Phenotypic Changes Research Articles

Roles and Mechanisms of miRNAs in Abdominal Aortic Aneurysm: Signaling Pathways and Clinical Insights.

Abdominal aortic aneurysm refers to a serious medical condition that can cause the irreversible expansion of the abdominal aorta, which can lead to ruptures that are associated with up to 80% mortality. Currently, surgical and interventional procedures are the only treatment options available for treating abdominal aortic aneurysm patients. In this review, we focus on the upstream and downstream molecules of the microRNA-related signaling pathways and discuss the roles, mechanisms, and targets of microRNAs in abdominal aortic aneurysm modulation to provide novel insights for precise and targeted drug therapy for the vast number of abdominal aortic aneurysm patients. Recent studies have highlighted that microRNAs, which are emerging as novel regulators of gene expression, are involved in the biological activities of regulating abdominal aortic aneurysms. Accumulating studies suggested that microRNAs modulate abdominal aortic aneurysm development through various signaling pathways that are yet to be comprehensively summarized. A total of six signaling pathways (NF-κB signaling pathway, PI3K/AKT signaling pathway, MAPK signaling pathway, TGF-β signaling pathway, Wnt signaling pathway, and P53/P21 signaling pathway), and a total of 19 miRNAs are intimately associated with the biological properties of abdominal aortic aneurysm through targeting various essential molecules. MicroRNAs modulate the formation, progression, and rupture of abdominal aortic aneurysm by regulating smooth muscle cell proliferation and phenotype change, vascular inflammation and endothelium function, and extracellular matrix remodeling. Because of the broad crosstalk among signaling pathways, a comprehensive analysis of miRNA-mediated signaling pathways is necessary to construct a well-rounded upstream and downstream regulatory network for future basic and clinical research of AAA therapy.

Adaptive attenuation of virulence in hypervirulent carbapenem-resistant Klebsiella pneumoniae.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

Transcriptomic and metabolomic profiling reveals molecular regulatory network involved in flower development and phenotypic changes in two Lonicera macranthoides varieties

Transcriptomic and metabolomic profiling reveals molecular regulatory network involved in flower development and phenotypic changes in two Lonicera macranthoides varieties

Tanshinol ameliorates imiquimod-induced psoriasis by inhibiting M1 macrophage polarization through suppression of the notch signaling pathway.

Psoriasis is a common immune-related chronic inflammatory skin disease, often accompanied by significant itching, and once diseased, the course of the disease lasts for most of the lifetime. Tanshinol (TAN) is an active ingredient of Salvia miltiorrhiza, which possesses pharmacological effects such as anti-inflammatory and antioxidant properties. However, the effects of TAN on psoriasis have not been widely reported. Therefore, the aim of this study was to investigate the therapeutic effects and mechanisms of TAN in psoriasis. An imiquimod (IMQ)-induced psoriasis mouse model was constructed and treated with different doses of TAN to observe the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. Further removal of macrophages or inhibition or activation of Notch signaling pathway was performed to examine the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. In addition, in vitro experiments verified that TAN regulates RAW264.7 macrophage polarization and cytokine secretion through the Notch pathway. The results showed that TAN alleviated IMQ-induced skin lesions and pathological phenotypes in psoriasis mice and inhibited Notch signaling pathway and M1-type macrophage polarization. Moreover, macrophage clearance and Notch signaling pathway activation inhibited the effect of TAN on psoriasis. Further in vitro experiments showed that Notch agonists reversed the effects of TAN on macrophage polarization and inflammatory cytokines. Collectively, these findings suggest that TAN may exert a therapeutic effect on psoriasis by inhibiting the Notch signaling pathway and thus M1-type macrophage polarization.

YQTL Pipeline: A structured computational workflow for large scale quantitative trait loci discovery and downstream visualization.

Quantitative trait loci (QTL) denote regions of DNA whose variation is associated with variations in quantitative traits. QTL discovery is a powerful approach to understand how changes in molecular and clinical phenotypes may be related to DNA sequence changes. However, QTL discovery analysis encompasses multiple analytical steps and the processing of multiple input files, which can be laborious, error prone, and hard to reproduce if performed manually. To facilitate and automate large-scale QTL analysis, we developed the yQTL Pipeline, where the 'y' indicates the dependent quantitative variable being modeled. Prior to the association test, the pipeline supports the calculation or the direct input of pre-defined genome-wide principal components and genetic relationship matrix when applicable. User-specified covariates can also be provided. Depending on whether familial relatedness exists among the subjects, genome-wide association tests will be performed using either a linear mixed-effect model or a linear model. The options to run an ANOVA model or testing the interaction with a covariate are also available. Using the workflow management tool Nextflow, the pipeline parallelizes the analysis steps to optimize run-time and ensure results reproducibility. In addition, a user-friendly R Shiny App is developed to facilitate result visualization. It can generate Manhattan and Miami plots of phenotype traits, genotype-phenotype boxplots, and trait-QTL connection networks. We applied the yQTL Pipeline to analyze metabolomics profiles of blood serum from the New England Centenarians Study (NECS) participants. A total of 9.1M SNPs and 1,052 metabolites across 194 participants were analyzed. Using a p-value cutoff 5e-8, we found 14,983 mQTLs associated with 312 metabolites. The built-in parallelization of our pipeline reduced the run time from ~90 min to ~26 min. Visualization using the R Shiny App revealed multiple mQTLs shared across multiple metabolites. The yQTL Pipeline is available with documentation on GitHub at https://github.com/montilab/yQTLpipeline.

The transcriptional landscape of cancer stem-like cell functionality in breast cancer

BackgroundCancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression.MethodsIn this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors.ResultsWe found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer.ConclusionThese results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.

Energy deprivation affects nitrogen assimilation and fatty acid biosynthesis leading to leaf chlorosis under waterlogging stress in the endangered Abies koreana.

Energy deprivation triggers various physiological, biochemical and molecular changes in plants under abiotic stress. We investigated the oxidative damages in the high altitude grown conifer Korean fir (Abies koreana) exposed to waterlogging stress. Our experimental results showed that waterlogging stress led to leaf chlorosis, 35days after treatment. A significant decrease in leaf fresh weight, chlorophyll and sugar content supported this phenotypic change. Biochemical analysis showed a significant increase in leaf proline, lipid peroxidase and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical content of waterlogged plants. To elucidate the molecular mechanisms, we conducted RNA-sequencing (RNA-seq) and de novo assembly. Using RNA-seq analysis approach and filtering (P<0.05 and false discovery rate<0.001), we obtained 134 unigenes upregulated and 574 unigenes downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis placed the obtained differentially expressed unigenes in α-linoleic pathway, fatty acid degradation, glycosis, glycolipid metabolism and oligosaccharide biosynthesis process. Mapping of unigenes with Arabidopsis using basic local alignment search tool for nucleotides showed several critical genes in photosynthesis and carbon metabolism downregulated. Following this, we found the repression of multiple nitrogen (N) assimilation and nucleotide biosynthesis genes including purine metabolism. In addition, waterlogging stress reduced the levels of polyunsaturated fatty acids with a concomitant increase only in myristic acid. Together, our results indicate that the prolonged snowmelt may cause inability of A. koreana seedlings to lead the photosynthesis normally due to the lack of root intercellular oxygen and emphasizes a detrimental effect on the N metabolic pathway, compromising this endangered tree's ability to be fully functional under waterlogging stress.

Invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis

IntroductionA dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. MethodsCecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT−/−) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. ResultsWithin iNKT−/− mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT−/− mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT−/− mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. ConclusionsThe observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

The shaping of mRNA translation plasticity by RNA G-quadruplexes in cancer progression and therapy resistance.

Translational reprogramming in response to oncogenic signaling or microenvironmental stress factors shapes the proteome of cancer cells, enabling adaptation and phenotypic changes underlying cell plasticity, tumor progression and response to cancer therapy. Among the mechanisms regulating translation are RNA G-quadruplexes (RG4s), non-canonical four-stranded structures whose conformational modulation by small molecule ligands and RNA-binding proteins affects the expression of cancer proteins. Here, we discuss the role of RG4s in the regulation of mRNA translation by focusing on paradigmatic examples showing their contribution to adaptive mechanisms of mRNA translation in cancer.

Double-negative T cells have a reparative role after experimental severe ischemic acute kidney injury.

T cells mediate organ injury and repair. A proportion of unconventional kidney T cells called double-negative (DN) T cells (TCR+ CD4- CD8-), with anti-inflammatory properties, were previously demonstrated to protect from early injury in moderate experimental acute kidney injury (AKI). However, their role in repair after AKI has not been studied. We hypothesized that DN T cells mediate repair after severe AKI. C57B6 mice underwent severe (40 min) unilateral ischemia-reperfusion injury (IRI). Kidney DN T cells were studied by flow cytometry and compared with gold-standard anti-inflammatory CD4+ regulatory T cells (Tregs). In vitro effects of DN T cells and Tregs on renal tubular epithelial cell (RTEC) repair after injury were quantified with live-cell analysis. DN T cells, Tregs, CD4, or vehicle were adoptively transferred after severe AKI. Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-sinistrin. Fibrosis was assessed with Masson's trichrome staining. Profibrotic genes were measured with qRT-PCR. Percentages and the numbers of DN T cells substantially decreased during repair phase after severe AKI, as well as their activation and proliferation. Both DN T cells and Tregs accelerated RTEC cell repair in vitro. Post-AKI transfer of DN T cells reduced kidney fibrosis and improved GFR, as did Treg transfer. DN T cell transfer lowered transforming growth factor (TGF)β1 and α-smooth muscle actin (αSMA) expression. DN T cells reduced effector-memory CD4+ T cells and IL-17 expression. DN T cells undergo quantitative and phenotypical changes after severe AKI, accelerate RTEC repair in vitro as well as improve GFR and renal fibrosis in vivo. DN T cells have potential as immunotherapy to accelerate repair after AKI.NEW & NOTEWORTHY Double-negative (DN) T cells (CD4- CD8-) are unconventional kidney T cells with regulatory abilities. Their role in repair from acute kidney injury (AKI) is unknown. Kidney DN T cell population decreased during repair after ischemic AKI, in contrast to regulatory T cells (Tregs) which increased. DN T cell administration accelerated tubular repair in vitro, while after severe in vivo ischemic injury reduced kidney fibrosis and increased glomerular filtration rate (GFR). DN T cell infusion is a potential therapeutic agent to improve outcome from severe AKI.

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Evolutionary adaptations generally reverse phenotypic plasticity to restore ancestral phenotypes during new environment adaptation in cattle.

Phenotype plasticity and evolution adaptations are the two main ways in which allow populations to deal with environmental changes, but the potential relationship between them remains controversial. Using a reciprocal transplant approach with cattle adapted to the Tibetan Plateau and adjacent lowlands, we aim to investigate the relative contributions of evolutionary processes and phenotypic plasticity in driving both phenotypic and transcriptomic changes under natural conditions. We observed that while numerous genetic transcriptomic changes were evident during the forward adaptation to highland environments, plastic changes predominantly facilitate the transformation of transcriptomes into a preferred state when Tibetan cattle are reintroduced to lowland habitats. Genes with ancestral plasticity are generally reversed by evolutionary adaptations and show a closer expression level to the ancestral stage in evolved Tibetan cattle. A similar trend was also observed at the phenotypes level, with a majority of biochemical and hemorheology phenotypes showing a tendency to revert to their ancestral patterns, suggesting the restoration of ancestral expression levels is a widespread evolutionary trend during adaptation. The findings of our study contribute to the debate regarding the relative contributions of plasticity and genetic changes in mammal environment adaptation. Furthermore, we highlight that the restoration of ancestral phenotypes represents a general pattern in cattle new environment adaptation.

Non-canonical mRNA translation initiation in cell stress and cancer.

The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.

Environment-induced heritable variations are common in Arabidopsis thaliana

Parental or ancestral environments can induce heritable phenotypic changes, but whether such environment-induced heritable changes are a common phenomenon remains unexplored. Here, we subject 14 genotypes of Arabidopsis thaliana to 10 different environmental treatments and observe phenotypic and genome-wide gene expression changes over four successive generations. We find that all treatments caused heritable phenotypic and gene expression changes, with a substantial proportion stably transmitted over all observed generations. Intriguingly, the susceptibility of a genotype to environmental inductions could be predicted based on the transposon abundance in the genome. Our study thus challenges the classic view that the environment only participates in the selection of heritable variation and suggests that the environment can play a significant role in generating of heritable variations.

SGLT2 inhibition eliminates senescent cells and alleviates pathological aging.

It has been reported that accumulation of senescent cells in various tissues contributes to pathological aging and that elimination of senescent cells (senolysis) improves age-associated pathologies. Here, we demonstrate that inhibition of sodium-glucose co-transporter 2 (SGLT2) enhances clearance of senescent cells, thereby ameliorating age-associated phenotypic changes. In a mouse model of dietary obesity, short-term treatment with the SGLT2 inhibitor canagliflozin reduced the senescence load in visceral adipose tissue and improved adipose tissue inflammation and metabolic dysfunction, but normalization of plasma glucose by insulin treatment had no effect on senescent cells. Canagliflozin extended the lifespan of mice with premature aging even when treatment was started in middle age. Metabolomic analyses revealed that short-term treatment with canagliflozin upregulated 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, enhancing immune-mediated clearance of senescent cells by downregulating expression of programmed cell death-ligand 1. These findings suggest that inhibition of SGLT2 has an indirect senolytic effect by enhancing endogenous immunosurveillance of senescent cells.

Aging promotes mast cell activation and antigen presenting capacity in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Aging is an independent and dominant risk factor for atherosclerosis and is associated with a low-grade chronic inflammation termed inflammaging. This age-induced pro-inflammatory environment may have great impact on plaque-residing immune cells, including mast cells. Mast cells have been found to accumulate in the human atherosclerotic plaque upon disease progression and have been associated with plaque instability. However, it is currently unknown whether aging drives the pro-atherogenic effects of mast cells in atherosclerosis. To assess the effects of aging on mast cell phenotype, we examined mast cell populations in young and old male Ldlr-/- mice fed either a chow (CD) or Western-type (WD) diet. We observed a 6-fold increase in the number of mast cells in the aged atherosclerotic aorta compared to young (Y-CD: 4.5±0.7 vs. O-CD: 279.4±9 cells, p&amp;lt;0.01; Y-WD: 45.3±15.2 vs. O-CD: 279.4±9 cells, p&amp;lt;0.01). Furthermore, we detected a marked increase in activation status with age as shown by the significantly augmented number of CD63+ mast cells in the atherosclerotic aorta (Y-CD: 1.8±0.6 vs. O-CD: 259.7±85.4 cells, p&amp;lt;0.01; Y-WD: 41.3±13.6 vs. O-CD: 259.7±85.4 cells, p&amp;lt;0.01). In line, we identified that aged bone-marrow derived mast cells displayed elevated basal CD63 expression compared to young cells, indicative of intrinsic low-grade activation. Moreover, we demonstrated that aging increases the amount of MHCII+ mast cells in atherosclerosis, allowing them to act as antigen-presenting cells, which correspondingly led to increased CD4+ T cell proliferation in vitro (young: 35.2±1.2% vs. old: 45.8±1.2%, p&amp;lt;0.001). Finally, single-cell RNA sequencing of human atherosclerotic plaques confirmed mast cell-specific expression of MHC-II orthologs. To conclude, we established that aging induces phenotypic changes of mast cells in atherosclerosis, regarding both activation status and antigen presenting capacity, which can contribute to disease progression.

MicroRNA-31-5p is upregulated during neointima formation and influences human coronary artery smooth muscle cell functions by regulating PIAS3 and DKK1

Abstract Funding Acknowledgements None. Background and purpose MicroRNAs (miRs) have emerged as potential therapeutic targets in cardiovascular disease, given their ability to modulate multiple signaling pathways and cellular processes. Neointima formation as an angioplasty-related complication remains a major issue calling for innovative therapeutic options and is predominantly mediated by phenotypic and functional changes of vascular smooth muscle cells. Here, we propose that targeting miR-31-5p might be an effective approach to selectively modulate smooth muscle cell functions to enhance vascular healing and regeneration. Methods C57J/BlL6 mice (n=6, 3 months, male) were subjected to wire-induced femoral artery injury. At days 10 and 21 after injury, femoral arteries were harvested for miRNA analysis. Expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (HCAEC) and -smooth muscle cells (HCASMC). Further, the effect of miR-31-5p on cellular functions was investigated on HCAECs and HCASMCs in vitro, including migration (scratch assay), proliferation (BrdU assay), and apoptosis. Possible targets of miR-31-5p were identified in silico by structured literature research and target binding prediction and confirmed on mRNA and protein levels. High-resolution microscopy analysis was used to investigate morphological changes. Results Expression analysis revealed a significant upregulation of miR-31-5p (p&amp;lt;0.0001) in murine femoral artery neointimal tissue at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in HCASMC (p&amp;lt;0,05) but not HCAEC following serum stimulation. On a functional level, miR-31-5p revealed dissenting effects on HCAEC and HCASMC. Whereas migration and proliferation are not altered in HCAECs, knockdown of miR-31-5p significantly reduced migration (p&amp;lt;0,01), proliferation (p&amp;lt;0,05), and increased apoptosis (p&amp;lt;0,05) in HCASMCs. Additionally, the knockdown of miR-31-5p influenced the phenotypical switch of HCASMC towards a contractile phenotype. Under these conditions, the release of cytokines like TNF-alpha and IL1-beta was also significantly reduced (p&amp;lt;0,05) in HCASMCs. Systematic in silico target screens suggested DKK1 and PIAS3 as potential direct targets. Indeed, knockdown of miR-31-5p resulted in a significant upregulation of DKK1 in HCASMC (p&amp;lt;0,05) on mRNA level and DKK1 (p&amp;lt;0,05) and PIAS3 (p&amp;lt;0,05) on mRNA and protein level. An overexpression resulted in a significant downregulation of DKK1 in HCASMC (p&amp;lt;0,05) on mRNA level. Conclusion In conclusion, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional neointima formation-promoting role in HCASMC rather than HCAEC. Mechanistically, DKK1 and PIAS3 as direct targets of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate HCASMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.

Identifying novel clinical phenotypes of acute respiratory distress syndrome using trajectories of daily fluid balance: a secondary analysis of randomized controlled trials

BackgroundPreviously identified phenotypes of acute respiratory distress syndrome (ARDS) could not reveal the dynamic change of phenotypes over time. We aimed to identify novel clinical phenotypes in ARDS using trajectories of fluid balance, to test whether phenotypes respond differently to different treatment, and to develop a simplified model for phenotype identification.MethodsFACTT (conservative vs liberal fluid management) trial was classified as a development cohort, joint latent class mixed models (JLCMMs) were employed to identify trajectories of fluid balance. Heterogeneity of treatment effect (HTE) for fluid management strategy across phenotypes was investigated. We also constructed a parsimonious probabilistic model using baseline data to predict the fluid trajectories in the development cohort. The trajectory groups and the probabilistic model were externally validated in EDEN (initial trophic vs full enteral feeding) trial.ResultsUsing JLCMM, we identified two trajectory groups in the development cohort: Class 1 (n = 758, 76.4% of the cohort) had an early positive fluid balance, but achieved negative fluid balance rapidly, and Class 2 (n = 234, 24.6% of the cohort) was characterized by persistent positive fluid balance. Compared to Class 1 patients, patients in Class 2 had significantly higher 60-day mortality (53.5% vs. 17.8%, p < 0.001), and fewer ventilator-free days (0 vs. 20, p < 0.001). A significant HTE between phenotypes and fluid management strategies was observed in the FACTT. An 8-variables model was derived for phenotype assignment.ConclusionsWe identified and validated two novel clinical trajectories for ARDS patients, with both prognostic and predictive enrichment. The trajectories of ARDS can be identified with simple classifier models.

Transcription profiling reveals tissue-specific metabolic pathways in the fat body and ovary of the diapausing mosquito Culex pipiens

The northern house mosquito, Culex pipiens, employs diapause as an essential survival strategy during winter, inducing important phenotypic changes such as enhanced stress tolerance, lipid accumulation, and extended longevity. During diapause, the cessation of reproductive development represents another distinctive phenotypic change, underlining the need for adjusted modulation of gene expressions within the ovary. Although considerable advancements in screening gene expression profiles in diapausing and non-diapausing mosquitoes, there remains a gap in tissue-specific transcriptomic profiling that could elucidate the complicated formation of diverse diapause features in Cx. pipiens. Here, we filled this gap by utilizing RNA sequencing, providing a detailed examination of gene expression patterns in the fat body and ovary during diapause compared to non-diapause conditions. Functional annotation of upregulated genes identified associations with carbohydrate metabolism, stress tolerance, immunity, and epigenetic regulation. The validation of candidate genes using quantitative real-time PCR verified the differentially expressed genes identified in diapausing mosquitoes. Our findings contribute novel insights into potential regulators during diapause in Cx. pipiens, thereby opening possible avenues for developing innovative vector control strategies.

Sdc4 deletion perturbs intervertebral disc matrix homeostasis and promotes early osteopenia in the aging mouse spine

Syndecan 4 (SDC4), a cell surface heparan sulfate proteoglycan, is known to regulate matrix catabolism by nucleus pulposus cells in an inflammatory milieu. However, the role of SDC4 in the aging spine has never been explored. Here we analyzed the spinal phenotype of Sdc4 global knockout (KO) mice as a function of age. Micro-computed tomography showed that Sdc4 deletion severely reduced vertebral trabecular and cortical bone mass, and biomechanical properties of vertebrae were significantly altered in Sdc4 KO mice. These changes in vertebral bone were likely due to elevated osteoclastic activity. The histological assessment showed subtle phenotypic changes in the intervertebral disc. Imaging-Fourier transform-infrared analyses showed a reduced relative ratio of mature collagen crosslinks in young adult nucleus pulposus (NP) and annulus fibrosus (AF) of KO compared to wildtype discs. Additionally, relative chondroitin sulfate levels increased in the NP compartment of the KO mice. Transcriptomic analysis of NP tissue using CompBio, an AI-based tool showed biological themes associated with prominent dysregulation of heparan sulfate GAG degradation, mitochondria metabolism, autophagy, endoplasmic reticulum (ER)-associated misfolded protein processes and ER to Golgi protein processing. Overall, this study highlights the important role of SDC4 in fine-tuning vertebral bone homeostasis and extracellular matrix homeostasis in the mouse intervertebral disc.