Impaired O2 unloading from stored blood results in diffusion-limited O2 release at tissues: evidence from human kidneys.

ABSTRACT: The volume of oxygen drawn from systemic capillaries down a partial pressure gradient is determined by the oxygen content of red blood cells (RBCs) and their oxygen-unloading kinetics, although the latter is assumed to be rapid and, therefore, not a meaningful factor. Under this paradigm, oxygen transfer to tissues is perfusion-limited. Consequently, clinical treatments to optimize oxygen delivery aim at improving blood flow and arterial oxygen content, rather than RBC oxygen handling. Although the oxygen-carrying capacity of blood is increased with transfusion, studies have shown that stored blood undergoes kinetic attrition of oxygen release, which may compromise overall oxygen delivery to tissues by causing transport to become diffusion-limited. We sought evidence for diffusion-limited oxygen release in viable human kidneys, normothermically perfused with stored blood. In a cohort of kidneys that went on to be transplanted, renal respiration correlated inversely with the time-constant of oxygen unloading from RBCs used for perfusion. Furthermore, the renal respiratory rate did not correlate with arterial O2 delivery unless this factored the rate of oxygen-release from RBCs, as expected from diffusion-limited transport. To test for a rescue effect, perfusion of kidneys deemed unsuitable for transplantation was alternated between stored and rejuvenated RBCs of the same donation. This experiment controlled oxygen-unloading, without intervening ischemia, holding all non-RBC parameters constant. Rejuvenated oxygen-unloading kinetics improved the kidney's oxygen diffusion capacity and increased cortical oxygen partial pressure by 60%. Thus, oxygen delivery to tissues can become diffusion-limited during perfusion with stored blood, which has implications in scenarios, such as ex vivo organ perfusion, major hemorrhage, and pediatric transfusion. This trial was registered at www.clinicaltrials.gov as #ISRCTN13292277.

T cell-mediated Immune response and correlates of inflammation and their relationship with COVID-19 clinical severity: not an intuitive guess.

BACKGROUND: Predictors of the outcome of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection remain to be fully determined. We evaluated selected viral characteristics and immunological responses that might predict and/or correlate to the clinical outcome of COVID-19. METHODS: For individuals developing divergent clinical outcomes, the magnitude and breadth of T cell-mediated responses were measured within 36 h of symptom onset. Peripheral Blood Mononuclear Cells (PBMCs) were subjected to in vitro stimulation with SARS-CoV-2-based peptides. In addition, SARS-CoV-2 sequences were generated by metagenome, and HLA typing was performed using Luminex technology. FINDINGS: CD4+ T cell activation was negatively correlated with SARS-CoV-2 basal viral load in patients with severe COVID-19 (p = 0·043). The overall cellular immune response, as inferred by the IFN-γ signal, was higher at baseline for patients who progressed to mild disease compared to patients who progressed to severe disease (p = 0·0044). Subjects with milder disease developed higher T cell responses for MHC class I and II-restricted peptides (p = 0·033). INTERPRETATION: Mounting specific cellular immune responses in the first days after symptom onset, as inferred by IFN-γ magnitude in the ELISPOT assay, may efficiently favor a positive outcome. In contrast, progression to severe COVID-19 was accompanied by stronger cellular immune responses, higher CD4 + T cell activation, and a higher number of in silico predicted high-affinity class I HLA alleles.

Ethical Principles Do Not Support Mandatory Preanesthesia Pregnancy Screening Tests: A Narrative Review.

Respect for patient autonomy is a pillar of medical ethics, manifested predominantly through informed consent. Mandatory (routine) nonconsented preoperative urine pregnancy testing does not adequately respect patient autonomy, is potentially coercive, and has the potential to cause harm medically, psychologically, socially, and financially. Inaccuracies in pregnancy testing can result in false-positive and false-negative results, especially in early pregnancy. There is substantial scientific evidence that anesthesia is not harmful to the fetus, raising the question of whether pregnancy testing provides substantial benefit to the patient. Not performing a preanesthesia pregnancy test has not been associated with significant medicolegal consequences. We review the ethical implications of mandatory preanesthesia pregnancy testing in light of these facts.

sp3 -Rich Heterocycle Synthesis on DNA: Application to DNA-Encoded Library Production.

DNA encoded library (DEL) synthesis represents a convenient means to produce, annotate and store large collections of compounds in a small volume. While DELs are well suited for drug discovery campaigns, the chemistry used in their production must be compatible with the DNA tag, which can limit compound class accessibility. As a result, most DELs are heavily populated with peptidomimetic and sp2 -rich molecules. Herein, we show that sp3 -rich mono- and bicyclic heterocycles can be made on DNA from ketochlorohydrin aldol products through a reductive amination and cyclization process. The resulting hydroxypyrrolidines possess structural features that are desirable for DELs and target a distinct region of pharmaceutically relevant chemical space.

Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

Dolutegravir-associated resistance mutations after first-line treatment failure in Brazil.

BACKGROUND: Since January 2017, the recommended first-line antiretroviral regimen in Brazil is the fixed-dose combination of tenofovir plus lamivudine with dolutegravir (TL + D). According to the literature, integrase resistance-associated mutations (INRAMs) are rarely found upon virologic failure to first-line dolutegravir plus two nucleoside reverse transcriptase inhibitors. We evaluated the HIV antiretroviral genotypic resistance profile of patients referred for genotyping in the public health system who failed first-line TL + D after at least six months of therapy on or before December 31, 2018. METHODS: HIV Sanger sequences of the pol gene were generated from plasma of patients with confirmed virologic failure to first-line TL + D in the Brazilian public health system before December 31, 2018. RESULTS: One hundred thirteen individuals were included in the analysis. Major INRAMs were detected in seven patients (6.19%), four with R263K, one with G118R, one with E138A, and one with G140R. Four patients with major INRAMs also had the K70E and M184V mutations in the RT gene. Sixteen (14.2%) additional individuals presented minor INRAMs, and five (4,42%) patients had both major and minor INRAMS. Thirteen (11.5%) patients also presented mutations in the RT gene selected by tenofovir and lamivudine, including four with both the K70E and M184V mutations and four with only M184V. The integrase mutations L101I and T124A, which are in the in vitro pathway for integrase inhibitor resistance, were found in 48 and 19 patients, respectively. Mutations not related to TL + D, thus probable transmitted resistance mutations (TDR), were present in 28 patients (24.8%): 25 (22.1%) to nucleoside reverse transcriptase inhibitors, 19 (16.8%) to non-nucleoside reverse transcriptase inhibitors, and 6 (5.31%) to protease inhibitors. CONCLUSIONS: In marked contrast to previous reports, we report a relatively high frequency of INRAMs among selected patients failing first-line TL + D in the public health system in Brazil. Possible reasons for this discrepancy include delays in detecting virologic failure, patients inadvertently on dolutegravir monotherapy, TDR, and/or infecting subtype.

Computer image analysis with artificial intelligence: a practical introduction to convolutional neural networks for medical professionals.

Artificial intelligence tools, particularly convolutional neural networks (CNNs), are transforming healthcare by enhancing predictive, diagnostic, and decision-making capabilities. This review provides an accessible and practical explanation of CNNs for clinicians and highlights their relevance in medical image analysis. CNNs have shown themselves to be exceptionally useful in computer vision, a field that enables machines to 'see' and interpret visual data. Understanding how these models work can help clinicians leverage their full potential, especially as artificial intelligence continues to evolve and integrate into healthcare. CNNs have already demonstrated their efficacy in diverse medical fields, including radiology, histopathology, and medical photography. In radiology, CNNs have been used to automate the assessment of conditions such as pneumonia, pulmonary embolism, and rectal cancer. In histopathology, CNNs have been used to assess and classify colorectal polyps, gastric epithelial tumours, as well as assist in the assessment of multiple malignancies. In medical photography, CNNs have been used to assess retinal diseases and skin conditions, and to detect gastric and colorectal polyps during endoscopic procedures. In surgical laparoscopy, they may provide intraoperative assistance to surgeons, helping interpret surgical anatomy and demonstrate safe dissection zones. The integration of CNNs into medical image analysis promises to enhance diagnostic accuracy, streamline workflow efficiency, and expand access to expert-level image analysis, contributing to the ultimate goal of delivering further improvements in patient and healthcare outcomes.

Kidney transplantation from expanded criteria donors: an increased risk of urinary complications - the UriNary Complications Of Renal Transplant (UNyCORT) study.

OBJECTIVES: To assess the impact of expanded criteria donors (ECD) on urinary complications in kidney transplantation. PATIENTS AND METHODS: The UriNary Complications Of Renal Transplant (UNyCORT) is a cohort study based on the French prospective Données Informatisées et VAlidées en Transplantation/Computerized and VAlidated Data in Transplantation (DIVAT) cohort. Data were extracted between 1 January 2002 and 1 January 2018 with 1-year minimum follow-up, in relation to 44 pre- and postoperative variables. ECD status was included according to United Network for Organ Sharing (UNOS) definition. The primary outcome of the UNyCORT study was the association between the donor's ECD/standard criteria donors (SCD) status and urinary complications at 1 year in uni- and multivariate analysis. Sub-group analysis, stratified analysis on ECD/SCD donor's status and transplant failure analysis were then conducted. RESULTS: Between 1 January 2002 and 1 January 2018, 10 279 kidney transplants in adult recipients were recorded within the DIVAT network. A total of 8559 (83.4%) donors were deceased donors and 1699 (16.6%) were living donors (LD). Among donation after circulatory death (DCD) donors, 224 (2.85%) were uncontrolled DCD and 93 (1.09%) were controlled DCD donors. A total of 3617 (43.9%) deceased donors were ECD. The overall urological complication rate was 16.26%. The donor's ECD status was significantly associated with an increased risk of urological complications at 1 year in multivariate analysis (odds ratio: 1.50, 95% CI 1.31-1.71; P < 0.001) and especially with stenosis and ureteric fistulae at 1 year. There is no association with LD, uncontrolled and controlled DCD. The placement of an endo-ureteric stent was beneficial in preventing urinary complications in all donors and particularly in ECD donors. CONCLUSION: The donor's ECD status is associated with a higher likelihood of stenosis and ureteric fistulae at 1 year. Recipients of grafts from ECD donors should probably be considered for closer urological monitoring and systematic preventive measures.

Assessment of Mitochondrial Function and Oxygen Consumption Measured During Ex Vivo Normothermic Machine Perfusion of Injured Pig Kidneys Helps to Monitor Organ Viability.

Donor kidney assessment may improve organ utilisation. Normothermic Machine Perfusion (NMP) has the potential to facilitate this advance. The mechanism of action is not yet determined and we aimed to assess mitochondrial function during NMP. Anaesthetised pigs (n = 6) had one kidney clamped for 60 min. The healthy contralateral kidney was removed and underwent NMP for 8 h (healthy control (HC), n = 6). Following 60 min warm ischaemia the injured kidney underwent HMP for 24 h, followed by NMP for 8 h (n = 6). Mitochondria were extracted from fresh tissue for analysis. Injured kidneys were analysed as two separate groups (IMa, n = 3 and IMb, n = 3). Renal resistance was higher (0.39ï, ± 0.29 vs. 1.65ï, ± 0.85; p = 0.01) and flow was lower (55ï, ± 28 vs. 7ï, ± 4; p = 0.03) during HMP in IMb than IMa. NMP blood flow was higher in IMa versus IMb (2-way ANOVA; p < 0.001) After 60 min NMP, O2 consumption was significantly lower in IMb versus IMa (p ≤ 0.002). State-3 respiration was significantly different between the groups (37ï, ± 19 vs. 24ï, ± 14 vs. 10ï, ± 8; nmolO2/min/mg; p = 0.049). Lactate levels were significantly lower in IMa versus IMb (p = 0.028). Mitochondrial respiration levels during NMP may be suggestive of kidney viability. Oxygen consumption, renal blood flow and lactate can differentiate severity of kidney injury during NMP.

Immunogenicity of personalized dendritic-cell therapy in HIV-1 infected individuals under suppressive antiretroviral treatment: interim analysis from a phase II clinical trial.

BACKGROUND: We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. METHODS: PBMCs were obtained from 10 HIV+ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient's HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients' cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4+ and CD8+ T-cells. RESULTS: The protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4+ and CD8+ T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4+ T-cells. The number of candidates that increased in vitro the cytokine levels in CD4+ and CD8+ T cells upon stimulation with Gag peptides from baseline to day 15 and from baseline to day 30 and day 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. CONCLUSIONS: MDDC had a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment. Trial registration NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829 , posted November 11th, 2016).

Normothermic machine perfusion of kidneys: current strategies and future perspectives.

PURPOSE OF REVIEW: This review aims to summarize the latest original preclinical and clinical articles in the setting of normothermic machine perfusion (NMP) of kidney grafts. RECENT FINDINGS: Kidney NMP can be safely translated into the clinical routine and there is increasing evidence that NMP may be beneficial in graft preservation especially in marginal kidney grafts. Due to the near-physiological state during NMP, this technology may be used as an ex-vivo organ assessment and treatment platform. There are reports on the application of mesenchymal stromal/stem cells, multipotent adult progenitor cells and microRNA during kidney NMP, with first data indicating that these therapies indeed lead to a decrease in inflammatory response and kidney injury. Together with the demonstrated possibility of prolonged ex-vivo perfusion without significant graft damage, NMP could not only be used as a tool to perform preimplant graft assessment. Some evidence exists that it truly has the potential to be a platform to treat and repair injured kidney grafts, thereby significantly reducing the number of declined organs. SUMMARY: Kidney NMP is feasible and can potentially increase the donor pool not only by preimplant graft assessment, but also by ex-vivo graft treatment.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25 mg would be tested, starting with 10 mg in cohort 1. No significant difference between Mirococept at 10 mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80 mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3 hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80 mg (equivalent to 120 mg in human kidney) that provides a basis for further clinical development.

Micellar Buchwald-Hartwig Coupling of Aryl and Heteroarylamines for the Synthesis of DNA-Encoded Libraries.

DNA-encoded libraries are a very efficient means of identifying ligands for protein targets in high throughput. To fully maximize their use, it is essential to be able to carry out efficient reactions on DNA-conjugated substrates. Arylamines are privileged motifs in druglike molecules, and methods for their incorporation into DNA-encoded libraries are highly desirable. One of the preferred methods for their preparation, the Buchwald-Hartwig coupling, does not perform well on DNA conjugates using current approaches. We report the application of our recently developed micellar technology for on-DNA chemistry to the Buchwald-Hartwig reaction. Optimization of conditions led to a robust, high-yielding method for the synthesis of DNA-conjugated aryl and heteroarylamines, which is broad in substrate scope for both the arylamine and the DNA-conjugated aryl halide and is fully compatible with DNA-encoding and decoding procedures. This method will enable the preparation of diverse, high-fidelity libraries of biarylamines.

Functional Group Tolerance of a Micellar on-DNA Suzuki-Miyaura Cross-Coupling Reaction for DNA-Encoded Library Design.

DNA-encoded libraries (DELs) offer great promise for the discovery of new ligands for proteins. Many current reactions used for DEL synthesis do not proceed efficiently over a wide range of substrates. Combining a diverse array of multicomponent reactions with micellar-promoted Suzuki-Miyaura cross-coupling provides a strategy for synthesizing highly diverse DELs with exceptionally high fidelity. These results demonstrate that the micellar Suzuki-Miyaura reaction has exceptional functional group tolerance and broad applicability.

Serological and molecular epidemiology of the Dengue, Zika and Chikungunya viruses in a risk area in Brazil.

BACKGROUND: The co-circulation of types of arbovirus in areas where they are endemic increased the risk of outbreaks and limited the diagnostic methods available. Here, we analyze the epidemiological profile of DENV, CHIKV and ZIKV at the serological and molecular level in patients with suspected infection with these arboviruses in the city of Juazeiro do Norte, Ceará, Brazil. METHODS: In 2016, the Central Public Health Laboratory (LACEN) of Juazeiro do Norte received 182 plasma samples from patients who visited health facilities with symptoms compatible with arbovirus infection. The LACEN performed serological tests for detection of IgM/IgG to DENV and CHIKV. They then sent these samples to the Retrovirology Laboratory of the Federal University of São Paulo and Faculty of Medical of the ABC where molecular analyses to confirm the infection by DENV, ZIKV and CHIKV were performed. The prevalence of IgM/IgG antibodies and of infections confirmed by RT-qPCR were presented with 95% confidence interval. RESULTS: In serologic analysis, 125 samples were positive for antibodies against CHIKV and all were positive for antibodies against DENV. A higher prevalence of IgG against CHIKV (63.20% with 95% CI: 45.76-70.56) than against DENV (95.05% with 95% CI: 78.09-98.12) was observed. When the samples were submitted to analysis by RT-qPCR, we observed the following prevalence: mono-infection by ZIKV of 19.23% (95% CI: 14.29-34.82) patients, mono-infection by CHIKV of 3.84% (95% CI: 2.01-5.44) and co-infection with ZIKV and CHIKV of 1.09% (95% CI: 0.89-4.56). CONCLUSION: The serologic and molecular tests performed in this study were effective in analyzing the epidemiological profile of DENV, CHIKV and ZIKV in patients with suspected infection by these arboviruses in the city of Juazeiro do Norte, Ceará/Brazil.

Allograft nephrectomy versus nonallograft nephrectomy after failed renal transplantation: a systematic review by updated meta-analysis.

There is limited evidence regarding the impact of allograft nephrectomy (AN) on the long-term outcome of subsequent kidney re-transplantation compared with no prior allograft nephrectomy. The aim of the present study was to conduct a systematic review and meta-analysis to estimate the accumulation of evidence over time. Primary outcomes were 5-year graft and patient survival. Cochrane library, Google scholar, PubMed, Medline and Embase were systematically searched. Meta-analysis was conducted using both fixed- and random-effects models. Study quality was assessed in duplicate using the Newcastle-Ottawa scale. Sixteen studies were included, with a total of 2256 patients. All included studies were retrospective and comparative. There was no significant difference in 5-year graft survival (GS) [Hazard Ratio (HR) = 1.11, 95% Confidence Intervals (CI): 0.89, 1.38, P = 0.37, I2  = 10%) or in 5-year patient survival (PS; HR = 0.70, 95% CI: 0.45, 1.10, P = 0.12, I2  = 0%]. Patients in the AN cohort were significantly younger than patients in the nonallograft nephrectomy (NAN) cohort by one year. Prior allograft nephrectomy was associated with a significantly higher risk of delayed graft function (DGF), acute rejection, primary nonfunction (PNF), per cent of panel reactive antibodies (% PRA) and allograft loss of the subsequent transplant. Although, DGF, % PRA, acute rejection and primary nonfunction rates were significantly higher in the AN cohort, allograft nephrectomy prior to re-transplantation had no significant association with five-year graft and patient survival.

Development of ex situ normothermic reperfusion as an innovative method to assess pancreases after preservation.

Static cold storage (SCS) is the standard method for pancreas preservation prior to transplantation; however, it does not permit organ assessment. Normothermic reperfusion (NR) is utilized clinically for other organs to assess viability. Our aim was to develop NR using normothermic machine perfusion technique to simulate reperfusion at the time of transplantation, enabling evaluation of oxygenated hypothermic machine perfusion (HMPO2) as a newer strategy to optimize pancreas preservation. 13 porcine pancreases procured after circulatory death were divided into 3 groups: 4 pancreases preserved using SCS, and 2 groups preserved by HMPO2 (n = 4 and n = 5, differing by type of preservation solution). Duration of perfusion or cold storage was 6 hours before the 1-hour assessment using NR. Outcome measures were perfusion characteristics, biochemistry and change in tissue water mass as oedema assessment. During NR, the HMPO2 groups demonstrated better perfusion characteristics, normal macroscopic appearances, decreased water mass and one HMPO2 group demonstrated a response to glucose stimulation. Conversely, the SCS group showed an increased water mass and developed early macroscopic appearances of oedema, interstitial haemorrhage and minimal portal outflow. This study suggests that ex situ assessment of pancreases by NR is promising, and that HMPO2 may be better than SCS.

Ischemia-Reperfusion Injuries Assessment during Pancreas Preservation.

Maintaining organ viability between donation and transplantation is of critical importance for optimal graft function and survival. To date in pancreas transplantation, static cold storage (SCS) is the most widely practiced method of organ preservation. The first experiments in ex vivo perfusion of the pancreas were performed at the beginning of the 20th century. These perfusions led to organ oedema, hemorrhage, and venous congestion after revascularization. Despite these early hurdles, a number of factors now favor the use of perfusion during preservation: the encouraging results of HMP in kidney transplantation, the development of new perfusion solutions, and the development of organ perfusion machines for the lung, heart, kidneys and liver. This has led to a resurgence of research in machine perfusion for whole organ pancreas preservation. This review highlights the ischemia-reperfusion injuries assessment during ex vivo pancreas perfusion, both for assessment in pre-clinical experimental models as well for future use in the clinic. We evaluated perfusion dynamics, oedema assessment, especially by impedance analysis and MRI, whole organ oxygen consumption, tissue oxygen tension, metabolite concentrations in tissue and perfusate, mitochondrial respiration, cell death, especially by histology, total cell free DNA, caspase activation, and exocrine and endocrine assessment.

High Fidelity Suzuki-Miyaura Coupling for the Synthesis of DNA Encoded Libraries Enabled by Micelle Forming Surfactants.

DNA encoded chemical libraries provide a highly efficient means of screening vast numbers of small molecules against an immobilized protein target. Their potential is currently restricted by the constraints of carrying out library synthesis in the presence of attached DNA tags, for which a limited number of reactions and substrates can be used. Even established reactions, such as Suzuki-Miyaura couplings, do not give efficient coupling reactions across a wide range of substrates and can lead to significant DNA degradation. We developed an efficient protocol for carrying out Suzuki-Miyaura couplings on DNA tagged substrates that proceeds with unprecedented efficiency to the desired biaryl products (>98% on average with no detectable DNA degradation) across a wide range of drug-like substrates using a micellar promoted process with commercial TPGS-750-M surfactant. We have demonstrated the applicability of this method in DEL synthesis by preparing a prototypical two-dimensional 36-member library employing the Suzuki-Miyaura coupling methodology as the final library synthesis step. This work shows, for the first time, that standard micellar surfactants can promote reactions for encoded library synthesis, leading to libraries of exceptional fidelity, and demonstrates the potential to expand the range of accessible DNA compatible chemistry.