Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA.

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses.

Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1ß (IL-1ß), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-ß and IL-1ß dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.

Balance between the cell viability and death in 3D.

Cell death is a phenomenon, frequently perceived as an absolute event for cell, tissue and the organ. However, the rising popularity and complexity of such 3D multicellular 'tissue building blocks' as heterocellular spheroids, organoids, and 'assembloids' prompts to revise the definition and quantification of cell viability and death. It raises several questions on the overall viability of all the cells within 3D volume and on choosing the appropriate, continuous, and non-destructive viability assay enabling for a single-cell analysis. In this review, we look at cell viability and cell death modalities with attention to the intrinsic features of such 3D models as spheroids, organoids, and bioprints. Furthermore, we look at emerging and promising methodologies, which can help define and understand the balance between cell viability and death in dynamic and complex 3D environments. We conclude that the recent innovations in biofabrication, biosensor probe development, and fluorescence microscopy can help answer these questions.

MORN motif-containing protein OsMORN1 and OsMORN2 are crucial for rice pollen viability and cold tolerance.

The pollen viability directly affects the pollination process and the ultimate grain yield of rice. Here, we identified that the MORN motif-containing proteins, OsMORN1 and OsMORN2, had a crucial role in maintaining pollen fertility. Compared with the wild type (WT), the pollen viability of the osmorn1 and osmorn2 mutants was reduced, and pollen germination was abnormal, resulting in significantly lower spikelet fertility, seed-setting rate, and grain yield per plant. Further investigation revealed that OsMORN1 was localized to the Golgi apparatus and lipid droplets. Lipids associated with pollen viability underwent alterations in osmorn mutants, such as the diacylglyceride (18:3_18:3) was 5.1-fold higher and digalactosyldiacylglycerol (18:2_18:2) was 5.2-fold lower in osmorn1, while the triacylglycerol (TG) (16:0_18:2_18:3) was 8.3-fold higher and TG (16:0_18:1_18:3) was 8.5-fold lower in osmorn2 than those in WT. Furthermore, the OsMORN1/2 was found to be associated with rice cold tolerance, as osmorn1 and osmorn2 mutants were more sensitive to chilling stress than WT. The mutants displayed increased hydrogen peroxide accumulation, reduced antioxidant enzyme activities, elevated malondialdehyde content, and a significantly decreased seedling survival rate. Lipidomics analysis revealed distinct alterations in lipids under low temperature, highlighting significant changes in TG (18:2_18:3_18:3) and TG (18:4_18:2_18:2) in osmorn1, TG (16:0_18:2_18:2) and PI (17:2_18:3) in osmorn2 compared to the WT. Therefore, it suggested that OsMORN1 and OsMORN2 regulate both pollen viability and cold tolerance through maintaining lipid homeostasis.

Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?

BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].

A comprehensive preanalytical protocol for fresh solid tumor biospecimens.

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Single-cell tracking as a tool for studying EMT-phenotypes.

This article demonstrates that label-free single-cell video tracking is a useful approach for in vitro studies of Epithelial-Mesenchymal Transition (EMT). EMT is a highly heterogeneous process, involved in wound healing, embryogenesis and cancer. The process promotes metastasis, and increased understanding can aid development of novel therapeutic strategies. The role of EMT-associated biomarkers depends on biological context, making it challenging to compare and interpret data from different studies. We demonstrate single-cell video tracking for comprehensive phenotype analysis. In this study we performed single-cell video tracking on 72-h long recordings. We quantified several behaviours at a single-cell level during induced EMT in MDA-MB-468 cells. This revealed notable variations in migration speed, with different dose-response patterns and varying distributions of speed. By registering cell morphologies during the recording, we determined preferred paths of morphological transitions. We also found a clear association between migration speed and cell morphology. We found elevated rates of cell death, diminished proliferation, and an increase in mitotic failures followed by re-fusion of sister-cells. The method allows tracking of phenotypes in cell lineages, which can be particularly useful in epigenetic studies. Sister-cells were found to have significant similarities in their speeds and morphologies, illustrating the heritability of these traits.

Lys-63-specific deubiquitinase BRCC36 enhances the sensitivity of multiple myeloma cells to lenalidomide by inhibiting lysosomal degradation of cereblon.

Multiple myeloma (MM) is the second most common hematological tumor in adults. Immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide (Len), are effective drugs for the treatment of multiple myeloma. Len can recruit IKZF1 and IKZF3 to cereblon (CRBN), a substrate receptor of the cullin 4-RING E3 ligase (CRL4), promote their ubiquitination and degradation, and finally inhibit the proliferation of myeloma cells. However, MM patients develop resistance to IMiDs over time, leading to disease recurrence and deterioration. To explore the possible approaches that may enhance the sensitivity of IMiDs to MM, in this study, we used the proximity labeling technique TurboID and quantitative proteomics to identify Lys-63-specific deubiquitinase BRCC36 as a CRBN-interacting protein. Biochemical experiments demonstrated that BRCC36 in the BRISC complex protects CRBN from lysosomal degradation by specifically cleaving the K63-linked polyubiquitin chain on CRBN. Further studies found that a small-molecule compound SHIN1, which binds to BRISC complex subunit SHMT2, can upregulate CRBN by elevating BRCC36. The combination of SHIN1 and Len can further increase the sensitivity of MM cells to IMiDs. Therefore, this study provides the basis for the exploration of a possible strategy for the SHIN1 and Len combination treatment for MM.

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling.

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2 at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.NEW & NOTEWORTHY Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields.

The NF-κB/FXR/TonEBP pathway protects renal medullary interstitial cells against hypertonic stress.

Farnesoid X receptor (FXR), a ligand-activated transcription factor, plays an important role in maintaining water homeostasis by up-regulating aquaporin 2 (AQP2) expression in renal medullary collecting ducts; however, its role in the survival of renal medullary interstitial cells (RMICs) under hypertonic conditions remains unclear. We cultured primary mouse RMICs and found that the FXR was expressed constitutively in RMICs, and that its expression was significantly up-regulated at both mRNA and protein levels by hypertonic stress. Using luciferase and ChIP assays, we found a potential binding site of nuclear factor kappa-B (NF-κB) located in the FXR gene promoter which can be bound and activated by NF-κB. Moreover, hypertonic stress-induced cell death in RMICs was significantly attenuated by FXR activation but worsened by FXR inhibition. Furthermore, FXR increased the expression and nuclear translocation of hypertonicity-induced tonicity-responsive enhance-binding protein (TonEBP), the expressions of its downstream target gene sodium myo-inositol transporter (SMIT), and heat shock protein 70 (HSP70). The present study demonstrates that the NF-κB/FXR/TonEBP pathway protects RMICs against hypertonic stress.

m6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

Viability of Chlamydia trachomatis in different anatomical sites - a systematic review & meta-analysis.

BACKGROUND: Modern assays for the detection of Chlamydia trachomatis (CT) rely on nucleic acid amplification testing (NAAT) of DNA or ribosomal RNA. However, it is also known that both viable ("living") & non-viable ("dead") CT can be detected by NAAT. Multiple laboratory techniques to measure CT viability have emerged. METHODS: We searched PubMed, EMBASE, Scopus and Dimensions as well as conference abstracts for entries between January 2000 to May 2023. We included any studies that measured CT viability among NAAT-positive samples. Viability assays include enhanced cell culture, direct fluorescent antibody (DFA), messenger RNA (mRNA) detection via digital droplet PCR (ddPCR), viability PCR (V-PCR) & real-time PCR measuring RNA-to-DNA ratio (RDR) (e.g. InSignia®). A meta-analysis was performed on the proportions of non-viable CT by anatomical site. RESULTS: We screened 31,342 records and included 16 studies in the analysis. The pooled proportions of non-viable CT by site were: 33% (95%CI 19-47%) in rectal swabs (eight studies), 17% (95%CI 7-27%) in cervical swabs (six studies), 15% (95%CI 6-25%) in vaginal swabs (six studies) and 11% (95%CI 9-17%) in urine/urethral swabs (two studies). CONCLUSION: All included studies found that a proportion of NAAT-detected CT is non-viable. The findings have far-reaching implications for screening programs and studies evaluating new STI tests and antimicrobial regimens.

Identification of the Candidate mGlu2 Allosteric Modulator THRX-195518 through In Silico Method and Evaluation of Its Neuroprotective Potential against Glutamate-Induced Neurotoxicity in SH-SY5Y Cell Line.

Glutamate (Glu) toxicity has been an important research topic in toxicology and neuroscience studies. In vitro and in vivo studies have shown that Group II metabotropic Glu2 (mGlu2) activators have cell viability effects. This study aims to determine a candidate ligand with high mGlu2 allosteric region activity among cytotoxicity-safe molecules using the in silico positioning method and to evaluate its cell viability effect in vitro. We investigated the candidate molecule's cell viability effect on the SH-SY5Y human neuroblastoma cell line by MTT analysis. In the study, LY 379268 (agonist) and JNJ-46281222 (positive allosteric modulator; PAM) were used as control reference molecules. Drug bank screening yielded THRX-195518 (docking score being -12.4 kcal/mol) as a potential novel drug candidate that has a high docking score and has not been mentioned in the literature so far. The orthosteric agonist LY 379268 exhibited a robust protective effect in our study. Additionally, our findings demonstrate that JNJ-46281222 and THRX-195518, identified as activating the mGlu2 allosteric region through in silico methods, preserve cell viability against Glu toxicity. Therefore, our study not only emphasizes the positive effects of this compound on cell viability against Glu toxicity but also sheds light on the potential of THRX-195518, acting as a mGlu2 PAM, based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) data, as a candidate drug molecule. These findings underscore the potential utility of THRX-195518 against both neurotoxicity and Central Nervous System (CNS) disorders, providing valuable insights.

Exopolysaccharides from the Green Microalga Strain Coelastrella sp. BGV-Isolation, Characterization, and Assessment of Anticancer Potential.

Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a new Bulgarian strain of the green microalga Coelastrella sp. BGV were investigated. A fast and convenient method of precipitation with cold ethanol was used to isolate EPS from the culture medium. The chemical characteristics of the isolated EPS were examined by colorimetric and spectrophotometric analyses, HPSEC-RID and HPLC-UV chromatography, and FT-IR spectroscopy. The results showed that the isolated EPS sample consists of three carbohydrate fractions with different molecular weights (11.5 × 104 Da, 30.7 × 104 Da, and 72.4 × 104 Da, respectively) and contains 7.14 (w/w%) protein. HPLC-UV analysis revealed the presence of galactose and fucose. The total uronic acid content in the sample was 4.5 (w/w%). The IR-FT spectrum of EPS revealed the presence of various functional groups typical of a polysaccharide (or proteoglycan) composed primarily of neutral sugars. The anticancer potential of the obtained EPS was assessed using cell lines with cancerous and non-cancerous origins as in vitro experimental models. The results of the performed MTT assay showed that EPS reduced the viability of the cervical and mammary carcinoma cell lines HeLa and MCF-7, while the control non-cancer cell lines BALB/3T3 and HaCaT were less affected. The HeLa cell line showed the highest sensitivity to the effects of EPS and was therefore used for further studies of its anticancer potential. The ability of EPS to inhibit cancer cell migration was demonstrated by wound-healing (scratch) assay. The cell cycle FACS analysis indicated that the EPS treatment induced significant increases in the sub G1 cell population and decreases of the percentages of cells in the G1, S, and G2-M phases, compared to the control. The fluorescent microscopy studies performed using three different staining methods in combination with Annexin V-FITC flow cytometric analysis clearly demonstrate the ability of EPS to induce cancer cell death via the apoptosis pathway. Moreover, an altered pattern and intensity of the immunocytochemical staining for the apoptosis- and proliferation-related proteins p53, bcl2, and Ki67 was detected in EPS-treated HeLa cancer cells as compared to the untreated controls. The obtained results characterize the new local strain of green microalgae Coelastrella sp. BGV as a producer of EPS with selective antitumor activity and provide an opportunity for further studies of its pharmacological and biotechnological potential.

The Autophagic and Apoptotic Death of Forebrain Neurons of Rats with Global Brain Ischemia Is Diminished by the Intranasal Administration of Insulin: Possible Mechanism of Its Action.

Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.

Abdominal Normothermic Regional Perfusion after Donation after Circulatory Death Improves Pancreatic Islet Isolation Yield.

Abdominal Normothermic Regional Perfusion (aNRP) is an in-situ normothermic oxygenated donor perfusion technique before procurement during controlled donation after circulatory death (cDCD) procedures and allows for organ quality evaluation. There are few data on the effect of aNRP on pancreatic islet isolation and subsequent transplantation outcomes. We aim to evaluate the impact of aNRP on cDCD pancreatic islet isolation and transplantation. A retrospective analysis was performed on pancreatic islet isolation outcomes from aNRP, cDCD, and Donation after Brain death (DBD) pancreases. Isolations were compared to previous donor age (60-75) matched isolations. Islet function was asses by a dynamic Glucose Stimulated Insulin Secretion (dGSIS). Donor baseline characteristics did not differ among groups. Isolations from aNRP pancreases (471,739 IEQ [655,435 - 244,851]) yielded more islets compared to cDCD (218,750 IEQ [375,951 - 112,364, p<0.01) and to DBD (206,522 IEQ [385,544 - 142,446, p=0.03) pancreases. dGSIS tests in seven aNRP islet preparations showed a mean stimulation index of 4.91, indicating good functionality. Bilirubin and alanine aminotransferase during aNRP correlated with islet yield (r2=0.685, p=0.002; r2=0.491, p=0.016 respectively). Islet isolation after aNRP in cDCD donors results in a high islet yield with viable functional islets. aNRP could increase the utilization of pancreases for islet transplantation.

Assessment of liver graft quality during hypothermic oxygenated perfusion: the first international validation study.

BACKGROUND AND AIM: While it is currently assumed that liver assessment is only possible during normothermic machine perfusion (NMP), there is uncertainty regarding a reliable and quick prediction of graft injury during ex situ hypothermic oxygenated perfusion (HOPE). We therefore intended to test, in an international liver transplant cohort, recently described mitochondrial injury biomarkers measured during HOPE before liver transplantation. STUDY DESIGN: Perfusate samples of human livers from 10 centers in 7 countries with HOPE-experience were analyzed for released mitochondrial compounds, i.e. flavin mononucleotide (FMN), NADH, purine derivates and inflammatory markers. Perfusate FMN was correlated with graft loss due to primary non-function or symptomatic non-anastomotic biliary strictures (NAS), and kidney failure, as well as liver injury after transplantation. Livers deemed unsuitable for transplantation served as negative control. RESULTS: We collected 473 perfusate samples of human DCD (n=315) and DBD livers (n=158). Fluorometric assessment of FMN in perfusate was validated by mass spectrometry (R=0.7011,p<0.0001). Graft loss due to primary non-function or cholangiopathy was predicted by perfusate FMN values (c-statistic mass spectrometry 0.8418 (95%CI 0.7466-0.9370,p<0.0001), c-statistic fluorometry 0.7733 (95%CI 0.7006-0.8461,p<0.0001). Perfusate FMN values were also significantly correlated with symptomatic NAS and kidney failure, and superior in prediction of graft loss when compared to conventional scores derived from donor and recipient parameters, such as the donor risk index and the balance of risk score. Mitochondrial FMN values in liver tissues of non-utilized livers were low, and inversely correlated to high perfusate FMN values and purine metabolite release. CONCLUSIONS: This first international study validates the predictive value of the mitochondrial co-factor FMN, released from complex I during HOPE, and may therefore contribute to a better risk stratification of injured livers before implantation. IMPACT AND IMPLICATIONS: Analysis of 473 perfusates, collected from 10 international centers during hypothermic oxygenated perfusion (HOPE), revealed that mitochondria derived flavin mononucleotide (FMN) values in perfusate is predictive for graft loss, cholangiopathy, and kidney failure after liver transplantation. This result is of high clinical relevance, as recognition of graft quality is urgently needed to improve the safe utilization of marginal livers. Ex-situ machine perfusion approaches, such as HOPE, are therefore likely to increase the number of useable liver grafts.

Early-Life Silver Spoon Improves Survival and Breeding Performance of Adult Zebra Finches.

AbstractDevelopmental plasticity allows organisms to increase the fit between their phenotype and their early-life environment. The extent to which such plasticity also enhances adult fitness is not well understood, however, particularly when early-life and adult environments differ substantially. Using a cross-factorial design that manipulated diet at two life stages, we examined predictions of major hypotheses-silver spoon, environmental matching, and thrifty phenotype-concerning the joint impacts of early-life and adult diets on adult morphology/display traits, survival, and reproductive allocation. Overall, results aligned with the silver spoon hypothesis, which makes several predictions based on the premise that development in poor-quality environments constrains adult performance. Males reared and bred on a low-protein diet had lower adult survivorship than other male treatment groups; females' survivorship was higher than males' and not impacted by early diet. Measures of allocation to reproduction primarily reflected breeding diet, but where natal diet impacted reproduction, results supported the silver spoon. Both sexes showed reduced expression of display traits when reared on a low-protein diet. Results accord with other studies in supporting the relevance of the silver spoon hypothesis to birds and point to significant ramifications of sex differences in early-life viability selection on the applicability/strength of silver spoon effects.

Cardiorespiratory fitness is associated with estimates of myocardial perfusion: influence of age and sex.

Cardiorespiratory fitness (CRF) and the subendocardial viability ratio (SEVR) decline with age and predict future cardiovascular disease (CVD) events in a sex-dependent manner. However, the relation between CRF and SEVR in apparently healthy males and females across the age span is largely unknown. We hypothesized higher CRF is associated with greater SEVR in older females but not in males. Two-hundred sixty-two (126 M/136 F, age range 20-84 yr) participants underwent measures of CRF (maximal O2 consumption, V̇o2max) and SEVR (pulse wave analysis, PWA). A two-way analysis of variance (ANOVA) was used to examine differences in baseline characteristics between younger (<45 yr) and middle-aged and older (MA/O, ≥45 yr) males and females. Bivariate correlations assessed the relation between CRF, SEVR, and age in males and females. Partial correlations adjusted for CVD risk factors and medications. MA/O females had the lowest CRF and SEVR compared with all other groups (P < 0.05, both). SEVR was negatively correlated with age (r = -0.29) and positively correlated with CRF (r = 0.53) in females (P < 0.05, both) that persisted after controlling for CVD risk factors and medications (P < 0.05, all). SEVR was correlated with CRF in males only after adjusting for CVD risk factors and medications (r = 0.26, P < 0.05). These findings collectively demonstrate higher CRF is associated with greater SEVR in males and females after adjusting for CVD risk factors and medications, therefore highlighting subtle sex-specific nuances that warrant further investigation.NEW & NOTEWORTHY Cardiorespiratory fitness (CRF) and the subendocardial viability ratio (SEVR) are independent predictors of mortality and decline with age. However, the sex-specific relationship between CRF and SEVR with aging in adult males and females is unknown. Our findings demonstrate higher CRF is associated with greater age-related SEVR in males and females, after adjusting for traditional cardiovascular disease (CVD) risk factors and medications. However, subtle sex-related nuances exist in the relationship between SEVR and CRF that require further investigation.