Scap structures highlight key role for rotation of intertwined luminal loops in cholesterol sensing.

The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.

Retrospective on Cholesterol Homeostasis: The Central Role of Scap.

Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.

Proteostatic Tactics in the Strategy of Sterol Regulation.

In eukaryotes, the synthesis and uptake of sterols undergo stringent multivalent regulation. Both individual enzymes and transcriptional networks are controlled to meet changing needs of the many sterol pathway products. Regulation is tailored by evolution to match regulatory constraints, which can be very different in distinct species. Nevertheless, a broadly conserved feature of many aspects of sterol regulation is employment of proteostasis mechanisms to bring about control of individual proteins. Proteostasis is the set of processes that maintain homeostasis of a dynamic proteome. Proteostasis includes protein quality control pathways for the detection, and then the correction or destruction, of the many misfolded proteins that arise as an unavoidable feature of protein-based life. Protein quality control displays not only the remarkable breadth needed to manage the wide variety of client molecules, but also extreme specificity toward the misfolded variants of a given protein. These features are amenable to evolutionary usurpation as a means to regulate proteins, and this approach has been used in sterol regulation. We describe both well-trod and less familiar versions of the interface between proteostasis and sterol regulation and suggest some underlying ideas with broad biological and clinical applicability.

Cholesterol Homeostatic Regulator SCAP-SREBP2 Integrates NLRP3 Inflammasome Activation and Cholesterol Biosynthetic Signaling in Macrophages.

Cholesterol metabolism has been linked to immune functions, but the mechanisms by which cholesterol biosynthetic signaling orchestrates inflammasome activation remain unclear. Here, we have shown that NLRP3 inflammasome activation is integrated with the maturation of cholesterol master transcription factor SREBP2. Importantly, SCAP-SREBP2 complex endoplasmic reticulum-to-Golgi translocation was required for optimal activation of the NLRP3 inflammasome both in vitro and in vivo. Enforced cholesterol biosynthetic signaling by sterol depletion or statins promoted NLPR3 inflammasome activation. However, this regulation did not predominantly depend on changes in cholesterol homeostasis controlled by the transcriptional activity of SREBP2, but relied on the escort activity of SCAP. Mechanistically, NLRP3 associated with SCAP-SREBP2 to form a ternary complex which translocated to the Golgi apparatus adjacent to a mitochondrial cluster for optimal inflammasome assembly. Our study reveals that, in addition to controlling cholesterol biosynthesis, SCAP-SREBP2 also serves as a signaling hub integrating cholesterol metabolism with inflammation in macrophages.

Structural advances in sterol-sensing domain-containing proteins.

The sterol-sensing domain (SSD) is present in several membrane proteins that function in cholesterol metabolism, transport, and signaling. Recent progress in structural studies of SSD-containing proteins, such as sterol regulatory element-binding protein (SREBP)-cleavage activating protein (Scap), Patched, Niemann-Pick disease type C1 (NPC1), and related proteins, reveals a conserved core that is essential for their sterol-dependent functions. This domain, by its name, 'senses' the presence of sterol substrates through interactions and may modulate protein behaviors with changing sterol levels. We summarize recent advances in structural and mechanistic investigations of these proteins and propose to divide them to two classes: M for 'moderator' proteins that regulate sterol metabolism in response to membrane sterol levels, and T for 'transporter' proteins that harbor inner tunnels for cargo trafficking across cellular membranes.

STAT3 activation of SCAP-SREBP-1 signaling upregulates fatty acid synthesis to promote tumor growth.

SCAP plays a central role in controlling lipid homeostasis by activating SREBP-1, a master transcription factor in controlling fatty acid (FA) synthesis. However, how SCAP expression is regulated in human cancer cells remains unknown. Here, we revealed that STAT3 binds to the promoter of SCAP to activate its expression across multiple cancer cell types. Moreover, we identified that STAT3 also concurrently interacts with the promoter of SREBF1 gene (encoding SREBP-1), amplifying its expression. This dual action by STAT3 collaboratively heightens FA synthesis. Pharmacological inhibition of STAT3 significantly reduces the levels of unsaturated FAs and phospholipids bearing unsaturated FA chains by reducing the SCAP-SREBP-1 signaling axis and its downstream effector SCD1. Examination of clinical samples from patients with glioblastoma, the most lethal brain tumor, demonstrates a substantial co-expression of STAT3, SCAP, SREBP-1, and SCD1. These findings unveil STAT3 directly regulates the expression of SCAP and SREBP-1 to promote FA synthesis, ultimately fueling tumor progression.

PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability.

The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.

The role of KLF2 in regulating hepatic lipogenesis and blood cholesterol homeostasis via the SCAP/SREBP pathway.

Liver steatosis is a common metabolic disorder resulting from imbalanced lipid metabolism, which involves various processes such as de novo lipogenesis, fatty acid uptake, fatty acid oxidation, and VLDL secretion. In this study, we discovered that KLF2, a transcription factor, plays a crucial role in regulating lipid metabolism in the liver. Overexpression of KLF2 in the liver of db/db mice, C57BL/6J mice, and Cd36-/- mice fed on a normal diet resulted in increased lipid content in the liver. Additionally, transgenic mice (ALB-Klf2) that overexpressed Klf2 in the liver developed liver steatosis after being fed a normal diet. We found that KLF2 promotes lipogenesis by increasing the expression of SCAP, a chaperone that facilitates the activation of SREBP, the master transcription factor for lipogenic gene expression. Our mechanism studies revealed that KLF2 enhances lipogenesis in the liver by binding to the promoter of SCAP and increasing the expression of genes involved in fatty acid synthesis. Reduction of KLF2 expression led to a decrease in SCAP expression and a reduction in the expression of SREBP1 target genes involved in lipogenesis. Overexpression of KLF2 also increased the activation of SREBP2 and the mRNA levels of its downstream target SOAT1. In C57BL/6J mice fed a high-fat diet, overexpression of Klf2 increased blood VLDL secretion, while reducing its expression decreased blood cholesterol levels. Our study emphasizes the novelty that hepatic KLF2 plays a critical role in regulating lipid metabolism through the KLF2/SCAP/SREBPs pathway, which is essential for hepatic lipogenesis and maintaining blood cholesterol homeostasis.

N-glycosylation of SCAP exacerbates hepatocellular inflammation and lipid accumulation via ACSS2-mediated histone H3K27 acetylation.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a widely expressed membrane glycoprotein that acts as an important modulator of lipid metabolism and inflammatory stress. N-glycosylation of SCAP has been suggested to modulate cancer development, but its role in nonalcoholic steatohepatitis (NASH) is poorly understood. In this study, the N-glycosylation of SCAP was analyzed by using sequential trypsin proteolysis and glycosidase treatment. The liver cell lines expressing wild-type and N-glycosylation sites mutated SCAP were constructed to investigate the N-glycosylation role of SCAP in regulating inflammation and lipid accumulation as well as the underlying mechanisms. The hepatic SCAP protein levels were significantly increased in C57BL/6J mice fed with Western diet and sugar water (WD + SW) and diabetic db/db mice, which exhibited typical liver steatosis and inflammation accompanied with hyperglycemia. In vitro, the enhanced N-glycosylation by high glucose increased the protein stability of SCAP and hence increased its total protein levels, whereas the ablation of N-glycosylation significantly decreased SCAP protein stability and alleviated lipid accumulation and inflammation in hepatic cell lines. Mechanistically, SCAP N-glycosylation increased not only the SREBP-1-mediated acetyl-CoA synthetase 2 (ACSS2) transcription but also the AMPK-mediated S659 phosphorylation of ACCS2 protein, causing the enhanced ACSS2 levels in nucleus and hence increasing the histone H3K27 acetylation (H3K27ac), which is a key epigenetic modification associated with NASH. Modulating ACSS2 expression or its location in the nuclear abolished the effects of SCAP N-glycosylation on H3K27ac and lipid accumulation and inflammation. In conclusion, SCAP N-glycosylation aggravates inflammation and lipid accumulation through enhancing ACSS2-mediated H3K27ac in hepatocytes.NEW & NOTEWORTHY N-glycosylation of SCAP exacerbates inflammation and lipid accumulation in hepatocytes through ACSS2-mediated H3K27ac. Our data suggest that SCAP N-glycosylation plays a key role in regulating histone H3K27 acetylation and targeting SCAP N-glycosylation may be a new strategy for treating nonalcoholic steatohepatitis (NASH).

Microglial SCAP deficiency protects against diabetes-associated cognitive impairment through inhibiting NLRP3 inflammasome-mediated neuroinflammation.

Hyperglycemia-induced pathological microglial responses and subsequent neuronal damage are notable characteristics of diabetes-associated cognitive impairment (DACI). Cholesterol accumulation in the brain is a prevalent consequence of diabetes mellitus (DM), exacerbating pathological microglial responses. Regarding disordered glucose and lipid metabolism, the Sterol Regulatory Element-Binding Protein (SREBP) cleavage-activating protein (SCAP), a cholesterol sensor, exhibits increased expression and abnormal translocation from the endoplasmic reticulum to the Golgi, amplifying the inflammatory response. Therefore, we hypothesized that overexpression of microglia-SCAP and cholesterol accumulation in DM mice could induce pathological microglial responses associated with DACI. Our type 2 DM mice model presented an abnormal increase in microglial SCAP expression. The functional loss of microglia-specific SCAP in DM mice improved cognitive impairment, neuronal synaptic plasticity deficits, and abnormal microglial responses. Mechanistically, the accumulated SCAP directly bound to and enhanced the activation of the microglial-specific inflammatory amplifier, NLRP3 inflammasome, in Golgi, thereby increasing pathological microglial responses and promoting neuronal damage. These findings indicate an important regulatory axis of microglial responses from SCAP to the NLRP3 inflammasome pathway in microglia. These underscore the crosstalk between cholesterol disorders and pathological microglial responses, offering a promising avenue for pharmaceutical interventions in DACI.

The Clinical Significance and the Potential Regulatory Mechanism of the LncRNA OIP5-AS1 in Paediatric Severe Community-Acquired Pneumonia Blood Through the MiR-150-5p/PDCD4 Axis.

BACKGROUND: This study aimed to elucidate the clinical significance and regulatory mechanism of the long non-coding RNA OIP5-AS1 in severe community-acquired pneumonia (SCAP) among paediatric patients. METHODS: qRT-PCR was used to assess the mRNA levels of OIP5-AS1. ROC curve analysis was used to assess the diagnostic significance of OIP5-AS1. Short-term prognostic significance was evaluated through Kaplan-Meier survival. An in vitro cell model was developed using LPS-induced MRC-5 cells. CCK-8, flow cytometry, and ELISA were conducted to measure cell viability, apoptosis, and inflammatory factor levels. The association between miR-150-5p and PDCD4 was confirmed through DLR assays. RESULTS: Elevated OIP5-AS1 were observed in paediatric patients with SCAP, which enabled effective differentiation from healthy individuals. High expression of OIP5-AS1 correlated with reduced survival rates. OIP5-AS1 knockdown attenuated cell viability suppression and the promotion of apoptosis and inflammatory factors induced by LPS. However, this attenuation was reversed by reduced levels of miR-150-5p. miR-150-5p was identified as a target of PDCD4 and OIP5-AS1. CONCLUSION: Increased OIP5-AS1 levels show potential as a valuable diagnostic and prognostic biomarker for paediatric patients with SCAP. This study illustrates its role in regulating cell viability, apoptosis, and the inflammatory response via the miR-150-5p/PDCD4 axis, acting as a ceRNA.

Clinical profile analysis and nomogram for predicting in-hospital mortality among elderly severe community-acquired pneumonia patients: a retrospective cohort study.

BACKGROUND: Severe community-acquired pneumonia is one of the most lethal forms of CAP with high mortality. For rapid and accurate decisions, we developed a mortality prediction model specifically tailored for elderly SCAP patients. METHODS: The retrospective study included 2365 elderly patients. To construct and validate the nomogram, we randomly divided the patients into training and testing cohorts in a 70% versus 30% ratio. The primary outcome was in-hospital mortality. Univariate and multivariate logistic regression analyses were used in the training cohort to identify independent risk factors. The robustness of this model was assessed using the C index, ROC and AUC. DCA was employed to evaluate the predictive accuracy of the model. RESULTS: Six factors were used as independent risk factors for in-hospital mortality to construct the prediction model, including age, the use of vasopressor, chronic renal disease, neutrophil, platelet, and BUN. The C index was 0.743 (95% CI 0.719-0.768) in the training cohort and 0.731 (95% CI 0.694-0.768) in the testing cohort. The ROC curves and AUC for the training cohort and testing cohort (AUC = 0.742 vs. 0.728) indicated a robust discrimination. And the calibration plots showed a consistency between the prediction model probabilities and observed probabilities. Then, the DCA demonstrated great clinical practicality. CONCLUSIONS: The nomogram incorporated six risk factors, including age, the use of vasopressor, chronic renal disease, neutrophil, platelet and BUN, which had great predictive accuracy and robustness, while also demonstrating clinical practicality at ICU admission.

The Role of SCAP/SREBP as Central Regulators of Lipid Metabolism in Hepatic Steatosis.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide at an alarming pace, due to an increase in obesity, sedentary and unhealthy lifestyles, and unbalanced dietary habits. MASLD is a unique, multi-factorial condition with several phases of progression including steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Sterol element binding protein 1c (SREBP1c) is the main transcription factor involved in regulating hepatic de novo lipogenesis. This transcription factor is synthesized as an inactive precursor, and its proteolytic maturation is initiated in the membrane of the endoplasmic reticulum upon stimulation by insulin. SREBP cleavage activating protein (SCAP) is required as a chaperon protein to escort SREBP from the endoplasmic reticulum and to facilitate the proteolytic release of the N-terminal domain of SREBP into the Golgi. SCAP inhibition prevents activation of SREBP and inhibits the expression of genes involved in triglyceride and fatty acid synthesis, resulting in the inhibition of de novo lipogenesis. In line, previous studies have shown that SCAP inhibition can resolve hepatic steatosis in animal models and intensive research is going on to understand the effects of SCAP in the pathogenesis of human disease. This review focuses on the versatile roles of SCAP/SREBP regulation in de novo lipogenesis and the structure and molecular features of SCAP/SREBP in the progression of hepatic steatosis. In addition, recent studies that attempt to target the SCAP/SREBP axis as a therapeutic option to interfere with MASLD are discussed.

Decoding the multifaceted signatures and transcriptomic characteristics of stem cells derived from apical papilla and dental pulp of human supernumerary teeth.

Supernumerary teeth are advantaged sources for high-quality stem cell preparation from both apical papilla (SCAP-Ss) and dental pulp (DPSCs). However, the deficiency of the systematic and detailed comparison of the biological and transcriptomic characteristics of the aforementioned stem cells largely hinders their application in regenerative medicine. Herein, we collected supernumerary teeth for SCAP-S and DPSC isolation and identification by utilizing multiple biological tests (e.g., growth curve, cell cycle and apoptosis, adipogenic and osteogenic differentiation, and quantitative real-time polymerase chain reaction). Furthermore, we took advantage of transcriptome sequencing and multifaceted bioinformatic analyses to dissect the similarities and diversities between them. In this study, we found that SCAP-Ss and DPSCs showed indistinctive signatures in morphology and immunophenotypes, whereas with diversity in cell vitality and multi-lineage differentiation as well as gene expression profiling and differentially expressed genes-associated gene ontology and signaling pathways. Collectively, our data indicated the diversity of the multifaceted signatures of human supernumerary teeth-derived stem cells both at the cellular and molecular levels, which also supplied new references for SCAP-Ss serving as splendid alternative stem cell sources for regenerative medicine purposes.

The human dental apical papilla promotes spinal cord repair through a paracrine mechanism.

Traumatic spinal cord injury is an overwhelming condition that strongly and suddenly impacts the patient's life and her/his entourage. There are currently no predictable treatments to repair the spinal cord, while many strategies are proposed and evaluated by researchers throughout the world. One of the most promising avenues is the transplantation of stem cells, although its therapeutic efficiency is limited by several factors, among which cell survival at the lesion site. In our previous study, we showed that the implantation of a human dental apical papilla, residence of stem cells of the apical papilla (SCAP), supported functional recovery in a rat model of spinal cord hemisection. In this study, we employed protein multiplex, immunohistochemistry, cytokine arrays, RT- qPCR, and RNAseq technology to decipher the mechanism by which the dental papilla promotes repair of the injured spinal cord. We found that the apical papilla reduced inflammation at the lesion site, had a neuroprotective effect on motoneurons, and increased the apoptosis of activated macrophages/ microglia. This therapeutic effect is likely driven by the secretome of the implanted papilla since it is known to secrete an entourage of immunomodulatory or pro-angiogenic factors. Therefore, we hypothesize that the secreted molecules were mainly produced by SCAP, and that by anchoring and protecting them, the human papilla provides a protective niche ensuring that SCAP could exert their therapeutic actions. Therapeutic abilities of the papilla were demonstrated in the scope of spinal cord injury but could very well be beneficial to other types of tissue.

Identification of a degradation signal at the carboxy terminus of SREBP2: A new role for this domain in cholesterol homeostasis.

Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterol-sensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2's proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1's stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity.

TTC39B destabilizes retinoblastoma protein promoting hepatic lipogenesis in a sex-specific fashion.

BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.

The role of cholesterol binding in the control of cholesterol by the Scap-Insig system.

Scap and Insig, two proteins embedded in the membrane of the endoplasmic reticulum (ER), regulate the synthesis of cholesterol in animal cells by forming a dimer in the presence of high concentrations of cholesterol. Cryo-electron microscopic structures for the Scap-Insig dimer show a sterol-binding site at the dimer interface, but none of the structures include cholesterol itself. Here, a molecular docking approach developed to characterise cholesterol binding to the transmembrane (TM) regions of membrane proteins is used to characterise cholesterol binding to sites on the TM surface of the dimer and to the interfacial binding site. Binding of cholesterol is also observed at sites on the extra-membranous luminal domains of Scap, but the properties of these sites suggest that they will be unoccupied in vivo. Comparing the structure of Scap in the dimer with that predicted by AlphaFold for monomeric Scap suggests that dimer formation could result in relocation of TM helix 7 of Scap and of the loop between TM6 and 7, and that this could be the key change on Scap that signals that there is a high concentration of cholesterol in the ER.

Clinical profile analysis and nomogram for predicting in-hospital mortality among elderly severe community-acquired pneumonia patients with comorbid cardiovascular disease: a retrospective cohort study.

BACKGROUND: Researchers have linked cardiovascular disease (CVD) with advancing age; however, how it drives disease progression in elderly severe community acquired pneumonia (SCAP) patients is still unclear. This study aims to identify leading risk predictors of in-hospital mortality in elderly SCAP patients with CVD, and construct a comprehensive nomogram for providing personalized prediction. PATIENTS AND METHODS: The study retrospectively enrolled 2365 elderly patients identified SCAP. Among them, 413 patients were found to have CVD. The LASSO regression and multivariate logistic regression analysis were utilized to select potential predictors of in-hospital mortality in elderly SCAP patients with CVD. By incorporating these features, a nomogram was then developed and subjected to internal validations. Discrimination, calibration, and clinical use of the nomogram were assessed via C-index, calibration curve analysis, and decision plot. RESULTS: Compared with patients without CVD, elderly SCAP patients with CVD had a significant poor outcome. Further analysis of the CVD population identified 7 independent risk factors for in-hospital mortality in elderly SCAP patients, including age, the use of vasopressor, numbers of primary symptoms, body temperature, monocyte, CRP and NLR. The nomogram model incorporated these 7 predictors showed sufficient predictive accuracy, with the C-index of 0.800 (95% CI 0.758-0.842). High C-index value of 0.781 was obtained in the internal validation via bootstrapping validation. Moreover, the calibration curve indicative a good consistency of risk prediction, and the decision curve manifested that the nomogram had good overall net benefits. CONCLUSION: An integrated nomogram was developed to facilitate the personalized prediction of in-hospital mortality in elderly SCAP patients with CVD.

Exosomes derived from stem cells from the apical papilla alleviate inflammation in rat pulpitis by upregulating regulatory T cells.

AIM: To evaluate the effects of exosomes derived from stem cells from the apical papilla (SCAP-Exos) in rats with experimentally induced pulpitis and the effects of SCAP-Exos on the conversion of regulatory T cells (Tregs) and methylation status of the Foxp3 locus in Tregs in vitro. METHODOLOGY: SCAP-Exos were isolated and identified using transmission electron microscopy, western blotting, and nanoparticle tracking analysis. Lipopolysaccharide was used to experimentally induced pulpitis in rats, and the effects of SCAP-Exos on the rats with pulpitis were detected using haematoxylin-eosin staining and immunofluorescence staining. CD4+ CD25- T cells were treated with different doses of SCAP-Exos, and flow cytometric analysis was used to assess the effects of SCAP-Exos on Treg proliferation and conversion. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the expression of interleukin 10 (IL-10). MethylTarget® technology was used to measure the methylation level of the Foxp3 locus in T cells. The expression levels of ten-eleven-translocation (Tet) 1, Tet2, and Tet3 in T cells were detected by real-time PCR and western blotting. RESULTS: SCAP-Exos had an elliptical vesicle-like structure with a diameter of approximately 143.7 nm and expressed the exosomal markers Alix and CD9. SCAP-Exo administration increased Treg accumulation in the inflamed dental pulp and alleviated inflammation in the dental pulp in vivo. SCAP-Exos promoted Treg conversion in vitro. Mechanistically, SCAP-Exos promoted Tet2-mediated Foxp3 demethylation to maintain the stable expression of Foxp3. CONCLUSIONS: SCAP-Exos promoted Treg conversion and effectively alleviated inflammation in the dental pulp of rats. This study shows that SCAP-Exos can regulate the local immune microenvironment to favour tissue regeneration, thus providing a potential novel strategy utilising SCAP-Exos as a cell-free approach to treat early inflammation of dental pulp in immature permanent teeth in the clinic.