Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

A randomised trial of oral prednisone for cystic fibrosis pulmonary exacerbation treatment.

BACKGROUND: Elevated markers of systemic and pulmonary inflammation are associated with failure to recover lung function following pulmonary exacerbations in people with cystic fibrosis (pwCF). Our aim was to determine whether adjuvant oral prednisone treatment would improve recovery of forced expiratory volume in 1 s (FEV1) % pred in CF pulmonary exacerbations not responding to antibiotic therapy. METHODS: This was a randomised, double-blind, placebo-controlled trial in pwCF treated with intravenous antibiotics for a pulmonary exacerbation. At day 7, those who had not returned to >90% baseline FEV1 % pred were randomised to adjuvant prednisone 1 mg·kg-1 twice daily (maximum 60 mg·day-1) or placebo for 7 days. The primary outcome was the difference in proportion of subjects who recovered >90% baseline FEV1 % pred at day 14 of i.v. antibiotic therapy. RESULTS: 173 subjects were enrolled, with 76 randomised. 50% of subjects in the prednisone group recovered baseline FEV1 on day 14 compared with 39% of subjects in the placebo group (difference of 11%, 95% CI -11-34%; p=0.34). The mean±sd change in FEV1 % pred from day 7 to day 14 was 6.8±8.8% predicted in the prednisone group and 4.6±6.9% predicted in the placebo group (mean difference 2.2% predicted, 95% CI -1.5-5.9%; p=0.24). Time to subsequent exacerbation was not prolonged in prednisone-treated subjects (hazard ratio 0.83, 95% CI 0.45-1.53; p=0.54). CONCLUSIONS: This study failed to detect a difference in FEV1 % pred recovery between adjuvant oral prednisone and placebo treatment in pwCF not responding at day 7 of i.v. antibiotic therapy for pulmonary exacerbations.

DNA lesion bypass and the stochastic dynamics of transcription-coupled repair.

DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.

Enzymatic conversion of human blood group A kidneys to universal blood group O.

ABO blood group compatibility restrictions present the first barrier to donor-recipient matching in kidney transplantation. Here, we present the use of two enzymes, FpGalNAc deacetylase and FpGalactosaminidase, from the bacterium Flavonifractor plautii to enzymatically convert blood group A antigens from the renal vasculature of human kidneys to 'universal' O-type. Using normothermic machine perfusion (NMP) and hypothermic machine perfusion (HMP) strategies, we demonstrate blood group A antigen loss of approximately 80% in as little as 2 h NMP and HMP. Furthermore, we show that treated kidneys do not bind circulating anti-A antibodies in an ex vivo model of ABO-incompatible transplantation and do not activate the classical complement pathway. This strategy presents a solution to the donor organ shortage crisis with the potential for direct clinical translation to reduce waiting times for patients with end stage renal disease.

Doubling the life of Cu/ZnO methanol synthesis catalysts via use of Si as a structural promoter to inhibit sintering.

Cu/ZnO/Al2O3 catalysts used to synthesize methanol undergo extensive deactivation during use, mainly due to sintering. Here, we report on formulations wherein deactivation has been substantially reduced by the targeted use of a small quantity of a Si-based promoter, resulting in accrued activity benefits that can exceed a factor of 1.8 versus unpromoted catalysts. This enhanced stability also provides longer lifetimes, up to double that of prior generation catalysts. Detailed characterization of a library of aged catalysts has allowed the most important deactivation mechanisms to be established and the chemical state of the silicon promoter to be identified. We show that silicon is incorporated within the ZnO lattice, providing a pronounced improvement in the hydrothermal stability of this component. These findings have important implications for sustainable methanol production from H2 and CO2.

Sequential mutations in exponentially growing populations.

Stochastic models of sequential mutation acquisition are widely used to quantify cancer and bacterial evolution. Across manifold scenarios, recurrent research questions are: how many cells are there with n alterations, and how long will it take for these cells to appear. For exponentially growing populations, these questions have been tackled only in special cases so far. Here, within a multitype branching process framework, we consider a general mutational path where mutations may be advantageous, neutral or deleterious. In the biologically relevant limiting regimes of large times and small mutation rates, we derive probability distributions for the number, and arrival time, of cells with n mutations. Surprisingly, the two quantities respectively follow Mittag-Leffler and logistic distributions regardless of n or the mutations' selective effects. Our results provide a rapid method to assess how altering the fundamental division, death, and mutation rates impacts the arrival time, and number, of mutant cells. We highlight consequences for mutation rate inference in fluctuation assays.

Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. RESULTS: Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Normothermic machine perfusion versus static cold storage in donation after circulatory death kidney transplantation: a randomized controlled trial.

Kidney transplantation is the optimal treatment for end-stage renal disease, but it is still severely limited by a lack of suitable organ donors. Kidneys from donation after circulatory death (DCD) donors have been used to increase transplant rates, but these organs are susceptible to cold ischemic injury in the storage period before transplantation, the clinical consequence of which is high rates of delayed graft function (DGF). Normothermic machine perfusion (NMP) is an emerging technique that circulates a warmed, oxygenated red-cell-based perfusate through the kidney to maintain near-physiological conditions. We conducted a randomized controlled trial to compare the outcome of DCD kidney transplants after conventional static cold storage (SCS) alone or SCS plus 1-h NMP. A total of 338 kidneys were randomly allocated to SCS (n = 168) or NMP (n = 170), and 277 kidneys were included in the final intention-to-treat analysis. The primary endpoint was DGF, defined as the requirement for dialysis in the first 7 d after transplant. The rate of DGF was 82 of 135 (60.7%) in NMP kidneys versus 83 of 142 (58.5%) in SCS kidneys (adjusted odds ratio (95% confidence interval) 1.13 (0.69-1.84); P = 0.624). NMP was not associated with any increase in transplant thrombosis, infectious complications or any other adverse events. A 1-h period of NMP at the end of SCS did not reduce the rate of DGF in DCD kidneys. NMP was demonstrated to be feasible, safe and suitable for clinical application. Trial registration number: ISRCTN15821205 .

Organ and cell-specific biomarkers of Long-COVID identified with targeted proteomics and machine learning.

BACKGROUND: Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID. METHODS: A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase. RESULTS: Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID. CONCLUSIONS: Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.

Honoring the gift: The transformative potential of transplant-declined human organs.

For decades, transplantation has been a life-saving treatment for those fortunate enough to gain access. Nevertheless, many patients die waiting for an organ and countless more never make it onto the waitlist because of a shortage of donor organs. Concurrently, thousands of donated organs are declined for transplant each year because of concerns about poor outcomes post-transplant. The decline of any donated organ-even if medically justified-is tragic for both the donor family and potential recipients. In this Personal Viewpoint, we discuss the need for a new mindset in how we honor the gift of organ donation. We believe that the use of transplant-declined human organs in translational research has the potential to hasten breakthrough discoveries in a multitude of scientific and medical areas. More importantly, such breakthroughs will allow us to properly value every donated organ. We further discuss the many practical challenges that such research presents and offer some possible solutions based on experiences in our own research laboratories. Finally, we share our perspective on what we believe are the necessary next steps to ensure a future where every donated organ realizes its full potential to impact the lives of current and future patients.

Cystic fibrosis related diabetes (CFRD) in the era of modulators: A scoping review.

Cystic fibrosis-related diabetes (CFRD) is a common complication of CF that increases in incidence as patients age. Poor glycemic control has been shown to negatively impact lung function and weight, resulting in higher risk of recurrent pulmonary exacerbations. With the advent of highly effective modulator therapies (HEMT), patients with CF are living longer and healthier lives. Consequently, CFRD and its microvascular complications are rising in prominence, becoming one of the most urgent clinical concerns. As HEMT were developed with the primary focus of improving pulmonary outcomes, it is not clear from the original phase III studies what the short- or long-term benefits of modulators might be on CFRD development and trajectory. In this review, we will examine the pathophysiology of CFRD, summarize and synthesize the available evidence of HEMT impact on CFRD and describe the emerging research needs in this field.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

De novo SIX2 activation in human kidneys treated with neonatal kidney stem/progenitor cells.

During development, nephron structures are derived from a SIX2+ stem cell population. After 36 weeks of gestation, these cells are exhausted, and no new nephrons are formed. We have previously described a non-invasive strategy to isolate and expand the native SIX2+ kidney stem cells from the urine of preterm neonates, named neonatal kidney stem/progenitor cells (nKSPC). Here, we investigated the safety and feasibility of administering nKSPC into human kidneys discarded for transplantation during normothermic machine perfusion (NMP) and evaluated the regenerative and immunomodulatory potential of nKSPC treatment. We found that nKSPC administration during NMP is safe and feasible. Interestingly, nKSPC induced the de novo expression of SIX2 in proximal tubular cells of the donor kidneys and upregulated regenerative markers such as SOX9 and VEGF. This is the first time that SIX2 re-expression is observed in adult human kidneys. Moreover, nKSPC administration significantly lowered levels of kidney injury biomarkers and reduced inflammatory cytokine levels via the tryptophan-IDO-kynurenine pathway. In conclusion, nKSPC is a novel cell type to be applied in kidney-targeted cell therapy, with the potential to induce an endogenous regenerative process and immunomodulation.

The Effects of Free Heme on Functional and Molecular Changes During Ex Vivo Normothermic Machine Perfusion of Human Kidneys.

Normothermic machine perfusion (NMP) is a technique of kidney preservation designed to restore cellular metabolism after cold ischemia. Kidneys are perfused with an oxygenated banked red blood cell (RBC) based solution for 1h at 36°C. During NMP, RBCs can become damaged, releasing free heme into the perfusate. This can act as a damage-associated molecular pattern (DAMP) activating inflammatory signalling pathways. The aim of this study was to measure the levels of free heme during NMP, assess the effect on kidney function and determine any association with inflammatory and stress related gene expression. Levels of free heme were measured in perfusate samples from a series of donation after circulatory death (DCD) kidneys undergoing NMP as part of a randomised controlled trial (RCT). The age of RBCs and levels of free heme were correlated with perfusion parameters. Changes in gene expression were analysed in a series of kidneys declined for transplantation using the NanoString nCounter Organ Transplant Panel and qRT-PCR. Older units of RBCs were associated with higher levels of free heme and levels increased significantly during NMP (Pre 8.56 ± 7.19µM vs 26.29 ± 15.18µM, P<0.0001). There was no association with levels of free heme and perfusion parameters during NMP (P > 0.05). Transcriptional and qPCR analysis demonstrated the upregulation of differentially expressed genes associated with apoptosis (FOS and JUN), inflammatory cytokines (IL-6, SOCS3, ATF3), chemokines (CXCL8, CXCL2, CC3/L1) and oxidative stress (KLF4) after NMP. However, these did not correlate with levels of free heme (P >0.05). A significant amount of free heme can be detected in the perfusate before and after NMP particularly when older units of red cells are used. Although transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways these were not associated with high levels of free heme.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

A digital pathology tool for quantification of color features in histologic specimens.

In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin-embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue-based analyses.

Extracellular vesicles in kidney transplantation: a state-of-the-art review.

Kidney transplantation is the optimal treatment for patients with kidney failure; however, early detection and timely treatment of graft injury remain a challenge. Precise and noninvasive techniques of graft assessment and innovative therapeutics are required to improve kidney transplantation outcomes. Extracellular vesicles (EVs) are lipid bilayer-delimited particles with unique biosignatures and immunomodulatory potential, functioning as intermediaries of cell signalling. Promising evidence exists for the potential of EVs to develop precision diagnostics of graft dysfunction, and prognostic biomarkers for clinician decision making. The inherent targeting characteristics of EVs and their low immunogenic and toxicity profiles combined with their potential as vehicles for drug delivery make them ideal targets for development of therapeutics to improve kidney transplant outcomes. In this review, we summarize the current evidence for EVs in kidney transplantation, discuss common methodological principles of EV isolation and characterization, explore upcoming innovative approaches in EV research, and discuss challenges and opportunities to enable translation of research findings into clinical practice.