In the non-hibernation period, much like in mice, heat shock factor 1, activated by elevated body temperature (Tb) during the wake period, initiated Per2 transcription in the liver, thereby contributing to the synchronization of the peripheral circadian clock to the Tb cycle. Our analysis of the hibernation period revealed that Per2 mRNA levels were reduced during deep torpor, yet Per2 transcription was momentarily elevated by heat shock factor 1, which was activated in response to elevated body temperature during interbout arousal. Although, we found that the mRNA of the Bmal1 core clock gene displayed non-cyclical expression during the interbout arousal phases. Because circadian rhythm relies on negative feedback loops controlled by clock genes, these findings indicate that the liver's peripheral circadian clock is inactive during hibernation.
The synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from the Kennedy pathway hinges on choline/ethanolamine phosphotransferase 1 (CEPT1) activity in the endoplasmic reticulum (ER), and choline phosphotransferase 1 (CHPT1) activity in the Golgi apparatus for PC production. Despite the synthesis of PC and PE by CEPT1 and CHPT1 in the ER and Golgi, the question of whether these products exhibit different cellular functions has not been formally addressed. By creating CEPT1 and CHPT1 knockout U2OS cell lines using CRISPR editing, we investigated the differential contributions of these enzymes to the feedback regulation of nuclear CTPphosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis and lipid droplet (LD) biogenesis. While CHPT1-knockout cells demonstrated a 50% reduction in phosphatidylcholine synthesis, CEPT1-knockout cells experienced a more substantial 80% reduction in phosphatidylethanolamine synthesis, along with a 50% decrease in phosphatidylcholine synthesis. The constitutive localization of CCT protein on the inner nuclear membrane and nucleoplasmic reticulum, coupled with its dephosphorylation, resulted from posttranscriptional induction of its expression following CEPT1 knockout. The activated CCT phenotype, characteristic of CEPT1-KO cells, was circumvented by the addition of PC liposomes, which re-introduced end-product inhibition. Moreover, the study confirmed that CEPT1 was situated in close proximity to cytoplasmic lipid droplets, and the elimination of CEPT1 resulted in an accumulation of smaller cytoplasmic lipid droplets, as well as an increase in nuclear lipid droplets enriched with CCT. On the contrary, the elimination of CHPT1 had no effect on CCT regulation or the generation of lipid droplets. Similarly, CEPT1 and CHPT1 share equal involvement in PC synthesis; nonetheless, exclusively PC generated by CEPT1 within the endoplasmic reticulum governs the regulation of CCT and the creation of cytoplasmic and nuclear lipid droplets.
Epithelial cell-cell junction integrity is regulated by MTSS1, a membrane-interacting scaffolding protein, which also acts as a tumor suppressor in a wide range of carcinomas. Through its I-BAR domain, MTSS1 interacts with phosphoinositide-rich membranes, subsequently enabling its ability to discern and create negative membrane curvature in laboratory tests. However, the intricate pathways by which MTSS1 localizes to intercellular junctions in epithelial cells and sustains their structural integrity remain unexplained. In cultured Madin-Darby canine kidney cell monolayers, we leverage electron microscopy and live-cell imaging to provide evidence that epithelial cell adherens junctions incorporate lamellipodia-like, dynamic actin-based membrane folds exhibiting high negative membrane curvature along their outer borders. Cell-cell junctions were found to exhibit dynamic actin-rich protrusions where BioID proteomics and imaging experiments showed MTSS1 interacting with the WAVE-2 complex, an activator of the Arp2/3 complex. Suppression of Arp2/3 or WAVE-2 activity led to impeded actin filament formation at adherens junctions, diminished membrane protrusion dynamics at the junctions, and ultimately, a breakdown of epithelial structure. bioactive calcium-silicate cement The results, taken as a whole, support a model wherein MTSS1, located on the membrane, alongside the WAVE-2 and Arp2/3 complexes, facilitates the formation of dynamic actin protrusions resembling lamellipodia, thus upholding the integrity of intercellular junctions in epithelial monolayers.
The transition from acute to chronic post-thoracotomy pain is theorized to be associated with the activation and polarized differentiation of astrocytes, including A1, A2, and A-pan subtypes. Crucial for A1 astrocyte polarization are the astrocyte-neuron and microglia interactions involving the C3aR receptor. Using a rat model of thoracotomy pain, this study examined the role of C3aR in astrocytes in mediating post-thoracotomy pain, specifically focusing on the induction of A1 receptor expression.
A thoracotomy model of pain was established using rats. The mechanical withdrawal threshold was determined to gauge pain responses. To induce A1, lipopolysaccharide (LPS) was injected into the peritoneal cavity. To reduce C3aR expression in vivo within astrocytes, the intrathecal injection of AAV2/9-rC3ar1 shRNA-GFAP was applied. YD23 chemical The methods used to assess the expression of linked phenotypic markers before and after the intervention comprised RT-PCR, western blotting, co-immunofluorescence, and single-cell RNA sequencing.
Downregulation of C3aR was shown to successfully inhibit LPS-induced activation of A1 astrocytes, resulting in lower expression of C3, C3aR, and GFAP, proteins that elevate from acute to chronic pain states. This effect effectively reduced both mechanical withdrawal thresholds and the occurrence of chronic pain. A higher number of A2 astrocytes were activated in the model group that evaded chronic pain. The observed increase in A2 astrocytes following LPS exposure was contingent upon the downregulation of C3aR. By knocking down C3aR, the activation of M1 microglia, which was triggered by LPS or thoracotomy, was reduced.
Our investigation found a correlation between C3aR-induced A1 polarization and the persistence of discomfort after a thoracotomy. A1 activation, impeded by C3aR downregulation, yields a rise in anti-inflammatory A2 activation and a decrease in pro-inflammatory M1 activation, potentially playing a role in the development of chronic post-thoracotomy pain.
Our research found that C3aR activation, leading to A1 cell polarization, is a contributing factor to persistent post-thoracotomy pain. C3aR downregulation curbs A1 activation, thus promoting anti-inflammatory A2 activation and mitigating pro-inflammatory M1 activation, which might be a part of the mechanism causing chronic post-thoracotomy pain.
The principal reason behind the diminished rate of protein synthesis within atrophied skeletal muscle is, for the most part, a mystery. Eukaryotic elongation factor 2 (eEF2) encounters impeded ribosome binding, consequent to threonine 56 phosphorylation by eukaryotic elongation factor 2 kinase (eEF2k). Using a rat hind limb suspension (HS) model, researchers investigated perturbations in the eEF2k/eEF2 pathway across different phases of disuse muscle atrophy. Two different aspects of eEF2k/eEF2 pathway malregulation were found, characterized by a substantial (P < 0.001) upregulation of eEF2k mRNA expression as early as day one of heat stress (HS) and an increase in eEF2k protein levels post 3 days of heat stress (HS). To determine the role of Cav11 in calcium-dependent eEF2k activation, we embarked on this investigation. Three days of heat stress caused a pronounced elevation in the ratio of T56-phosphorylated to total eEF2. BAPTA-AM treatment completely reversed this elevation, while nifedipine treatment led to a significant 17-fold decrease (P < 0.005). By combining pCMV-eEF2k transfection in C2C12 cells with small molecule administration, eEF2k and eEF2 activity was modulated. Importantly, pharmacologic induction of eEF2 phosphorylation led to elevated phosphorylated ribosomal protein S6 kinase (T389) and the reinstatement of overall protein synthesis within the HS rat population. Disuse muscle atrophy is characterized by the up-regulation of the eEF2k/eEF2 pathway, which is facilitated by calcium-dependent activation of eEF2k, often involving Cav11. In vitro and in vivo investigations demonstrate the influence of the eEF2k/eEF2 pathway on ribosomal protein S6 kinase activity and the expression of key atrophy biomarkers, including muscle atrophy F-box/atrogin-1 and muscle RING finger-1, as revealed by the study.
Organophosphate esters (OPEs) are ubiquitously found within the atmospheric environment. bioinspired reaction Yet, the atmospheric oxidation pathway for OPEs is not thoroughly scrutinized. Density functional theory (DFT) was used to investigate the tropospheric ozonolysis of diphenyl phosphate (DPhP), a representative organophosphate, along with the corresponding adsorption mechanisms on the surface of titanium dioxide (TiO2) mineral aerosols and the subsequent oxidation of hydroxyl groups (OH) upon photolysis. The investigation also delved into the reaction mechanism, reaction kinetics, the adsorption mechanism, and the evaluation of the ecotoxicity of the transformation byproducts. At 298 Kelvin, reaction rate constants for O3, OH, TiO2-O3 and TiO2-OH are: 5.72 x 10⁻¹⁵ cm³/molecule s⁻¹, 1.68 x 10⁻¹³ cm³/molecule s⁻¹, 1.91 x 10⁻²³ cm³/molecule s⁻¹, and 2.30 x 10⁻¹⁰ cm³/molecule s⁻¹, respectively. DPhP's atmospheric breakdown, induced by ozone, happens rapidly, lasting only four minutes in the lower troposphere, contrasting markedly with the longer lifetime of hydroxyl radicals. Furthermore, the lower the altitude, the more pronounced the oxidation process becomes. TiO2 clusters accelerate the reaction of DPhP with hydroxyl radicals, but simultaneously inhibit the ozonolysis of the DPhP molecule. Finally, among the significant transformation products generated by this process are glyoxal, malealdehyde, aromatic aldehydes, and similar compounds, which are still environmentally hazardous. The investigation into OPEs' atmospheric governance has yielded these novel findings.