Original Phenotype Research Articles

Typical NF2 and LTZR1 mutations are retained in an immortalized human schwann cell model of schwannomatosis

Human SCs play a primary role in SWN, a rare genetic disorder in which patients develop multiple schwannomas. So that, their isolation and immortalization could represent an irreplaceable tool to investigate the disease etiopathology. Although few clones of tumoural SCs have been obtained, unfortunately they present genetic, morphological and biological characteristics that do not fully represent the original cells. Herein we isolated, characterized and immortalized primary SCs from human schwannomas. Our immortalized human SCs present typical NF2 and LTZR1 genetic mutations of SWN and retain original phenotype characteristics, representing a valuable tool for further genetic, functional and biomolecular in vitro studies.

346 Investigating the potential of incorporating indirect genetic effects into genetic evaluations of dairy calf disease traits

Abstract The goal of infectious disease control is often local eradication, but this is theoretically impossible to achieve based on classical quantitative genetic theory. Current methods focus on the susceptibility of individuals to disease and assume that exposure to a pathogen is 1) constant over time, 2) equal among individuals, and (3) due entirely to the environment. For this reason, it is likely that conventional genetic methods are capturing only a fraction of genetic variation in disease occurrence. The incorporation of epidemiological theory into quantitative genetics provides an opportunity to better determine the level of genetic variation in infectious disease traits. This stems from the ability to include the positive feed-back dynamics of infectious disease transmission. From an epidemiolocal perspective, both susceptibility and infectivity also have indirect genetic effects (IGE), because the genotype of one individual impacts the risk of infection of other individuals, and this can drastically affect the rate and direction of response to selection. Epidemiological models unravel the genetic heterogeneity in both susceptibility and infectivity traits, and account for the impact each animal has on its contemporaries compared with conventional quantitative genetic approaches. From a calf health perspective, there is a shift towards group housing dairy calves, primarily due to welfare and social acceptability. In turn, this will lead to increased animal interactions from a young age. Therefore, the aim of this study was to investigate the potential of incorporating IGE into current quantitative approaches to determine its impact on selection potential. Our study looked at two common infectious diseases in calves on dairy farms: respiratory problems (RESP) and diarrhea (DIAR). Producer-recorded data comprised of 19,445 records collected on 34 herds that group housed calves between 2007 and 2020. Calves were allocated into pen groups based on birth dates and herd specific housing practices. Original phenotypes were split into 10 records, each representing a week of life, to determine when exactly calves became sick and infective. Several scenarios were investigated with respect to the time an animal was infective for following a disease case (1 or 2 wk), and the maximum difference in weeks between animal birth dates to be included within the same pen (2 to 5 wk). Variance components were estimated using a generalized linear mixed model fitting a complementary-log-log link function with offset for exposure. Initial heritability estimates for susceptibility on the observed scale for DIAR ranged from 0.03 to 0.07, and from 0.02 to 0.03 for RESP across scenarios. While heritability estimates for infectivity are currently being investigated, the results for susceptibility highlight the potential for incorporating IGE into genetic evaluation of disease traits and provide an introduction towards incorporating epidemiological theory into classical quantitative approaches to better respect the transmission dynamics of infections.

An Extended Application of the Fast Multi-Locus Ridge Regression Algorithm in Genome-Wide Association Studies of Categorical Phenotypes.

Categorical (either binary or ordinal) quantitative traits are widely observed to measure count and resistance in plants. Unlike continuous traits, categorical traits often provide less detailed insights into genetic variation and possess a more complex underlying genetic architecture, which presents additional challenges for their genome-wide association studies. Meanwhile, methods designed for binary or continuous phenotypes are commonly used to inappropriately analyze ordinal traits, which leads to the loss of original phenotype information and the detection power of quantitative trait nucleotides (QTN). To address these issues, fast multi-locus ridge regression (FastRR), which was originally designed for continuous traits, is used to directly analyze binary or ordinal traits in this study. FastRR includes three stages of continuous transformation, variable reduction, and parameter estimation, and it can computationally handle categorical phenotype data instead of link functions introduced or methods inappropriately used. A series of simulation studies demonstrate that, compared with four other continuous or binary or ordinal approaches, including logistic regression, FarmCPU, FaST-LMM, and POLMM, the FastRR method outperforms in the detection of small-effect QTN, accuracy of estimated effect, and computation speed. We applied FastRR to 14 binary or ordinal phenotypes in the Arabidopsis real dataset and identified 479 significant loci and 76 known genes, at least seven times as many as detected by other algorithms. These findings underscore the potential of FastRR as a very useful tool for genome-wide association studies and novel gene mining of binary and ordinal traits.

Segregation GWAS to linearize a non-additive locus with incomplete penetrance: an example of horn status in sheep

BackgroundThe objective of this study was to introduce a genome-wide association study (GWAS) in conjunction with segregation analysis on monogenic categorical traits. Genotype probabilities calculated from phenotypes, mode of inheritance and pedigree information, are expressed as the expected allele count (EAC) (range 0 to 2), and are inherited additively, by definition, unlike the original phenotypes, which are non-additive and could be of incomplete penetrance. The EAC are regressed on the single nucleotide polymorphism (SNP) genotypes, similar to an additive GWAS. In this study, horn phenotypes in Merino sheep are used to illustrate the advantages of using the segregation GWAS, a trait believed to be monogenic, affected by dominance, sex-dependent expression and likely affected by incomplete penetrance. We also used simulation to investigate whether incomplete penetrance can cause prediction errors in Merino sheep for horn status.ResultsEstimated penetrance values differed between the sexes, where males showed almost complete penetrance, especially for horned and polled phenotypes, while females had low penetrance values for the horned status. This suggests that females homozygous for the ‘horned allele’ have a horned phenotype in only 22% of the cases while 78% will be knobbed or have scurs. The GWAS using EAC on 4001 animals and 510,174 SNP genotypes from the Illumina Ovine high-density (600k) chip gave a stronger association compared to using actual phenotypes. The correlation between the EAC and the allele count of the SNP with the highest –log10(p-value) was 0.73 in males and 0.67 in females. Simulations using penetrance values found by the segregation analyses resulted in higher correlations between the EAC and the causative mutation (0.95 for males and 0.89 for females, respectively), suggesting that the most predictive SNP is not in full LD with the causative mutation.ConclusionsOur results show clear differences in penetrance values between males and female Merino sheep for horn status. Segregation analysis for a trait with mutually exclusive phenotypes, non-additive inheritance, and/or incomplete penetrance can lead to considerably more power in a GWAS because the linearized genotype probabilities are additive and can accommodate incomplete penetrance. This method can be extended to any monogenic controlled categorical trait of which the phenotypes are mutually exclusive.

Balancing the efforts of chart review and gains in PRS prediction accuracy: An empirical study

ObjectivePhenotypic misclassification in genetic association analyses can impact the accuracy of PRS-based prediction models. The bias reduction method proposed by Tong et al. (2019) has demonstrated its efficacy in reducing the effects of bias on the estimation of association parameters between genotype and phenotype while minimizing variance by employing chart reviews on a subset of the data for validating phenotypes, however its improvement of subsequent PRS prediction accuracy remains unclear. Our study aims to fill this gap by assessing the performance of simulated PRS models and estimating the optimal number of chart reviews needed for validation. MethodsTo comprehensively assess the efficacy of the bias reduction method proposed by Tong et al. in enhancing the accuracy of PRS-based prediction models, we simulated each phenotype under different correlation structures (an independent model, a weakly correlated model, a strongly correlated model) and introduced error-prone phenotypes using two distinct error mechanisms (differential and non-differential phenotyping errors). To facilitate this, we used genotype and phenotype data from 12 case-control datasets in the Alzheimer’s Disease Genetics Consortium (ADGC) to produce simulated phenotypes. The evaluation included analyses across various misclassification rates of original phenotypes as well as quantities of validation set. Additionally, we determined the median threshold, identifying the minimal validation size required for a meaningful improvement in the accuracy of PRS-based predictions across a broad spectrum. ResultsThis simulation study demonstrated that incorporating chart review does not universally guarantee enhanced performance of PRS-based prediction models. Specifically, in scenarios with minimal misclassification rates and limited validation sizes, PRS models utilizing debiased regression coefficients demonstrated inferior predictive capabilities compared to models using error-prone phenotypes. Put differently, the effectiveness of the bias reduction method is contingent upon the misclassification rates of phenotypes and the size of the validation set employed during chart reviews. Notably, when dealing with datasets featuring higher misclassification rates, the advantages of utilizing this bias reduction method become more evident, requiring a smaller validation set to achieve better performance. ConclusionThis study highlights the importance of choosing an appropriate validation set size to balance between the efforts of chart review and the gain in PRS prediction accuracy. Consequently, our study establishes a valuable guidance for validation planning, across a diverse array of sensitivity and specificity combinations.

Mice microglia cytokine profile changes under the influence of HSV-1

Introduction. Today, prevalence of neurodegenerative diseases increases. In recent years, more studies have revealed a new knowledge about the role of microglia in the development of these diseases. Animal experiments showed that peripheral inflammation causes activation of microglia in brain. All this points to the essential role of the cells in the development of neurodegeneration. Under the influence of various factors, microglia can change the phenotype and participate in both repair and damage to brain cells. Chronic herpesvirus infection caused by HSV-1 is another known factor in the development of neurodegenerative pathology. However, the exact pathogenetic mechanisms are still unknown, nevertheless, studying the virus effect on microglia has great potential. The goal of our study in this connection was to assess the effect of HSV-1 on microglia polarization in mouse strain with normal susceptibility to this virus and in strain which is more resistant to the action of HSV-1. For this purpose, changes in the cytokine profile were detected. A comparison of interstrain differences in the expression of cytokine genes was also compared in control groups. Materials and methods. The study involved infecting C57BL/6 and BALB/c mice with herpes simplex virus type 1, an isolation of microglia was based on separation steps using a discontinuous gradient density, the cytokine profile was assessed by gene expression levels using a real-time reverse transcription PCR. To calculate the relative fold gene expression of samples the 2–ΔΔCt method was used. Statistical significance was determined using the Mann–Whitney U-test. Results. There were found no interstrain differences in cytokine gene expression in control groups of different mouse strains. At the same time, gene expression differed in the experimental groups: in BALB/c mice, the expression of genes for both pro-inflammatory and anti-inflammatory cytokines increased; in C57BL/6 mice, a slight increase in the expression of IL-1β genes was observed. Conclusion. The data indicate the formation of different microglial phenotypes after HSV-1 infection in different mouse strains. Apparently, in BALB/c mice there is a switch from the pro-inflammatory M1 phenotype of microglia to the anti-inflammatory M2 phenotype, while in C57BL/6 mice the attenuation of the infectious process occurs through a return to the original M0 phenotype.

Clinicopathological analysis of gastric adenocarcinoma with elevated serum alpha-fetoprotein and enteroblastic differentiation

Objective: To investigate the immunophenotypic and molecular biological characteristics of patients with elevated serum alpha-fetoprotein (AFP) and enteroblastic differentiated gastric adenocarcinoma (GAED). Methods: The clinicopathological data of 13 patients with elevated serum AFP and GAED admitted to Shanxi Cancer Hospital from 2018 to 2020 were collected. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were used to analyze the immune markers and molecular biological characteristics of the pathological tissues of the patients. Kaplan-Meier method and log rank test were used for survival analysis. Results: Among the 13 patients with GAED, 12 were male and 1 was female, aged 41-70 years, with a median age of 64 years. The lesions were mainly located in the gastric antrum (5 cases) and gastric body (4 cases). IHC results showed that the tumor embryonic protein (AFP, SALL4, GPC3), intestinal epithelial differentiation protein (CDX-2, CD10), and some original intestinal epithelial phenotype markers (OCT3/4, Claudin6) were expressed in the tumor tissues. Combined application of multiple markers can reduce the rate of missed diagnosis. Among the 13 patients, 12 had at least one mutation (1 mutation: 1 case, 2-5 mutations: 3 cases, 6-15 mutations: 8 cases), and 1 case was not detected. The gene with the highest mutation frequency was TP53 (10 cases), and other mutant genes included EPHB1 (3 cases), ATRX (2 cases), EPHA5 (2 cases), GATA3 (2 cases), LRP1B (2 cases) and MAP2K4 (2 cases) were also detected. Three of the 13 patients had structural variations, which were C14orf177-GNAS, AIM1-FGFR3, and EPHA6-ROS1 gene rearrangements. All 13 patients had copy number variation, and 11 patients had copy number variation of more than 2 genes. The common amplification genes were IRS2 (5 cases), PTEN (5 cases), GNAS (4 cases), CCNE1 (3 cases), CEBPA (3 cases), PCK1 (3 cases) and ERBB2 (2 cases). The common deletion genes were SOX2 (5 cases) and MYC (5 cases). Among the 13 patients, 4 died, and 2 of the dead patients had liver metastasis. There were 4 patients with disease-free survival and 5 patients with disease progression, including 3 cases of abdominal metastasis and 2 cases of liver metastasis. The 3-year survival rate of patients was 65.9 %, and the 3-year progression-free survival rate was 30.7 %. Gene LRP1B point mutation was associated with poor prognosis (P＜0.001). There was no significant improvement in the prognosis of patients treated with immunotherapy compared with those treated with chemotherapy alone (P=0.595), but the prognosis of patients treated with postoperative chemotherapy or postoperative chemotherapy plus immunotherapy was better than that of patients treated with surgery alone (P＜0.05). Conclusions: Elevated serum AFP with GAED is a highly invasive tumor with unique molecular characteristics, often accompanied by multiple molecular events. TP53 mutation is the most common type of gene mutation. In addition, some cases are accompanied by HER2 amplification and gene rearrangement.

Neutrophils disrupt B-1a cell homeostasis by targeting Siglec-G to exacerbate sepsis.

B-1a cells, an innate-like cell population, are crucial for pathogen defense and the regulation of inflammation through their release ofnatural IgM and IL-10. In sepsis, B-1a cell numbers are decreasedin the peritoneal cavity as they robustly migrate to the spleen. Within the spleen, migrating B-1a cells differentiate into plasma cells, leading to alterations in their original phenotype and functionality. We discovered a key player, sialic acid-binding immunoglobulin-like lectin-G (Siglec-G), which is expressed predominantly on B-1a cells and negatively regulates B-1a cell migration to maintain homeostasis. Siglec-G interacts with CXCR4/CXCL12 to modulate B-1a cell migration. Neutrophils aid B-1a cell migration via neutrophil elastase (NE)-mediated Siglec-G cleavage. Human studies revealed increased NEexpression in septic patients. We identified an NE cleavage sequence in silico, leading to the discoveryof a decoy peptide that protects Siglec-G, preserves peritoneal B-1a cells, reduces inflammation, and enhances sepsis survival. The role of Siglec-G in inhibiting B-1a cell migration to maintain their inherent phenotype and function is compromised by NE in sepsis, offering valuable insights into B-1a cell homeostasis. Employing a small decoy peptide to prevent NE-mediated Siglec-G cleavage has emerged as a promising strategy to sustain peritoneal B-1a cell homeostasis, alleviate inflammation, and ultimately improve outcomes in sepsis patients.

LGR5 Modulates Differentiated Phenotypes of Chondrocytes Through PI3K/AKT Signaling Pathway.

Tissue engineering is increasingly viewed as a promising avenue for functional cartilage reconstruction. However, chondrocyte dedifferentiation during in vitro culture remains an obstacle for clinical translation of tissue engineered cartilage. Re-differentiated induction have been employed to induce dedifferentiated chondrocytes back to their original phenotype. Regrettably, these strategies have been proven to be only moderately effective. To explore underlying mechanism, RNA transcriptome sequencing was conducted on primary chondrocytes (P0), dedifferentiated chondrocytes (P5), and redifferentiated chondrocytes (redifferentiation-induction of P5, P5.R). Based on multiple bioinformatics analysis, LGR5 was identified as a target gene. Subsequently, stable cell lines with LGR5 knocking-down and overexpression were established using P0 chondrocytes. The phenotypic changes in P1 and P5 chondrocytes with either LGR5 knockdown or overexpression were assessed to ascertain the potential influence of LGR5 dysregulation on chondrocyte phenotypes. Regulatory mechanism was then investigated using bioinformatic analysis, protein-protein docking, immunofluorescence co-localization and immunoprecipitation. The current study found that dysregulation of LGR5 can significantly impact the dedifferentiated phenotypes of chondrocytes (P5). Upregulation of LGR5 appears to activate the PI3K/AKT signal via increasing the phosphorylation levels of AKT (p-AKT1). Moreover, the increase of p-AKT1 may stabilize β-catenin and enhance the intensity of Wnt/β-catenin signal, and help to restore the dedifferentated phenotype of chondrocytes. LGR5 can modulate the phenotypes of chondrocytes in P5 passage through PI3K/AKT signaling pathway.

ComorbidPGS: An R Package Assessing Shared Predisposition between Phenotypes Using Polygenic Scores

Introduction: Polygenic score (PGS) is a valuable method for assessing the estimated genetic liability to a given outcome or genetic variability contributing to a quantitative trait. While polygenic risk scores are widely used for complex traits, their application in uncovering shared genetic predisposition between phenotypes, i.e., when genetic variants influence more than one phenotype, remains limited. Methods: We developed an R package, comorbidPGS, which facilitates a systematic evaluation of shared genetic effects among (cor)related phenotypes using PGSs. The comorbidPGS package takes as input a set of single nucleotide polymorphisms along with their established effects on the original phenotype (Po), referred to as Po-PGS. It generates a comprehensive summary of effect(s) of Po-PGS on target phenotype(s) (Pt) with customisable graphical features. Results: We applied comorbidPGS to investigate the shared genetic predisposition between phenotypes defining elevated blood pressure (systolic blood pressure, SBP; diastolic blood pressure, DBP; pulse pressure) and several cancers (breast cancer; pancreatic cancer, PanC; kidney cancer, KidC; prostate cancer, PrC; colorectal cancer, CrC) using the European ancestry UK Biobank individuals and GWAS meta-analyses summary statistics from independent set of European ancestry individuals. We report a significant association between elevated DBP and the genetic risk of PrC (β [SE] = 0.066 [0.017], p value = 9.64 × 10−5), as well as between CrC PGS and both, lower SBP (β [SE] = −0.10 [0.029], p value = 3.83 × 10−4) and lower DBP (β [SE] = −0.055 [0.017], p value = 1.05 × 10−3). Our analysis highlights two nominally significant relationships for individuals with genetic predisposition to elevated SBP leading to higher risk of KidC (OR [95% CI] = 1.04 [1.0039–1.087], p value = 2.82 × 10−2) and PrC (OR [95% CI] = 1.02 [1.003–1.041], p value = 2.22 × 10−2). Conclusion: Using comorbidPGS, we underscore mechanistic relationships between blood pressure regulation and susceptibility to three comorbid malignancies. This package offers valuable means to evaluate shared genetic susceptibility between (cor)related phenotypes through polygenic scores.

Imaging Features of Arrhythmogenic Cardiomyopathies.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease characterized by replacement of ventricular myocardium with fibrofatty tissue, predisposing the patient to ventricular arrhythmias and/or sudden cardiac death. Most cases of ACM are associated with pathogenic variants in genes that encode desmosomal proteins, an important cell-to-cell adhesion complex present in both the heart and skin tissue. Although ACM was first described as a disease predominantly of the right ventricle, it is now acknowledged that it can also primarily involve the left ventricle or both ventricles. The original right-dominant phenotype is traditionally diagnosed using the 2010 task force criteria, a multifactorial algorithm divided into major and minor criteria consisting of structural criteria based on two-dimensional echocardiographic, cardiac MRI, or right ventricular angiographic findings; tissue characterization based on endomyocardial biopsy results; repolarization and depolarization abnormalities based on electrocardiographic findings; arrhythmic features; and family history. Shortfalls in the task force criteria due to the modern understanding of the disease have led to development of the Padua criteria, which include updated criteria for diagnosis of the right-dominant phenotype and new criteria for diagnosis of the left-predominant and biventricular phenotypes. In addition to incorporating cardiac MRI findings of ventricular dilatation, systolic dysfunction, and regional wall motion abnormalities, the new Padua criteria emphasize late gadolinium enhancement at cardiac MRI as a key feature in diagnosis and imaging-based tissue characterization. Conditions to consider in the differential diagnosis of the right-dominant phenotype include various other causes of right ventricular dilatation such as left-to-right shunts and variants of normal right ventricular anatomy that can be misinterpreted as abnormalities. The left-dominant phenotype can mimic myocarditis at imaging and clinical examination. Additional considerations for the differential diagnosis of ACM, particularly for the left-dominant phenotype, include sarcoidosis and dilated cardiomyopathy. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

Reactive changes of kisspeptin-producing hypothalamic neuroendocrine cells during hypogonadism and its replacement therapy by the kisspeptin analogue in rats

BACKGROUND: This study is devoted to the morphological substantiation of the model of male hypogonadism and to establishing the effectiveness of its replacement therapy at the level of the central link of the hypothalamic-pituitary-testicular axis using morphological methods. Information about reactive changes in neuroendocrine cells that synthesize the peptide kisspeptin, which regulates the production of gonadoliberin when modeling male and female hypogonadism, has not been described in the literature, which prevents the creation of a micro-morphological basis for the development of models of hypogonadism and the implementation of further preclinical studies of the effectiveness of its replacement therapy. The goal is to carry out a morphological analysis of kisspeptin-producing neuroendocrine cells of the hypothalamus in normal conditions, with experimental hypogonadism and after replacement therapy.
 AIM: To carry out a morphological analysis of kisspeptin-producing neuroendocrine cells of the hypothalamus in normal conditions, with experimental hypogonadism and after replacement therapy.
 MATERIALS AND METHODS: The objects of the study were 3 groups of adult male Wistar rats 6–8 months of age. In animals of the first and second groups, after anesthesia, total ischemia of both testicles was caused by ligating the left and right spermatic cord with the vascular bundle of the testicle for 60 minutes. Rats of the second group, a few minutes after restoration of testicular blood flow, were given replacement therapy by daily administration of a synthetic analogue of kisspeptin KS6 for 7 days. Control animals of the third group were subjected to sham surgery. After 10 days, all animals were sacrificed, their brains were removed and embedded in paraffin. Nissl-stained frontal histological sections of the most massive areas of the kisspeptin-producing nuclei of the hypothalamus-periventricular and arcuate-were examined using the Imagescope program (Electronic Analysis, Russia). The number of cell bodies of viable and dead neurons was counted (under the control of immunohistochemical identification of the caspase-3 antigen), and the area of the body, nucleus and cytoplasm of viable cells was calculated. Statistical processing of the data was carried out using the GraphPad PRISM (USA) program to determine the median, upper and lower quartiles. Differences were considered significant at p 0.01.
 RESULTS: Simulation of acute ischemia caused a significant increase in the number of dead neurons, a slight decrease in the number of viable neurons and a decrease in the area of their cytoplasm in both kisspeptin-producing nuclei. As a result of KS6 replacement therapy, most neuronal cell bodies retained their original phenotype, but the number of dead neurons was high in both experimental groups.
 CONCLUSIONS: Modeling of male hypogonadism using the method of bilateral acute testicular ischemia induces death and partially reversible degenerative changes in kisspeptin-producing neuroendocrine cells of the hypothalamus. Neuropeptide KS6 has a pronounced restorative effect on kisspeptin-producing neurons of the hypothalamus, which is due to its specific activating effect on endocrine cells of all parts of the hypothalamic-pituitary-testicular axis.

Cholangiocyte Organoids: The New Frontier in Regenerative Medicine for the Study and Treatment of Cholangiopathies.

Cholangiopathies include a group of chronic progressive disorders, affecting the cholangiocytes, the epithelial cells that line the biliary tree, leading to liver parenchymal fibrosis and eventually end-stage liver disease necessitating transplantation. Experimental modeling of these multifactorial cholestatic diseases faces challenges due to the lack of adequate experimental in vitro and in vivo models. A novel approach employs three-dimensional organoid systems that offer several advantages for modeling disease and testing drug response in vitro. Organoids mimic intercellular communication, replicate the architecture of organs, and maintain the cell's original phenotype. Cholangiocyte organoids provide an in vitro model to study the pathogenesis and pharmacotherapeutic treatment of cholangiopathies and show great promise for regenerative therapies. In particular, patient-derived organoids allow personalized medicine approaches and the study of individual disease characteristics. This review highlights the significance of cholangiocyte organoid models in advancing our understanding of cholangiopathies and driving advancements in regenerative medicine strategies.

N-Cadherin Targeted Melanin Nanoparticles Reverse the Endothelial-Mesenchymal Transition in Vascular Endothelial Cells to Potentially Slow the Progression of Atherosclerosis and Cancer.

Endothelial-mesenchymal transition (EndoMT) of vascular endothelial cells has recently been considered as a key player in the early progression of a variety of vascular and nonvascular diseases, including atherosclerosis, cancer, and organ fibrosis. However, current strategies attempting to identify pharmacological inhibitors to block the regulatory pathways of EndoMT suffer from poor selectivity, unwanted side effects, and a heterogeneous response from endothelial cells with different origins. Furthermore, EndoMT inhibitors focus on preventing EndoMT, leaving the endothelial cells that have already undergone EndoMT unresolved. Here, we report the design of a simple but powerful nanoparticle system (i.e., N-cadherin targeted melanin nanoparticles) to convert cytokine-activated, mesenchymal-like endothelial cells back to their original endothelial phenotype. We term this process "Reversed EndoMT" (R-EndoMT). R-EndoMT allows the impaired endothelial barriers to recover their quiescence and intactness, with significantly reduced leukocyte and cancer cell adhesion and transmigration, which could potentially stop atheromatous plaque formation and cancer metastasis in the early stages. R-EndoMT is achieved on different endothelial cell types originating from arteries, veins, and capillaries, independent of activating cytokines. We reveal that N-cadherin targeted melanin nanoparticles reverse EndoMT by downregulating an N-cadherin dependent RhoA activation pathway. Overall, this approach offers a different prospect to treat multiple EndoMT-associated diseases by designing nanoparticles to reverse the phenotypical transition of endothelial cells.

DAPredict: a database for drug action phenotype prediction.

The phenotypes of drug action, including therapeutic actions and adverse drug reactions (ADRs), are important indicators for evaluating the druggability of new drugs and repositioning the approved drugs. Here, we provide a user-friendly database, DAPredict (http://bio-bigdata.hrbmu.edu.cn/DAPredict), in which our novel original drug action phenotypes prediction algorithm (Yang,J., Zhang,D., Liu,L. etal. (2021) Computational drug repositioning based on the relationships between substructure-indication. Brief. Bioinformatics, 22, bbaa348) was embedded. Our algorithm integrates characteristics of chemical genomics and pharmacogenomics, breaking through the limitations that traditional drug development process based on phenotype cannot analyze the mechanism of drug action. Predicting phenotypes of drug action based on the local active structures of drugs and proteins can achieve more innovative drug discovery across drug categories and simultaneously evaluate drug efficacy and safety, rather than traditional one-by-one evaluation. DAPredict contains 305 981 predicted relationships between 1748 approved drugs and 454 ADRs, 83 117 predicted relationships between 1478 approved drugs and 178 Anatomical Therapeutic Chemicals (ATC). More importantly, DAPredict provides an online prediction tool, which researchers can use to predict the action phenotypic spectrum of more than 110 000 000 compounds (including about 168 000 natural products) and corresponding proteins to analyze their potential effect mechanisms. DAPredict can also help researchers obtain the phenotype-corresponding active structures for structural optimization of new drug candidates, making it easier to evaluate the druggability of new drug candidates and develop more innovative drugs across drug categories. Database URL: http://bio-bigdata.hrbmu.edu.cn/DAPredict/.

Virus-Specific CD8+ Stem Memory T Lymphocytes Display Enhanced Anti-Virus Responses

Virus specific memory CD8+ T cells can be divided into three subsets, stem memory (T SCM), central (T CM) and effector (T EM), but their lineage relationships and their ability to persist and confer protective immunity against virus infection are not well understood. Our results show that virus-specific CD8+T SCM have a greater capacity than T CM and T EM to persist in vivo and in vitro and are more efficient in mediating protective immunity against CMV infection and EBV infection because of their increased self-renew potential whether in transcriptome or in epigenome. We firstly expanded virus-specific T cells containing CD8+TSCM and CD8+TCM cells in vitro by stimulating healthy human PBMCs with cytokines and CMV (pp65 and IE1) and EBV (LMP2a and EBNA1) virus peptides. Then we fully evaluated the phenotypic and functional differences of diverse virus-specific memory T subsets and found that CD8+TSCM cells are a subset of low exhaustion, high migration and high secretion of IL-2. However, the expression of IFN-γand TNF-a and CD107a were lower compared with TEM cells, although comparable to CD8+TCM cells. We then evaluated the self-renewal and differentiation capacity of CD8+TSCM cells, compared with TCM or TEM cells. The results showed after stimulated with a-CD3/CD2/CD28 for 5 days, that CD8+TSCM cells possess around 50% of CD45RA+CD62L+ subpopulation cells. Meanwhile, CD8+TSCM cells constantly keep a higher expression of CD45RA+ cells than TCM and TEM cells in different proliferation generations. Notably, around 20% of TCM cells,5% of TEM cells and 10% of TEFF cells were found, suggesting that TSCM cells can reconstitute the whole memory T subsets. By contrast, the TCM cells mainly retained a central memory phenotype or differentiated into effector memory T cells and the TEM cells maintained its original phenotype. We also used the stem cell index to evaluate the stemness of different subgroups, and the results confirmed that the stemness evaluation of TSCM cells were significantly higher than TCM and TEM subsets. In vivo rein-fusion of different virus-specific T cell into irradidated NPG mice also showed that CD8+TSCM cells in vivo can differentiate into all memory T subsets in vivo while TCM cells mainly retained TEM and TCM subgroups at 4W post T cell infusion in mouse liver,lung and spleen(Figure 1A-G). To explore whether the antiviral ability of TSCM cells lasted longer than that of TCM and TEM cells. We first evaluated the apoptosis of different T cell subsets stimulated by AD169 virus and found less apoptosis rate was lower than TCM and TEM cells, suggesting persistent virus infection leads to damage to T cells and CD8+TSCM cells have a strong ability to resist virus damage, which may potentially lead to stronger and longer lasting antiviral effects. Based on this, we also evaluated the apoptosis level of virus-infected MRC-5 cells to evaluate whether CD8+TSCM cells exert a stronger anti-virus effect. The result showed that AD-169 infected MRC-5 cells co-incubated with CD8+TSCM had a higher proportion of apoptotic level than CD8+TCM cells. We also used the 5day-long-term infection model in vitro to evaluate the effect of virus infection on T cell differentiation and evaluated the change of virus copy number after long-term co-culture of different cell subsets. We found that sorted CD8+TSCM system can significantly reduce the virus copy number compared with the control group, while there is no significant difference among TCM and TEM cells (Figure1H,). Further analysis into survival ability of T cell subpopulations found that the proportion of CD3+ T cells in TSCM group were significantly higher and the CD8+TSCM group was dominated by TCM and TEM cells after 5-day stimulation but still kept about 22 percentage of TSCM phenotype. (Figure1I). In summary, CD8+ TSCM cells have a sustainable ability against CMV than TCM and TEM. To examine the in vivo activity of virus-specific CD8+TSCM cells. We used Raji-HLA*A0201-pp65-luciferase cells to construct tumor infiltration mouse model. The result showed that in contrast to control mice, we observed a reduction in bioluminescence over three weeks in mice treated with CD8+TSCM cells. Although mice treated with CD8+TCM and CD8+TEM cells retained a lower tumor size in the early two weeks post adoptive T infusion, they showed a rapid tumor progresses and event lead to shorter survival compared with CD8+TSCM group, indicating a sustained anti-CMV effect of CD8+TSCM cells.

Chronic Mitochondrial Dysfunction Renders Acute Myeloid Leukemia Resistant to Multipronged Inhibition of Mitochondrial Metabolism

Acute Myeloid Leukemia (AML) is a genetically diverse and aggressive myeloid neoplasm that awaits novel therapeutics to improve patient survival. Targeting AML metabolic aberrations as a genotype-agnostic strategy has shown promise, evidenced by the clinical success of venetoclax and the rapid pre-clinical development of other mitochondrial (mt) metabolism targeted therapies. However, the mechanism of how AML develops resistance to drugs targeting key mt metabolic pathways is not fully understood. We previously identified sirtuin 5 (SIRT5), a deacylase that regulates several pathways of mt metabolism in AML including oxidative phosphorylation and glutamine metabolism. In AML, it functions as a master metabolic regulator that is necessary for leukemic stem cell survival. Genetic or pharmacological inhibition of SIRT5 leads to rapid AML cell apoptosis in vitro and disease regression in multiple AML mouse models. However, a subset of AML cell lines and primary samples can survive SIRT5 inhibition, indicating an unusual intrinsic resistance to mt dysfunction. To provide a mechanistic explanation, we first compared the molecular features between SIRT5-dependent and independent cases. Among the 25 primary AML samples we tested, NPM1c mutation, but not FLT3-ITD, TP53, WT1, NRAS, or IDH1 mutations, is significantly enriched in SIRT5-independent samples. Accordingly, among the panel of 22 AML cell lines we tested, the only NPM1 mutant cell line (OCI-AML3) sits at the most SIRT5-independent extreme of the spectrum. NPM1 mutant AML cells have increased cytoplasmic mitochondrial DNA (mtDNA) and fragmented mt morphology, indicating chronic mt damage (Wu H et al. Cancer Discovery. 2021). Because SIRT5 exerts its metabolic maintenance functions within the mt, we hypothesize that AML cells that can adapt to chronic, sublethal mt dysfunction will be capable of proliferation without SIRT5. To test this, we took three orthogonal approaches: (1) introducing NPM1c mutation, (2) using low concentrations of ethidium bromide (EtBr) to selectively block mtDNA replication, or (3) genetically inducing chronic mt dysfunction by silencing the essential mt transcription factor TFAM in 3 AML cell lines that are heavily dependent on SIRT5 - OCI-AML2, CMK, and SKM-1. SIRT5-dependent cells expressing ectopic NPM1c exhibit mt dysfunction as evidenced by decreased oxygen consumption rate (OCR) measured using the Seahorse metabolic flux assay. Also, the lethal effect of SIRT5 depletion is rescued in these cells. Pharmacologically blocking NPM1c nuclear export with selinexor in the typically SIRT5-independent OCI-AML3 cells sensitizes them to the lethal effect of SIRT5 depletion. After culturing in 100 ng/mL EtBr for 3, 5, or 11 days, cells had significantly decreased mt abundance as measured by mtDNA content relative to nuclear DNA and had significantly decreased OCR and extracellular acidification rate (ECAR). SIRT5-dependent cells cultured in EtBr survived shRNA-induced SIRT5 depletion, in contrast to the rapid lethality of cells not cultured in EtBr. Additionally, cells initially cultured in EtBr and allowed to recover in the absence of EtBr assume their original phenotype of SIRT5 dependence. However, inhibiting specific subunits of the mt using potent complex I and III inhibitors (rotenone and antimycin A) shows no effect on SIRT5 dependence. Together these data suggest that mt damage, rather than loss of function in one pathway, is needed to convert SIRT5-dependent cells to SIRT5 independence. TFAM, a nuclearly coded transcription factor for mtDNA, is necessary for the replication and transcription of mt genes. We used cas13d-mediated knockdown of TFAM to deplete mtDNA and induce mt dysfunction. Experiments that investigate how this method of inducing mt dysfunction affects OCR, ECAR, and SIRT5-dependent cell line proliferation are currently underway and will be presented at the ASH conference. In summary, we present a mechanism of how NPM1 mutation-associated or experimentally induced chronic sublethal mt dysfunction renders AML resistant to therapies targeting vital mt metabolic pathways, such as SIRT5 inhibitors, demonstrating AML's metabolic flexibility. Experiments are scheduled to validate these findings in primary samples and additional metabolomic and transcriptional studies will be done to deduce the mechanism for SIRT5 dependence in AML.

Proposed diagnostic criteria for arrhythmogenic cardiomyopathy: European Task Force consensus report

Arrhythmogenic cardiomyopathy (ACM) is a heart muscle disease characterized by prominent “non-ischemic” myocardial scarring predisposing to ventricular electrical instability. Diagnostic criteria for the original phenotype, arrhythmogenic right ventricular cardiomyopathy (ARVC), were first proposed in 1994 and revised in 2010 by an international Task Force (TF). A 2019 International Expert report appraised these previous criteria, finding good accuracy for diagnosis of ARVC but a lack of sensitivity for identification of the expanding phenotypic disease spectrum, which includes left-sided variants, i.e., biventricular (ABVC) and arrhythmogenic left ventricular cardiomyopathy (ALVC). The ARVC phenotype together with these left-sided variants are now more appropriately named ACM. The lack of diagnostic criteria for the left ventricular (LV) phenotype has resulted in clinical under-recognition of ACM patients over the 4 decades since the disease discovery. In 2020, the “Padua criteria” were proposed for both right- and left-sided ACM phenotypes. The presently proposed criteria represent a refinement of the 2020 Padua criteria and have been developed by an expert European TF to improve the diagnosis of ACM with upgraded and internationally recognized criteria. The growing recognition of the diagnostic role of CMR has led to the incorporation of myocardial tissue characterization findings for detection of myocardial scar using the late‑gadolinium enhancement (LGE) technique to more fully characterize right, biventricular and left disease variants, whether genetic or acquired (phenocopies), and to exclude other “non-scarring” myocardial disease. The “ring-like’ pattern of myocardial LGE/scar is now a recognized diagnostic hallmark of ALVC. Additional diagnostic criteria regarding LV depolarization and repolarization ECG abnormalities and ventricular arrhythmias of LV origin are also provided. These proposed upgrading of diagnostic criteria represents a working framework to improve management of ACM patients.

Human amniotic membranes as an allogenic biological dressing for the treatment of burn wounds: Protocol for a randomized-controlled study

BackgroundBurn wounds pose significant challenges in medical treatment due to their devastating nature and resource-intensive requirements. Temporary coverage of burn wounds using synthetic or biological dressings allows for reepithelization before definitive skin grafting. Allogenic skin grafts have been widely used but come with drawbacks such as rejection and disease transmission. The use of amniotic membranes (AMs) offers a promising alternative for temporary coverage, as they possess biological properties that promote faster healing and improved scar quality. The various components of the amniotic membrane, including pluripotent stem cells, extracellular matrix proteins, and regenerative factors, contribute to cell growth, migration, and differentiation, as well as preservation of the original epithelial phenotype. ObjectiveReliable information on the treatment of burn wounds with AM is needed. The knowledge gained in this project may help to include this advantageous modern concept of biological dressings in clinical practice. The purpose of this study is to use human amniotic membranes from our in hospital laboratory, as an allogenic biological dressing after enzymatic debridement in superficial partial thickness, deep partial thickness or full thickness burn wounds. MethodsWe will include 30 patients in a randomized-controlled trial with each patient receiving the study intervention and the control intervention. Two 7 × 7 cm burn wound areas will be compared regarding percentage of skin graft take, healing time, healing percentage value and total healing time. Human amniotic membranes will be compared to allogenic skin grafts.

Substitution of self-reported measures for objectively assessed grip strength and slow walk in the Physical Frailty Phenotype: ramifications for validity

BackgroundFrailty assessment promises to identify older adults at risk for adverse consequences following stressors and target interventions to improve health outcomes. The Physical Frailty Phenotype (PFP) is a widely-studied, well validated assessment but incorporates performance-based slow walk and grip strength criteria that challenge its use in some clinical settings. Variants replacing performance-based elements with self-reported proxies have been proposed. Our study evaluated whether commonly available disability self-reports could be substituted for the performance-based criteria in the PFP while still identifying as “frail” the same subpopulations of individuals.MethodsParallel analyses were conducted in 3393 female and 2495 male Cardiovascular Health Study, Round 2 participants assessed in 1989–90. Candidate self-reported proxies for the phenotype’s “slowness” and “weakness” criteria were evaluated for comparable prevalence and agreement by mode of measurement. For best-performing candidates: Frailty status (3 + positive criteria out of 5) was compared for prevalence and agreement between the PFP and mostly self-reported versions. Personal characteristics were compared between those adjudicated as frail by (a) only a self-reported version; (b) only the PFP; (c) both, using bivariable analyses and multinomial logistic regression.ResultsSelf-reported difficulty walking ½ mile was selected as a proxy for the phenotype’s slowness criterion. Two self-reported weakness proxies were examined: difficulty transferring from a bed or chair or gripping with hands, and difficulty as just defined or in lifting a 10-pound bag. Prevalences matched to within 4% between self-reported and performance-based criteria in the whole sample, but in all cases the self-reported prevalence for women exceeded that for men by 11% or more. Cross-modal agreement was moderate, with by-criterion and frailty-wide Kappa statistics of 0.55–0.60 in all cases. Frail subgroups (a), (b), (c) were independently discriminated (p < 0.05) by race, BMI, and depression in women; by age in men; and by self-reported health for both.ConclusionsCommonly used self-reported disability items cannot be assumed to stand in for performance-based criteria in the PFP. We found subpopulations identified as frail by resultant phenotypes versus the original phenotype to systematically differ. Work to develop self-reported proxies that more closely replicate their objective phenotypic counterparts than standard disability self-reports is needed.