Aged hematopoietic stem cells (HSCs) show diminished capacity of self-renewal, skewed lineage output and compromised proteostasis. Ubiquitin proteasomal systems are critical for maintaining protein homeostasis. We show that the levels of Ube2g1, a E2 ubiquitinconjugating enzyme likely involved in clonal selection of HSCs, was elevated in aged murine and human HSCs. We hypothesized that elevated levels of Ube2g1 causally contribute to hematopoietic system aging. Elevated levels of Ube2g1 in young murine HSCs resulted in increased myeloid-to-lymphoid ratio and reduced naïve T-cells, both known hematopoietic aging hallmarks. Interestingly, the ubiquitination function of Ube2g1 didn’t primarily account for the observed phenotypes. Elevated levels of Ube2g1 affected global tyrosine phosphorylation, mediated through a Ube2g1-Shp2 axis, which correlated with impaired Tcell development and reduced HSC function. Our work identifies a novel connection between proteins involved in the regulation of ubiquitination and phosphorylation in HSCs that affect phenotypes linked to aging of HSCs.

Somatic mutations accumulate throughout life and can serve as endogenous markers to trace cellular lineage relationships. In hematopoietic stem cells (HSCs), aging is associated with functional decline and clonal skewing, yet how somatic mutational histories shape clonal architecture at single-cell resolution remains incompletely understood. Here, we leverage single-cell RNA sequencing from murine and human long-term HSCs to identify expressed somatic single-nucleotide variants and reconstruct mutation-based genealogies. To address technical noise inherent to single-cell transcriptomes, we implement a stringent joint variant filtering strategy that exploits daughter cell relationships and probabilistic modeling to distinguish true somatic events from artifacts. Using these high-confidence variants, we infer phylogenetic and clonal relationships among individual HSCs and uncover striking age-dependent differences in lineage structure. Whereas young HSCs show little evidence of hierarchical clonal organization, aged HSCs frequently exhibit structured genealogies consistent with clonal expansion. Integration of mutational, transcriptional, and signature-based analyses further reveals that genetically distinct clones in aged samples are transcriptionally divergent and enriched for pathways linked to aging and DNA damage responses. Together, our findings demonstrate that somatic mutations recovered from single-cell transcriptomes can resolve HSC clonal evolution and reveal age-associated alterations in stem cell dynamics. ### Competing Interest Statement The authors have declared no competing interest. DFG, CRC 1506 LaCaixa Health Research, DeepNiche800

High-dose chemotherapy and consecutive autologous stem cell transplantation (ASCT) remain the backbone of treatment for transplant-eligible patients of Multiple Myeloma (MM). However, patients are still at high risk of relapse or treatment-related complications. Hence, by understanding the function of hematopoietic stem and progenitor cells (HSPCs) from MM patients in more detail, transplant outcomes in MM patients might be further improved. We combine in our study functional analyses of the potential of HSPCs from newly diagnosed (NDMM) and chemotherapy treated MM patients in a xenotransplant model system with in depth single cells sequencing analysis to provide novel data that might inform clinical routine to improve the outcome of ASCT in MM. Our data demonstrate that (i) HSPCs from treated MM patients are indeed significantly impaired in their overall reconstitution potential and provide a reduced level of B-cells in comparison to HSPCs from age-matched healthy donors and NDMM patients. (ii) We further demonstrate that CD34+ HSPCs acquire a high-risk MM expression profile signature upon induction treatment, which likely adds to the risk of relapse. This high-risk MM expression profile signature relies within CD34+ HSPCs primarily in granulocyte/macrophage progenitors (GMPs), megakaryocyte Erythroid Progenitors (MEPs) and monocytes, while hematopoietic stem cells (HSCs) stay unaffected by transcriptional changes. These data suggest that the elimination of myeloid progenitors and more mature monocytes (likely by purification for HSCs) in HSPCs harvests from treated MM patients for subsequent ASCT might improve transplant outcomes by avoiding re-infusion of cells with a dysregulated and disease-linked transcriptional program.

OBJECTIVES Postoperative delirium (POD) is common, costly and associated with long-term morbidity and increased mortality. We conducted a cohort study to assess the contribution of cardiopulmonary bypass (CPB) to the development of POD by means of algorithm-based data processing.METHODS A database was compiled from 3 datasets of patients who underwent cardiac surgery between 2014 and 2019: intensive care unit discharge files, CPB protocols and medical quality management records. Following data extraction and structuring using novel algorithms, missing data were imputed. Ten independent imputations were analysed by multiple logistic regression with stepwise deletion of factors to arrive at a minimal adequate model.RESULTS POD was diagnosed in 456/3163 patients (14.4%). In addition to known demographic risk factors and comorbidities like male sex, age, carotid disease, acute kidney failure and diabetes mellitus, cardiopulmonary parameters like total blood volume at the CPB [adjusted odds ratio (AOR) 1.001; confidence interval (CI) 1.1001-1.002] were independent predictors of POD. Higher values of the minimal blood flow were associated with a lower risk of POD (AOR 0.993; CI 0.988-0.997). Flow rates at least 30% above target did emerge in the minimal adequate model as a potential risk factor, but the confidence interval suggested a lack of statistical significance (AOR 1.819; 95% CI: 0.955-3.463).CONCLUSIONS CPB data processing proved to be a useful tool for obtaining compact information to better identify the roles of individual operational states. Strict adherence to perfusion limits along with tighter control of blood flow and acid-base balance during CPB may help to further decrease the risk of POD. Postoperative delirium (POD) is a transient neurocognitive disorder in which the patient's awareness, attention and cognitive performance are acutely impaired.

OBJECTIVES:Elevated serum creatine kinase isoenzyme MB (CK-MB) levels indicate myocardial ischaemia and periprocedural myocardial injury during treatment of heart diseases. We established a method to predict CK-MB mass from activity data based on a prospective pilot study in order to simplify multicentre trials. METHODS:38 elective cardiac surgery patients without acute myocardial ischaemia and terminal renal failure were recruited. CK-MB mass and activity were determined in venous blood samples drawn preoperatively, postoperatively, 6 h post-op, and 12 h post-op. Linear regression and generalized additive models (GAMs) were applied to describe the relationship of mass and activity. Influences of demographic and perioperative factors on the fit of GAMs was evaluated. The agreement of predicted and measured CK-MB masses was assessed by Bland-Altman analyses. RESULTS:Linear regression provided an acceptable overall fit (r2 = 0.834) but showed deviances at low CK-MB levels. GAMs did not benefit from the inclusion of age, body mass index and surgical times. The minimal adequate model predicted CK-MB masses from activities, sex and sampling time with an r2 of 0.981. Bland-Altman analyses confirmed narrow limits of agreement (spread: 8.87 µg/l) and the absence of fixed (P = 0.41) and proportional (P = 0.21) biases. CONCLUSIONS:GAM-based modelling of CK-MB data in a representative patient cohort allowed to predict CK-MB masses from activities, sex and sampling time. This approach simplifies the integration of study centres with incompatible CK-MB data into multicentre trials in order to facilitate inclusion of CK-MB levels in statistical models.

The functional impact and cellular context of mosaic structural variants (mSVs) in normal tissues is understudied. Utilizing Strand-seq, we sequenced 1,133 single-cell genomes from 19 human donors of increasing age, and discovered the heterogeneous mSV landscapes of hematopoietic stem and progenitor cells. While mSVs are continuously acquired throughout life, expanded subclones in our cohort are confined to individuals >60. Cells already harboring mSVs are more likely to acquire additional somatic structural variants, including megabase-scale segmental aneuploidies. Capitalizing on comprehensive single-cell micrococcal nuclease digestion with sequencing reference data, we conducted high-resolution cell-typing for eight hematopoietic stem and progenitor cells. Clonally expanded mSVs disrupt normal cellular function by dysregulating diverse cellular pathways, and enriching for myeloid progenitors. Our findings underscore the contribution of mSVs to the cellular and molecular phenotypes associated with the aging hematopoietic system, and establish a foundation for deciphering the molecular links between mSVs, aging and disease susceptibility in normal tissues.

article: Effect of the analgesic metamizole (dipyrone) on volume balance and vasopressor use in cardiac surgery patients - Minerva Anestesiologica 2022 December;88(12):1073-4 - Minerva Medica - Journals

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Introduction: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. Methods: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. Results: Storage in HEPES-supplemented Dulbecco’s modified Eagle’s medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E−8 vs. 7.5E−8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. Conclusion: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

BACKGROUND:Pulsatile extracorporeal circulation (ECC) may improve perfusion of critical organs during cardiac surgery. This study analyzed the influence of the components of a minimal invasive ECC (MiECC) on the transfer of pulsatile energy into the pseudo-patient of a mock circulation.METHODS:An aortic model with human-like geometry and compliance was perfused by a diagonal pump. Surplus hemodynamic energy (SHE) was determined from flow and pressure data. Five adult-size oxygenator models and three sizes of cannulas were compared. Pulsatile pump settings were optimized, and parallel dual-pump configurations were evaluated.RESULTS:Oxygenator models showed up to twofold differences in pressure gradients and influenced SHE at flow rates up to 2.0 L min-1 . Adjustments of frequency, systole duration, and rotational speed gain significantly improved SHE compared with empirical settings, with SHE above 21% of mean arterial pressure at flow rates of 1.0 L min-1 to 1.5 L min-1 and SHE above 5% at 3.5 L min-1 . Small diameter cannula (15 Fr) limited SHE compared with larger cannula (21 Fr and 23 Fr). Two diagonal pumps did not provide higher SHE than a single pump, but permitted additional control over pulse pressure and SHE by varying the total fraction of pulsatile flow and the fraction of flow bypassing the oxygenator.CONCLUSIONS:Proper selection of components and optimizations of pump settings significantly improved pulse pressure and SHE of pulsatile MiECC. Surplus hemodynamic energy depended on flow rate with a maximum at 1.0 L min-1 -1.5 L min-1 . Pulsatile MiECC may specifically assist organ perfusion during phases of low flow.

BACKGROUND:Pulsatile perfusion during extracorporeal circulation is a promising concept to improve perfusion of critical organs. Clinical benefits are limited by the amount of pulsatile energy provided by standard pumps. The present study investigated the properties of a novel positive displacement blood pump in a mock circulation.METHODS:The pump was attached to an aortic model with a human-like geometry and compliance as a pseudo patient. Hemodynamic data were recorded while the pump settings were adjusted systematically.RESULTS:Using a regular oxygenator, maximum flow was 2.6 L/min at a pressure of 27 mm Hg and a frequency (F) of 90 bpm. Pulse pressure (PP; 28.9 mm Hg) and surplus hemodynamic energy (SHE; 26.1% of mean arterial pressure) were highest at F = 40 bpm. Flow and pressure profiles appeared sinusoid. Using a low-resistance membrane ventilator to assess the impact of back pressure, maximum flow was 4.0 L/min at a pressure of 58.6 mm Hg and F = 40 bpm. At F = 40 bpm, PP was 58.7 mm Hg with an SHE of 33.4%. SHE decreased with increasing flow, heart rate, and systolic percentage but surpassed 10% with reasonable settings.CONCLUSIONS:The present prototype achieved sufficient flow and pressure ranges only in the presence of a low-resistance membrane ventilator. It delivered supraphysiologic levels of pulse pressure and SHE. Further modifications are planned to establish this concept for adult pulsatile perfusion.

Background: The incidence of postoperative delirium (POD) following cardiac surgery is rising as a result of increased age and complexity of patients. The ensuing problems are not only costly, but are commonly associated with long-term morbidity and increased mortality. We conducted a case–control study to assess the contribution of cardiopulmonary bypass (CPB) to the development of POD.

Background:The restriction of hydroxyethylstarch (HES) necessitated changes in volume management in cardiac surgery, increasing the use of gelatin (GELA) and crystalloid (CRYS) mono strategies.Methods:This retrospective study evaluated the effects of changed volume replacement management to a GELA or CRYS mono therapy on mortality, acute kidney injury (AKI), blood loss, and transfusion in cardiac surgery patients with at least one coronary artery bypass grafting (CABG) at a university hospital. Three groups (HES n=938, GELA n=397, CRYS n=205) were derived from 1,540 patients with complete data sets. Data were analyzed by multiple regression models.Results:Patients had similar risk profiles, comorbidities, and preoperative routine diagnostics prior to surgery. No difference was observed in mortality and AKI. HES treated patients showed highest blood loss, followed by GELA while CRYS patients had the lowest (P<0.0001). Patients in the HES group had highest transfusion of packed red blood cells (PRBCs) and platelet concentrates (PCs), followed by GELA, whereas CRYS had the lowest (P<0.0001). Fresh frozen plasma (FFP) transfusion, administration of fibrinogen, and prothrombin complex concentrates (PCCs) were highest in HES group. CRYS showed the shortest time of mechanical ventilation (P<0.0001) and left the intensive care unit (ICU) significantly earlier (P<0.0001). Multivariable regression analysis found that colloid volume significantly predicted hospital mortality and renal replacement therapy (RRT), but not AKI.Conclusions:Administration of crystalloids without any colloid showed no differences in mortality or AKI, but less blood loss and transfusion. Colloids should be considered critically and further studies should investigate effects of GELA in cardiac surgery.

Hintergrund Bei der Grundlagenforschung zur pulmonal-arteriellen Hypertonie wurden funktionelle Organbadversuche zur Beurteilung vasodilatatorischer Medikamentenwirkungen und pathologische Untersuchungen an humanen Pulmonalarterien (PA) durchgeführt. Diese Studie vergleicht die funktionellen mit den strukturellen Ergebnissen.

Various vasodilator medications are used in the treatment of pulmonary arterial hypertension (PAH), such as endothelin receptor antagonists (ERA) or phosphodiesterase-5-(PDE-5-)inhibitors. In a human ex vivo model, we investigated whether the combination of two substance classes could achieve a higher effect or - without loss of vasodilatation - a lower dosage of the individual substances might be sufficient. We established an ex vivo organ bath model to evaluate the dose-dependent effects of ERA and PDE-5-inhibitors on pulmonary vessels harvested from patients who underwent surgery (lung resection/transplantation). We compared the combined use of both substance classes with administration of one class of drugs alone. Due to the limitations of the experimental design, it is not possible to extrapolate our results to the conditions in vivo. Nevertheless, organ bath proved to be helpful in evaluating the dose-dependent effects of ERA and PDE-5 inhibitors, which is not practical in everyday clinical practice. In this setting, the effectiveness of the combination therapy and the potential for dose reduction depended on the concentrations used and on the influence of previous illnesses on blood vessel function. This article describes the most important results of our experimental investigations and suggestions for future projects.

Conclusion:Our study demonstrates that the neuropeptide CGRP via signaling to its receptor RAMP1 promotes liver regeneration through controling YAP activity.This previously unknown signaling pathway might be highly relevant for patients with liver diseases.

Objectives: Pulsatile extracorporeal circulation (ECC) may improve organ perfusion in cardiac surgery. Some minimally invasive ECC (MiECC) systems provide optional pulsatile perfusion. Surplus hemodynamic energy (SHE) describes the gain in hemodynamic energy provided by pulsation over laminar flow. We investigated effects of oxygenators, arterial cannulae, and drive settings on SHE in a mock circulation.

PURPOSE:Medical combination therapy of pulmonary arterial hypertension (PAH) may alleviate the drawbacks of monotherapy by avoiding drug tolerance and by increasing effectiveness, as shown by the combination of ambrisentan and tadalafil (AMBITION trial). The present ex-vivo study evaluated the combination of the endothelin receptor antagonists (ERA) macitentan and bosentan with the phosphodiesterase-5 (PDE-5) inhibitor vardenafil in pulmonary arteries from patients suffering from terminal lung disease as a model of PAH. METHODS:Segments of the pulmonary vessels were excised from resected lungs of patients requiring lung transplantation (LTX). Contraction of pulmonary arteries (PA) was elicited by consecutive dose-response curves of endothelin-1 (ET-1) followed by norepinephrine (NE) to allow inhibition by different pathways. Forces were measured isometrically in an organ bath in the presence and absence of ERA and PDE-5 inhibitors and their combination. RESULTS:PA of 38 patients were examined between October 2016 and November 2019. Bosentan (1E-7 M) and macitentan (1E-8 M, 3E-8 M, 1E-7 M) inhibited ET-1 induced contractions, whereas vardenafil (1E-6 M, 3E-6 M, 1E-5 M) inhibited only the NE induced part of the contractions. Vardenafil enhanced bosentan-induced inhibition of vasoconstriction in a dose-dependent fashion. Combination effects exceeded single bosentan at 3E-6 M and 1E-5 M vardenafil, and they exceeded single vardenafil at the lower vardenafil concentrations. Macitentan showed a more pronounced inhibition than bosentan regardless of the lower concentrations. Accordingly, combination effects with vardenafil resembled those of macitentan alone. CONCLUSIONS:Macitentan and bosentan were potent antagonists of vasoconstriction in PA of LTX patients. The benefit of drug combinations was demonstrated at selected concentrations only owing to a narrow therapeutic range of vardenafil in this ex-vivo model. These results suggest the utility of drug combinations other than the established pair of ambrisentan and tadalafil in PAH treatment but also make a case for a further assessment of vasodilator properties of drugs complementing ERA.

Obesity has been described as a major factor of health risk in modern society. Next to intricately linked comorbidities like coronary artery disease or diabetes, an influence of obesity on regeneration after muscle injury has been described previously. However, the influence of obesity on tissue regeneration in a combined trauma, merging the more systemic influence of a blunt lung trauma and the local blunt muscle trauma, has not been investigated yet. Therefore, the aim of this study was to investigate the influence of obesity on regeneration in a mouse model that combined both muscle and thorax trauma. Using gene expression analysis, a focus was put on the structure as well as the organization of the extracellular matrix and on functional satellite cell physiology. An increased amount of debris in the lung of obese mice compared to normal weight mice up to 192 h after combined trauma based on visual assessment can be reported which is accompanied by a decreased response of Mmp2 in obese mice. Additionally, a delayed and elongated response of inhibitor genes like Timp1 has been revealed in obese mice. This elongated response to the trauma in obese mice can also be seen in plasma based on increased levels of pro-inflammatory chemo- and cytokines (IL-6, MCP-1, and IL 23) 192 h post trauma. In addition to changes in the lung, morphological analysis of the injured extensor iliotibialis anticus of the left hind leg in lean and diet-induced obese mice revealed deposition of fat in the regenerating muscle in obese animals hindering the structure of a compact muscle. Additionally, decreased activation of satellite cells and changes in organization and build-up of the ECM could be detected, finally leading to a decreased stability of the regenerated muscle in obese mice. Both factors contribute to an attenuated response to the trauma by obese mice which is reflected by a statistically significant decrease in muscle force of obese mice compared to lean mice 192 h post trauma induction.

INTRODUCTION:Pulsatile extracorporeal circulation may improve organ perfusion during cardiac surgery. Some minimally invasive extracorporeal circulation (MiECC) systems allow pulsatile perfusion. The present study investigated the influence of arterial tubing compliance on hemodynamic energy transfer into the patient.METHODS:Aortic models with adult human geometry were perfused in a mock circulation. A MiECC system was connected using either high-compliance silicone tubing or standard kit tubing. Energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE) were computed from flow and pressure data. Aortic models with physiological and sub-physiological compliance were tested to assess the influence of the pseudo-patient.RESULTS:Non-pulsatile flow did not generate SHE. SHE during pulsatile flow in the compliant aortic model was significantly higher with kit tubing compared to silicone tubing. Maximum SHE was achieved at 1.6 L/min with kit tubing (7.7% of mean arterial pressure) and with silicone tubing (4.9%). Using the low-compliance aortic model, SHE with kit tubing reached a higher maximum of 14.2% at 1.8 L/min compared to silicone tubing (11.8% at 1.5 L/min).CONCLUSIONS:Flexible arterial tubing did not preserve more hemodynamic energy from a pulsatile pump compared to standard kit tubing in a model of adult extracorporeal circulation. The pseudo-patient's compliance significantly affected the properties of the mock circulation.