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Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.

Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire lifespan. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover , they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters , regardless of the developmental stage, thereby contributing to protect cell identity.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)]. Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.

Pressure injuries in institutionalized older adults with reduced mobility represent a relevant clinical problem due to their impact on quality of life and health costs. Purpose: This study aimed to describe the behavior of blood flow and tissue temperature in the sacral region and the significance of interindividual conditions in the post-load recovery of tissue in the context of pressure injuries. Methods: An observational study was conducted in 55 older adults living in a geriatric institution. Photoplethysmography and temperature signals were recorded at four times: at baseline (preload) and 15 s, 5 min, and 10 min post-load. The perfusion index was calculated, and two dichotomous variables were defined, Return b (flow) and Return t (temperature), to evaluate the physiological capacity to return to baseline conditions. Associations with clinical, functional, and diagnostic variables were explored. Results: The perfusion index showed usefulness as an indicator of microvascular recovery, but with variability between individuals. Return b was significantly associated with BMI and diagnosis of cardiovascular disease, while t-return was associated with body temperature and basal perfusion index. No significant associations were observed with sex, age or functional scales, state of consciousness, or risk of pressure injury. Conclusions: The study shows heterogeneous physiological responses to external load influenced by individual characteristics. Flow and temperature monitoring allows a functional approximation of injury risk, although more complex models are required to understand the nonlinear dynamics of tissue responses.

Changes in levels of cytokines and chemokines have been proposed as possible biomarkers of tissue injury, including liver injury due to drugs. Recently, in acute drug-induced liver injury (DILI), we showed that 19 of 27 immune analytes were differentially expressed and that disparate patterns of immune responses were evident. Lower values of serum albumin (< 2.8 g/dL) and lower levels of only four analytes, namely, IL-9, IL-17, PDGF-bb, and RANTES, were highly predictive of early death [accuracy = 96%]. The goals of this study were to assess levels of the same 27 immune analytes in larger numbers of subjects to learn whether the earlier findings would be confirmed in new and larger cohorts of subjects, compared with a new cohort of healthy controls. We studied 127 subjects with acute DILI enrolled into the US DILIN. We also studied 118 subjects with severe acute liver injury of diverse etiologies, enrolled into the ALF SG registry of subjects. Controls comprised 63 de-identified subjects with no history of liver disease and normal liver tests. Analytes associated with poor outcomes [death before 6 months, n = 32 of the total of 232 non-acetaminophen (Apap) subjects], were lower serum albumin [2.6 vs 3.0 g/dL] and RANTES [6,458 vs 8,999 pg/mL] but higher levels of IL-6 [41 vs 18], IL-8 [78 vs 48], and MELD scores [30 vs 24]. Similar patterns were observed for outcome of death/liver transplant within 6 months. A model that included only serum albumin < 2.8 g/dL and RANTES below its median value of 11,349 had 83% (or 81%) accuracy for predicting early death (or early death/liver transplant) in 127 subjects from DILIN. No patterns of serum immune analytes were reflective of the etiologies of acute liver failure, but there were cytokine patterns that predicted prognosis in both acute DILI and ALF.

Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10−14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.

Objectives: Anterior cruciate ligament (ACL) tears are common in the adolescent athletic population. While ACL reconstruction (ACLR) enjoys largely good clinical results, there is clear variability in patient outcomes that cannot be easily explained by surgical technique. In particular, the occurrence of arthrofibrosis, muscular dysfunction, failure to return to sport and even post-traumatic osteoarthritis are difficult to predict based on demographic parameters. There is ample evidence in animal models that synovial inflammation plays a critical role in the development of joint dysfunction after ligament injury, however the role of synovial inflammation in a clinical ACL population has not been studied previously. Adolescents provide an ideal population in which to study post-ACL joint dysfunction as they typically have no pre-existing joint degeneration. We hypothesized that higher synovial immune cell infiltration at the time of ACLR would correlate with worse clinical outcomes (e.g., early loss of motion or arthrofibrosis). To investigate this hypothesis, flow cytometry of synovial samples provided a snapshot of the cellular composition of the synovium, scRNA-seq comprehensively examined a subset of those samples, and correlations with patient-reported clinical outcomes were analyzed. Methods: Patients aged 12-18 years undergoing primary ACLR were enrolled in an IRB-approved prospective study and demographic/injury information collected. At the time of surgery, an arthroscopic synovial biopsy from the prefemoral synovium was digested into a single cell suspension for immune profiling by multicolor flow cytometry and scRNA-seq. Flow cytometry data (n = 17) was acquired on a multi-channel flow cytometer and then analyzed by Principle Component Analysis (PCA) and hierarchical clustering to group the patients by their synovial immune cell profile. Some cells from 6 of the 17 samples were also used for scRNA-seq. Droplet barcoding of cells, RNA library construction, and sequencing of 41,842 cells total was followed generation of t-SNE and UMAP visualizations. Cells were identified by known cell type markers and prevalence analyzed via two-way ANOVA. Clinical outcomes were collected from 2-week, 6-week, 3-month, 6-month, and final postoperative clinical visits (range of motion, complications) and from patient surveys (IKDC, ACL-RSI). Statistical analysis of demographic/injury variables and clinical outcomes included mixed-effects longitudinal analysis, ANOVA, linear regression, and Pearson/Spearman correlations. Results: Seventeen patients were enrolled (9 female/8 male) with a mean follow-up of 8.9 ± months. Analysis of flow cytometry immune profiling revealed three clusters/immunotypes explaining 92.62% of the variation between patients (Fig. 1). There were no significant differences in age, sex, or concomitant meniscal/chondral injury between clusters. The immune cell profile of Type 3 indicated significantly higher immune cell infiltration into the synovium (45% ± 11%, p < 0.005), particularly of adaptive immune cells. Type 1 had lower immune cell infiltration (31.6% ± 3.8%) and adaptive immune contribution while Type 2 was intermediate. Type 1 also had a greater time between injury and surgery (median 99 days, 51-989; p < 0.05) than Type 2 (median 32 days, 21-38) and Type 3 (median 32 days, 13-42), and Type 1 synovium contained more mast cells (2.7 ± 0.78% of total cells, p < 0.05) than Type 2 (1.0 ± 0.39 %) and Type 3 (1.3 ± 0.82%). When a subset of these synovium tissues (n = 6) were analyzed via scRNA-seq, 25 cell types were identified overall (Fig. 2) with immunotype 3 trending towards more T cells and B cells, and a higher CD8+ to CD4+ T cell ratio. The overall immune profile pattern shown as a heatmap (Fig. 3) suggests that Type 3 synovium may experience higher adaptive inflammation compared to Type 1. The mean 6-month IKDC scores (n = 17, p = 0.99) were 77.93 ± 8.19 for Type 1, 78.55 ± 7.85 for Type 2, and 79.88 ± 14.44 for Type 3 (Fig. 4A). The mean 6-month ACL-RSI scores (n = 15, p = 0.33) were 78.58 ± 15.4 for Type 1, 72.33 ± 11.8 for Type 2, and 57.83 ± 26.4 for Type 3 (Fig. 4A). Knee range of motion was significantly worse for Type 3 in both extension and flexion (Fig. 4B-C, mixed effects p < 0.05), particularly in the 2-week to 3-month post-op range. Additionally, no complications occurred for patients in Type 1, one case of very mild loss of extension (< 3 degrees) was observed in Type 2, while significant complications occurred in Type 3 including one ACL re-rupture requiring revision and a separate case of arthrofibrosis with a 30-degreeflexion contracture requiring lysis of adhesions. Conclusions: Our pilot study provides the first analysis of immune cell behavior in the synovium of ACL injured knees at the time of ACLR. Three immunotypes were discovered. Type 1, the cluster with the lowest overall and adaptive inflammatory response corresponded to patients with a mean 3-month delay between injury and surgery. These patients also had the best clinical outcomes. Type 2 and Type 3 clusters included more acute surgeries (~ 1-month post-injury). However, Type 3 showed significantly higher inflammatory infiltrate, adaptive immune cell presence, and a prevalence of cytotoxic T cells. In turn, these patients had decreased early range of motion, lower ACL-RSI scores at 6 months, and higher incidence of surgical arthrofibrosis and graft failure. Thus, while time between injury and surgery is a contributor to inflammation levels at the time of ACLR, there are intrinsic, patient-specific differences in their acute inflammatory response that also contribute. Understanding these patient-specific differences in a larger prospective cohort might help guide targeted prevention strategies for arthrofibrosis, failure to return to sport, or even post-traumatic osteoarthritis.

Cigarette smoking is a major preventable cause of morbidity and mortality. While quitting smoking is the best option, switching from cigarettes to non-combustible alternatives (NCAs) such as e-vapor products is a viable harm reduction approach for smokers who would otherwise continue to smoke. A key challenge for the clinical assessment of NCAs is that self-reported product use can be unreliable, compromising the proper evaluation of their risk reduction potential. In this cross-sectional study of 205 healthy volunteers, we combined comprehensive exposure characterization with in-depth multi-omics profiling to compare effects across four study groups: cigarette smokers (CS), e-vapor users (EV), former smokers (FS), and never smokers (NS). Multi-omics analyses included metabolomics, transcriptomics, DNA methylomics, proteomics, and lipidomics. Comparison of the molecular effects between CS and NS recapitulated several previous observations, such as increased inflammatory markers in CS. Generally, FS and EV demonstrated intermediate molecular effects between the NS and CS groups. Stratification of the FS and EV by combustion exposure markers suggested that this position on the spectrum between CS and NS was partially driven by non-compliance/dual use. Overall, this study highlights the importance of in-depth exposure characterization before biological effect characterization for any NCA assessment study.

Emphysematous pyelonephritis (EP) is a severe complication of acute pyelonephritis caused by gas-producing bacteria, with high morbidity and mortality. Early recognition is essential, but the symptoms of EP are similar to those of severe acute pyelonephritis, complicating the diagnosis. Although computed tomography (CT) is the preferred diagnostic tool, its use is often limited in resource-constrained settings. Point-of-care ultrasound (POCUS) offers a rapid, accessible alternative for identifying signs of complicated pyelonephritis, including EP and obstructive pyelonephritis. Renal POCUS can be used to screen and risk stratify patients with severe acute pyelonephritis, optimizing CT use and reducing delays in diagnosing complications. We present cases from a hospital in Lima, Peru, illustrating how POCUS effectively facilitated the timely diagnosis and management of EP.