Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.

Ross procedure for acute infective endocarditis.

Surgical treatment of infective endocarditis remains a challenge, with concerns of optimal prosthesis selection and risks of recurrent infection remaining paramount. The pulmonary autograft has unique features which may make it the ideal aortic valve substitute, especially in infectious endocarditis. We describe strategic considerations and technical details in performing a Ross procedure in a young patient with acute aortic valve endocarditis.

Mitochondrial phosphatidylethanolamine synthesis affects mitochondrial energy metabolism and quiescence entry through attenuation of Snf1/AMPK signaling in yeast.

The Ups2-Mdm35 complex mediates intramitochondrial phosphatidylserine (PS) transport to facilitate mitochondrial phosphatidylethanolamine (PE) synthesis. In the present study, we found that ups2∆ yeast showed increased mitochondrial ATP production and enhanced quiescence (G0) entry in the post-diauxic shift phase. Transcriptomic and biochemical analyses revealed that the depletion of Ups2 leads to overactivation of the yeast AMPK homolog Snf1. Inactivation of Snf1 by depletion of an Snf1-activating kinase, Sak1 canceled the changes in mitochondrial ATP production and quiescence entry observed in ups2∆ cells. Furthermore, among the factors regulated by Snf1, upregulation of pyruvate carboxylase, Pyc1 and downregulation of acetyl-CoA carboxylase, Acc1, respectively, were sufficient to increase mitochondrial ATP production and quiescence entry. These results suggested that a normal PE synthesis mediated by Ups2-Mdm35 complex attenuates Snf1/AMPK activity, and that Snf1-mediated regulation of carbon metabolisms has great impacts on mitochondrial energy metabolism and quiescence entry. We also found that depletion of Ups2 together with the cell-cycle regulators Whi5 and Whi7, functional orthologs of the Rb1 tumor suppressor, caused a synthetic growth defect in yeast. Similarly, knockdown of PRELID3b, the human homolog of Ups2, decreased the viability of Rb1-deficient breast cancer cells, suggesting that PRELID3b is a potential target for cancer therapy.

Biofilm Formation and Virulence of Shigella flexneri Are Modulated by pH of Gastrointestinal Tract.

Shigella infection remains a public health problem in much of the world. Classic models of Shigella pathogenesis suggest that microfold epithelial cells in the small intestine are the preferred initial site of invasion. However, recent evidence supports an alternative model in which Shigella primarily infects a much wider range of epithelial cells that reside primarily in the colon. Here, we investigated whether the luminal pH difference between the small intestine and the colon could provide evidence in support of either model of Shigella flexneri pathogenesis. Because virulence factors culminating in cellular invasion are linked to biofilms in S. flexneri, we examined the effect of pH on the ability of S. flexneri to form and maintain adherent biofilms induced by deoxycholate. We showed that a basic pH (as expected in the small intestine) inhibited formation of biofilms and dispersed preassembled mature biofilms, while an acidic pH (similar to the colonic environment) did not permit either of these effects. To further elucidate this phenomenon at the molecular level, we probed the transcriptomes of biofilms and S. flexneri grown under different pH conditions. We identified specific amino acid (cysteine and arginine) metabolic pathways that were enriched in the bacteria that formed the biofilms but decreased when the pH increased. We then utilized a type III secretion system reporter strain to show that increasing pH reduced deoxycholate-induced virulence of S. flexneri in a dose-dependent manner. Taken together, these experiments support a model in which Shigella infection is favored in the colon because of the local pH differences in these organs.

Hybrid Double Valve Replacement for Multivalvular Disease With Severe Mitral Annular Calcification.

Severe mitral annular calcification poses a significant challenge, particularly in higher risk, frail, elderly patients with multivalvular disease. Both surgical and transcatheter options exist but remain fraught with risks of paravalvular leak, atrioventricular groove disruption, patient prosthesis mismatch, and left ventricular outflow tract obstruction. We present an innovative hybrid surgical approach to manage severe aortic and mitral valve disease in a Jehovah's Witness patient with severe circumferential mitral annular calcification.

Minimally Invasive Hybrid Approach to High Risk Mitral Disease With Severe Mitral Annular Calcification.

Severe circumferential mitral annular calcification (MAC) remains a significant challenge, complicating surgical treatment of mitral valve disease. Transcatheter treatment options are attractive; however, they remain fraught with risks of annular fixation, paravalvular leak, atrioventricular-groove disruption, and left-ventricular outflow tract obstruction. We describe a novel minimally invasive hybrid technique of transcatheter mitral valve replacement in a patient with severe circumferential MAC.

Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.

Transversus thoracis muscle plane block in cardiac surgery: a pilot feasibility study.

INTRODUCTION: Cardiac surgery patients often experience significant pain after median sternotomy. The transversus thoracis muscle plane (TTP) block is a newly developed, single-shot nerve block technique that provides analgesia for the anterior chest wall. In this double-blind pilot study, we assessed the feasibility of performing this novel block as an analgesic adjunct. METHODS: All patients aged 18-90 undergoing elective cardiac surgery were randomized to the block or standard care control group on admission to the intensive care unit after surgery. Under ultrasound guidance, patients in the block group received the TTP block with 20 mL of either 0.3% or 0.5% ropivacaine bilaterally, based on weight. The control group did not receive any injections. All blocks were performed by a single anesthesiologist, and data collection was performed by blinded assessors. The primary feasibility outcomes were rate of recruitment, adherence, and adverse events. The rate of recruitment was defined as the ratio of patients giving informed consent to the number of eligible patients who were approached to participate. Secondary outcomes included 12-hour and 24-hour Numeric Rating Scale (NRS) pain scores, 24-hour hydromorphone and acetaminophen requirements, time to extubation, time to first opioid administration, and patient satisfaction (on a yes/no questionnaire) at 24 hours. RESULTS: Twenty patients were approached for this study and 19 were enrolled. Eight patients received the intended intervention in each group. The recruitment rate was 95% of all approached eligible patients, and the adherence rate to treatment group was 94%. There were no block-related adverse events. The mean (SD) NRS pain scores at rest were 3.3 (3.2) in the block group vs 5.6 (3.2) in the control group at 12 hours. At 24 hours, the pain scores were 4.1 (3.9) vs 4.1 (3.3) in the block and control group, respectively. The mean (SD) 24-hour hydromorphone administration was 1.9 (1.1) mg in the block group vs 1.8 (0.9) mg in the control group. DISCUSSION: The TTP block is a novel pain management strategy poststernotomy. The results reveal a high patient recruitment, adherence, and satisfaction rate, and provide some preliminary data supporting safety. TRIAL REGISTRATION NUMBER: NCT03128346.

Minimally Invasive Periareolar Approach to Cor Triatriatum Repair.

Cor triatriatum sinister is an uncommon cardiac abnormality characterized by a membrane that divides the left atrium into two chambers. Definitive management requires surgical resection, traditionally through sternotomy. Minimally invasive reparative techniques are associated with reduced blood loss, shorter hospitalization, faster recovery time, and improved cosmesis with excellent patient satisfaction. We present a 29-year-old woman with cor triatriatum sinister and associated atrial septal defect who underwent successful minimally invasive repair through a right periareolar approach.

Dynamic Patient-Specific Three-Dimensional Simulation of Mitral Repair: Can We Practice Mitral Repair Preoperatively?

OBJECTIVE: Planned mitral repair strategies are generally established from preoperative echocardiography; however, specific details of the repair are often determined intraoperatively. We propose that three-dimensional printed, patient-specific, dynamic mitral valve models may help surgeons plan and trial all the details of a specific patient's mitral repair preoperatively. METHODS: Using preoperative echocardiography, segmentation, modeling software, and three-dimensional printing, we created dynamic, high-fidelity, patient-specific mitral valve models including the subvalvular apparatus. We assessed the accuracy of 10 patient mitral valve models anatomically and functionally in a heart phantom simulator, both objectively by blinded echocardiographic assessment, and subjectively by two mitral repair experts. After this, we attempted model mitral repair and compared the outcomes with postoperative echocardiography. RESULTS: Model measurements were accurate when compared with patients on anterior-posterior diameter, circumference, and anterior leaflet length; however, less accurate on posterior leaflet length. On subjective assessment, Likert scores were high at 3.8 ± 0.4 and 3.4 ± 0.7, suggesting good fidelity of the dynamic model echocardiogram and functional model in the phantom to the preoperative three-dimensional echocardiogram, respectively. Mitral repair was successful in all 10 models with significant reduction in mitral insufficiency. In two models, mitral repair was performed twice, using two different surgical techniques to assess which provided a better outcome. When compared with the actual patient mitral repair outcome, the repaired models compared favorably. CONCLUSIONS: Complex mitral valve modeling seems to predict an individual patient's mitral anatomy well, before surgery. Further investigation is required to determine whether deliberate preoperative practice can improve mitral repair outcomes.

Single cell analysis of Crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties.

BACKGROUND: Intestinal stem cells (ISCs) play indispensable roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn's disease (CD) affects properties of resident small intestinal stem cells remain uncertain. METHODS: CD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay. RESULTS: ISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCD-SIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISC-cluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs. CONCLUSIONS: aCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.

Contribution of ATOH1+ Cells to the Homeostasis, Repair, and Tumorigenesis of the Colonic Epithelium.

ATOH1 is a master transcription factor for the secretory lineage differentiation of intestinal epithelial cells (IECs). However, the comprehensive contribution of ATOH1+ secretory lineage IECs to the homeostasis, repair, and tumorigenesis of the intestinal epithelium remains uncertain. Through our ATOH1+ cell-lineage tracing, we show here that a definite number of ATOH1+ IECs retain stem cell properties and can form ATOH1+IEC-derived clonal ribbons (ATOH1+ICRs) under completely homeostatic conditions. Interestingly, colonic ATOH1+ IECs appeared to exhibit their stem cell function more frequently compared with those of the small intestine. Consistently, the formation of ATOH1+ICRs was significantly enhanced upon dextran sodium sulfate colitis-induced mucosal damage. In addition, colonic ATOH1+ IECs acquired tumor stem cell-like properties in the azoxymethane-DSS tumor model. Our results reveal an unexpected contribution of colonic ATOH1+ IECs to maintaining the stem cell population under both homeostatic and pathologic conditions and further illustrate the high plasticity of the crypt-intrinsic stem cell hierarchy.

Data showing proliferation and differentiation of intestinal epithelial cells under targeted depletion of Notch ligands in mouse intestine.

The data on the immunohistochemical analysis of conditional Notch ligand knockout mice is presented. Targeted deletion of Jag1, Dll1, Dll4, or Dll1 plus Dll4 in Lgr5+ve cells was induced by a Cre-mediated gene recombination, and differentiation or proliferation of the intestinal epithelial cells was examined by immunohistochemistry. These data are the extension of the data presented and discussed in the paper entitled "Indispensable role of non-canonical Notch signaling in the proliferation of Apc-deficient intestinal tumors" (Nakata et al., Submitted for publication) [1].

Indispensable role of Notch ligand-dependent signaling in the proliferation and stem cell niche maintenance of APC-deficient intestinal tumors.

Ligand-dependent activation of Notch signaling is required to maintain the stem-cell niche of normal intestinal epithelium. However, the precise role of Notch signaling in the maintenance of the intestinal tumor stem cell niche and the importance of the RBPJ-independent non-canonical pathway in intestinal tumors remains unknown. Here we show that Notch signaling was activated in LGR5+ve cells of APC-deficient mice intestinal tumors. Accordingly, Notch ligands, including Jag1, Dll1, and Dll4, were expressed in these tumors. In vitro studies using tumor-derived organoids confirmed the intrinsic Notch activity-dependent growth of tumor cells. Surprisingly, the targeted deletion of Jag1 but not RBPJ in LGR5+ve tumor-initiating cells resulted in the silencing of Hes1 expression, disruption of the tumor stem cell niche, and dramatic reduction in the proliferation activity of APC-deficient intestinal tumors in vivo. Thus, our results highlight the importance of ligand-dependent non-canonical Notch signaling in the proliferation and maintenance of the tumor stem cell niche in APC-deficient intestinal adenomas.

PGE2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells.

Intestinal epithelial cells (IECs) play an indispensable role in maintaining body fluid balance partly through their ability to regulate anion/fluid secretion. Yet in various inflammatory gastrointestinal diseases, over-secretion of anions results in symptoms such as severe diarrhoea. Endogenous mediators, such as vasoactive intestinal peptide or prostaglandin E2 (PGE2), regulate intestinal anion/fluid secretion, but their direct effect on purified human IECs has never been described in detail. Based on a previously described intestinal organoid swelling model, we established a 3D-scanner-assisted quantification method to evaluate the anion/fluid secretory response of cultured human IECs. Among various endogenous secretagogues, we found that PGE2 had the lowest EC50 value with regard to the induction of swelling of the jejunal and colonic organoids. This PGE2-mediated swelling response was dependent on environmental Cl- concentrations as well as on several channels and transporters as shown by a series of chemical inhibitor studies. The concomitant presence of various inflammatory cytokines with PGE2 failed to modulate the PGE2-mediated organoid swelling response. Therefore, the present study features PGE2 as a direct and robust mediator of anion/fluid secretion by IECs in the human intestine.