid Leukemia (AML) using OCI-AML2, OCI-AML3 and U937 cell lines as in vitro models. 10 miRNA were identified to be essential in our screen among all three cell lines: miR-19b-1, -19b-2, 29b-2, -302a, -3678, -3713, -3910-1, -4447, -4718 and -6795. By using weighted degrees of association, we identified pathway hubs that uniquely affect all 3 cell lines by integrating miRNA:mRNA networks using mirDIP and pathway analysis using pathDIP. Through the miRKo screen, network membership analyses and biological anticorrelation scoring through patient data analysis, we identified RRP2CA, RPS6KB-1, CREB1, RPM1A, MAPK10, MAP3K2, ITCH, FBX-W7, NR3C1 and XIAP as likely targets of the essential miRNA in AML; and signal transduction, apoptosis, TGF-beta signalling and MAPK signalling as candidate essential pathways in AML.

cancer biology↗