Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.

Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s.

LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.

Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic ß-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

LINE-1 Evasion of Epigenetic Repression in Humans.

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

A new human embryonic cell type associated with activity of young transposable elements allows definition of the inner cell mass.

There remains much that we do not understand about the earliest stages of human development. On a gross level, there is evidence for apoptosis, but the nature of the affected cell types is unknown. Perhaps most importantly, the inner cell mass (ICM), from which the foetus is derived and hence of interest in reproductive health and regenerative medicine, has proven hard to define. Here, we provide a multi-method analysis of the early human embryo to resolve these issues. Single-cell analysis (on multiple independent datasets), supported by embryo visualisation, uncovers a common previously uncharacterised class of cells lacking commitment markers that segregates after embryonic gene activation (EGA) and shortly after undergo apoptosis. The discovery of this cell type allows us to clearly define their viable ontogenetic sisters, these being the cells of the ICM. While ICM is characterised by the activity of an Old non-transposing endogenous retrovirus (HERVH) that acts to suppress Young transposable elements, the new cell type, by contrast, expresses transpositionally competent Young elements and DNA-damage response genes. As the Young elements are RetroElements and the cells are excluded from the developmental process, we dub these REject cells. With these and ICM being characterised by differential mobile element activities, the human embryo may be a "selection arena" in which one group of cells selectively die, while other less damaged cells persist.

Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes.

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing mRNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINEs use strategies to 'hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.

ZCCHC3 is a stress granule zinc knuckle protein that strongly suppresses LINE-1 retrotransposition.

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control.

Somatic retrotransposition in the developing rhesus macaque brain.

The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques (Macaca mulatta). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in ∼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4, a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution.

Long-term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co-administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus.

This study evaluates the sustained antidepressant-like effects and neurogenic potential of a 3-day intranasal co-administration regimen of galanin receptor 2 (GALR2) agonist M1145 and neuropeptide Y Y1 receptor (NPY1R) agonist [Leu31, Pro34]NPY in the ventral hippocampus of adult rats, with outcomes analyzed 3 weeks post-treatment. Utilizing the forced swimming test (FST), we found that this co-administration significantly enhances antidepressant-like behaviors, an effect neutralized by the GALR2 antagonist M871, highlighting the synergistic potential of these neuropeptides in modulating mood-related behaviors. In situ proximity ligation assay (PLA) indicated a significant increase in GALR2/NPYY1R heteroreceptor complexes in the ventral hippocampal dentate gyrus, suggesting a molecular basis for the behavioral outcomes observed. Moreover, proliferating cell nuclear antigen (PCNA) immunolabeling revealed increased cell proliferation in the subgranular zone of the dentate gyrus, specifically in neuroblasts as evidenced by co-labeling with doublecortin (DCX), without affecting quiescent neural progenitors or astrocytes. The study also noted a significant uptick in the number of DCX-positive cells and alterations in dendritic morphology in the ventral hippocampus, indicative of enhanced neuronal differentiation and maturation. These morphological changes highlight the potential of these agonists to facilitate the functional integration of new neurons into existing neural circuits. By demonstrating the long-lasting effects of a brief, 3-day intranasal administration of GALR2 and NPY1R agonists, our findings contribute significantly to the understanding of neuropeptide-mediated neuroplasticity and herald novel therapeutic strategies for the treatment of depression and related mood disorders, emphasizing the therapeutic promise of targeting neurogenesis and neuronal maturation processes.

Role of reactive oxygen species in the modulation of auxin flux and root development in Arabidopsis thaliana.

Reactive oxygen species (ROS) play a dual role in plant biology, acting as important signal transduction molecules and as toxic byproducts of aerobic metabolism that accumulate in cells upon exposure to different stressors and lead to cell death. In plants, root architecture is regulated by the distribution and intercellular flow of the phytohormone auxin. In this study, we identified ROS as an important modulator of auxin distribution and response in the root. ROS production is necessary for root growth, proper tissue patterning, cell growth, and lateral root (LR) induction. Alterations in ROS balance led to altered auxin distribution and response in SOD and RHD2 loss-of-function mutants. Treatment of Arabidopsis seedlings with additional sources of ROS (hydrogen peroxide) or an ROS production inhibitor (diphenylene iodonium) induced phenocopies of the mutants studied. Simultaneous application of auxin and ROS increased LR primordia induction, and PIN-FORMED protein immunolocalization further demonstrated the existing link between auxin and ROS in orchestrating cell division and auxin flux during root development. In Arabidopsis roots, genetic alterations in ROS balance led to defective auxin distribution and growth-related responses in roots. Exogenous hydrogen peroxide alters the establishment of the endogenous auxin gradient in the root meristem through regulation of PIN-FORMED polarity, while the simultaneous application of hydrogen peroxide and auxin enhanced LR induction in a dose- and position-dependent manner through activation of cell division.

Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

Risk of lung disease in the PI*SS genotype of alpha-1 antitrypsin: an EARCO research project.

BACKGROUND: The PI*S variant is one of the most prevalent mutations within alpha-1 antitrypsin deficiency (AATD). The risk of developing AATD-related lung disease in individuals with the PI*SS genotype is poorly defined despite its substantial prevalence. Our study aimed to characterize this genotype and its risk for lung disease and compare it with the PI*ZZ and PI*SZ genotypes using data from the European Alpha-1 antitrypsin Deficiency Research Collaboration international registry. METHOD: Demographic, clinical, functional, and quality of life (QoL) parameters were assessed to compare the PI*SS characteristics with the PI*SZ and PI*ZZ controls. A propensity score with 1:3 nearest-neighbour matching was performed for the most important confounding variables. RESULTS: The study included 1007 individuals, with PI*SS (n = 56; 5.6%), PI*ZZ (n = 578; 57.4%) and PI*SZ (n = 373; 37.0%). The PI*SS population consisted of 58.9% men, with a mean age of 59.2 years and a mean FEV1(% predicted) of 83.4%. Compared to PI*ZZ individuals they had less frequent lung disease (71.4% vs. 82.2%, p = 0.037), COPD (41.4% vs. 60%, p = 0.002), and emphysema (23.2% vs. 51.9%, p < 0.001) and better preserved lung function, fewer exacerbations, lower level of dyspnoea, and better QoL. In contrast, no significant differences were found in the prevalence of lung diseases between PI*SS and PI*SZ, or lung function parameters, exacerbations, dyspnoea, or QoL. CONCLUSIONS: We found that, as expected, the risk of lung disease associated with the PI*SS genotype is significantly lower compared with PI*ZZ, but does not differ from that observed in PI*SZ individuals, despite having higher serum AAT levels. TRIAL REGISTRATION: www. CLINICALTRIALS: gov (ID: NCT04180319).

The ancestral selection graph for a Λ-asymmetric Moran model.

Motivated by the question of the impact of selective advantage in populations with skewed reproduction mechanisms, we study a Moran model with selection. We assume that there are two types of individuals, where the reproductive success of one type is larger than the other. The higher reproductive success may stem from either more frequent reproduction, or from larger numbers of offspring, and is encoded in a measure Λ for each of the two types. Λ-reproduction here means that a whole fraction of the population is replaced at a reproductive event. Our approach consists of constructing a Λ-asymmetric Moran model in which individuals of the two populations compete, rather than considering a Moran model for each population. Provided the measure are ordered stochastically, we can couple them. This allows us to construct the central object of this paper, the Λ-asymmetric ancestral selection graph, leading to a pathwise duality of the forward in time Λ-asymmetric Moran model with its ancestral process. We apply the ancestral selection graph in order to obtain scaling limits of the forward and backward processes, and note that the frequency process converges to the solution of an SDE with discontinuous paths. Finally, we derive a Griffiths representation for the generator of the SDE and use it to find a semi-explicit formula for the probability of fixation of the less beneficial of the two types.

Assessing the reinforcement effect by response surface methodology of holocellulose from spent coffee grounds on biopolymeric films as food packaging materials.

The pollution caused by petroleum-derived plastic materials has become a major environmental problem that has encouraged the development of new compostable and environmentally friendly materials for food packaging based on biomodified polymers with household residues. This study aims to design, synthesize, and characterize a biobased polymeric microstructure film from polyvinyl alcohol and chitosan reinforced with holocellulose from spent coffee grounds for food-sustainable packaging. Chemical isolation with a chlorite-based solution was performed to obtain the reinforced holocellulose from the spent coffee ground, and the solvent casting method was used to obtain the films to study. Physicochemical and microscopic characterizations were conducted to identify and select the best formulations using a simplex-centroid design analysis. The response surface methodology results indicate that the new packaging material obtained with equal amounts of polymers and reinforced material (1:1:1) possesses the appropriate barrier properties and microstructural character to prevent water attack and hydrophobic behavior and thus could be used as an alternative for food packaging materials.

Genes involved in auxin biosynthesis, transport and signalling underlie the extreme adventitious root phenotype of the tomato aer mutant.

The use of tomato rootstocks has helped to alleviate the soaring abiotic stresses provoked by the adverse effects of climate change. Lateral and adventitious roots can improve topsoil exploration and nutrient uptake, shoot biomass and resulting overall yield. It is essential to understand the genetic basis of root structure development and how lateral and adventitious roots are produced. Existing mutant lines with specific root phenotypes are an excellent resource to analyse and comprehend the molecular basis of root developmental traits. The tomato aerial roots (aer) mutant exhibits an extreme adventitious rooting phenotype on the primary stem. It is known that this phenotype is associated with restricted polar auxin transport from the juvenile to the more mature stem, but prior to this study, the genetic loci responsible for the aer phenotype were unknown. We used genomic approaches to define the polygenic nature of the aer phenotype and provide evidence that increased expression of specific auxin biosynthesis, transport and signalling genes in different loci causes the initiation of adventitious root primordia in tomato stems. Our results allow the selection of different levels of adventitious rooting using molecular markers, potentially contributing to rootstock breeding strategies in grafted vegetable crops, especially in tomato. In crops vegetatively propagated as cuttings, such as fruit trees and cane fruits, orthologous genes may be useful for the selection of cultivars more amenable to propagation.

CACS, CCTA and mCAD-LT score in the pre-transplant assessment of coronary artery disease and the prediction of post-transplant cardiovascular events.

BACKGROUND: The optimal cardiovascular assessment of liver transplant (LT) candidates is unclear. We aimed to evaluate the performance of CT-based coronary tests (coronary artery calcium score [CACS] and coronary CT angiography [CCTA]) and a modification of the CAD-LT score (mCAD-LT, excluding family history of CAD) to diagnose significant coronary artery disease (CAD) before LT and predict the incidence of post-LT cardiovascular events (CVE). METHODS: We retrospectively analysed a single-centre cohort of LT candidates who underwent non-invasive tests; invasive coronary angiography (ICA) was performed depending on the results of non-invasive tests. mCAD-LT was calculated in all patients. RESULTS: Six-hundred-and-thirty-four LT candidates were assessed and 351 of them underwent LT. CACS, CCTA and ICA were performed in 245, 123 and 120 LT candidates, respectively. Significant CAD was found in 30% of patients undergoing ICA. The AUROCs of mCAD-LT (.722) and CCTA (.654) were significantly higher than that of CACS (.502) to predict the presence of significant CAD. Specificity of the tests ranged between 31% for CCTA and 53% for CACS. Among patients who underwent LT, CACS ≥ 400 and mCAD-LT were independently associated with the incidence of CVE; in patients who underwent CCTA before LT, significant CAD at CCTA also predicted post-LT CVE. CONCLUSION: In this cohort, mCAD-LT score and CT-based tests detect the presence of significant CAD in LT candidates, although they tend to overestimate it. Both mCAD-LT score and CT-based tests classify LT recipients according to their risk of post-LT CVE and can be used to improve post-LT risk mitigation.

Comparison of the SMLM technique and the MSSR algorithm in confocal microscopy for super-resolved imaging of cellulose fibres.

Nowadays, the use of super-resolution microscopy (SRM) is increasing globally due to its potential application in several fields of life sciences. However, a detailed and comprehensive guide is necessary for understanding a single-frame image's resolution limit. This study was performed to provide information about the structural organisation of isolated cellulose fibres from garlic and agave wastes through fluorophore-based techniques and image analysis algorithms. Confocal microscopy provided overall information on the cellulose fibres' microstructure, while techniques such as total internal reflection fluorescence microscopy facilitated the study of the plant fibres' surface structures at a sub-micrometric scale. Furthermore, SIM and single-molecule localisation microscopy (SMLM) using the PALM reconstruction wizard can resolve the network of cellulose fibres at the nanometric level. In contrast, the mean shift super-resolution (MSSR) algorithm successfully determined nanometric structures from confocal microscopy images. Atomic force microscopy was used as a microscopy technique for measuring the size of the fibres. Similar fibre sizes to those evaluated with SIM and SMLM were found using the MSSR algorithm and AFM. However, the MSSR algorithm must be cautiously applied because the selection of thresholding parameters still depends on human visual perception. Therefore, this contribution provides a comparative study of SRM techniques and MSSR algorithm using cellulose fibres as reference material to evaluate the performance of a mathematical algorithm for image processing of bioimages at a nanometric scale. In addition, this work could act as a simple guide for improving the lateral resolution of single-frame fluorescence bioimages when SRM facilities are unavailable.