Appearance Of Phenotype Research Articles

Technological evaluation and determination of the traits stability in breeding materials in the early generations

The experiment was carried out on an experimental field of Institute of Plant Genetic Resources “K. Malkov”, Sadovo during the period 2016 - 2018. Nineteen common winter wheat lines and variety Sadovo 1 were studied, under the controlled trial conditions. The purpose of the research is technological evaluation and determination of the stability of traits in breeding materials in the early generations. The analyzes were carried out in a laboratory to assess the technological qualities of the grain. For the characterization of the studied lines, the following traits were taken into account: sedimentation value (SV) - using a 2% solution of glacial acetic acid (Pumpyanskiy, 1971); fermentation number FN - Pelshenke test (PT) by mixing a sample with live yeast in two repetitions (Pelshenke et al., 1953). The traits stability of breeding material was evaluated by the stability variances σi2 and Si2 according to Shukla (1972), the equivalency Wi according to Wricke (1962) and the criterion of phenotypic stability (Ysi) according to Kang (1993). The program product IPCSSVKYSI (Interactive program for calculating Shukla’s stability index (Ysi), developed by Kang & Magari (1995) was used to determine the stability index. The highest value for the trait sedimentation value was reported for line RU 32/2072.73.74, followed by line RU 47/3504. With a high value of the trait fermentation number are the lines RU 32/2072.73.74, RU 91/1729; RU 93/1895 and RU 128/2900. The influence of the factors genotype, environment and their interaction on the phenotypic appearance of the studied traits has been significant. The following lines can be highlighted as valuable breeding materials: RU 32/2072,73,74 and RU 47/3504 (high value and stability of the traits of sedimentation value and fermentation number). RU 49/2300 (high value and stability of the trait sedimentation value). RU 91/1729 (high value and stability of the trait fermentation number) The listed genotypes can be successfully used in breeding and improvement work to create new and high-quality common winter wheat varieties.

Sensory Phenotypes Provide Insight Into the Natural Course of Diabetic Polyneuropathy.

The course of diabetic sensorimotor polyneuropathy (DSPN) development, from healthy status to overt DSPN, is poorly understood. We studied the characteristics and longitudinal appearance of lower-extremity sensory phenotypes (healthy, thermal hyperalgesia [TH], mechanical hyperalgesia [MH], and sensory loss [SL]) identified through quantitative sensory testing in individuals with diabetes. There was an increasing severity and patterned order of longitudinal appearance across healthy, TH, MH, and SL phenotypes. SL was most strongly associated with formal DSPN. Our findings provide insight into the natural history of DSPN. Sensory phenotyping can be implemented to identify high-risk individuals and those most likely to benefit from therapeutic interventions.

"Bet hedging" against climate change in developing and adult animals: roles for stochastic gene expression, phenotypic plasticity, epigenetic inheritance and adaptation.

Animals from embryos to adults experiencing stress from climate change have numerous mechanisms available for enhancing their long-term survival. In this review we consider these options, and how viable they are in a world increasingly experiencing extreme weather associated with climate change. A deeply understood mechanism involves natural selection, leading to evolution of new adaptations that help cope with extreme and stochastic weather events associated with climate change. While potentially effective at staving off environmental challenges, such adaptations typically occur very slowly and incrementally over evolutionary time. Consequently, adaptation through natural selection is in most instances regarded as too slow to aid survival in rapidly changing environments, especially when considering the stochastic nature of extreme weather events associated with climate change. Alternative mechanisms operating in a much shorter time frame than adaptation involve the rapid creation of alternate phenotypes within a life cycle or a few generations. Stochastic gene expression creates multiple phenotypes from the same genotype even in the absence of environmental cues. In contrast, other mechanisms for phenotype change that are externally driven by environmental clues include well-understood developmental phenotypic plasticity (variation, flexibility), which can enable rapid, within-generation changes. Increasingly appreciated are epigenetic influences during development leading to rapid phenotypic changes that can also immediately be very widespread throughout a population, rather than confined to a few individuals as in the case of favorable gene mutations. Such epigenetically-induced phenotypic plasticity can arise rapidly in response to stressors within a generation or across a few generations and just as rapidly be "sunsetted" when the stressor dissipates, providing some capability to withstand environmental stressors emerging from climate change. Importantly, survival mechanisms resulting from adaptations and developmental phenotypic plasticity are not necessarily mutually exclusive, allowing for classic "bet hedging". Thus, the appearance of multiple phenotypes within a single population provides for a phenotype potentially optimal for some future environment. This enhances survival during stochastic extreme weather events associated with climate change. Finally, we end with recommendations for future physiological experiments, recommending in particular that experiments investigating phenotypic flexibility adopt more realistic protocols that reflect the stochastic nature of weather.

Origin of CMS-PET1 cytotype in cultivated sunflower: A new insight

The vast majority of commercial sunflower hybrids worldwide are produced using cytoplasmic male sterility (CMS) of the PET1 type, resulting from the interspecific hybridization of Helianthus petiolaris with Helianthus annuus. Due to the fact that CMS-PET1 was not previously detected in wild sunflower, it was believed that this cytotype could arise during interspecific hybridization and is specific solely for cultivated sunflower. In this study, the open reading frame, orfH522, associated with the CMS-PET1 phenotype, was revealed for the first time in the 3′-flanking region of the mitochondrial atpA gene in wild H. annuus. An analysis of whole genome data from 1089 accessions showed that the frequency of occurrence of CMS-orfH522 in wild H. annuus populations is 3.58%, while in wild H. petiolaris populations, it is 1.26%. In general, the analysis demonstrated that PET1-CMS is a natural cytotype of H. annuus, and the appearance of the CMS phenotype in cultivated sunflowers is associated with the loss of stabilizing nuclear genes of fertility restorers, which occurred during interspecific hybridization. These data can explain the patterns of differential cytoplasmic and nuclear introgression occurring in wild sunflower and are useful for further evolutionary studies.

Isolation and culture of chicken bone marrow-derived CD34+ hematopoietic stem and progenitor cells and induced differentiation to myeloid cells

Hematopoietic stem and progenitor cell (HSPC) research will help elucidate the pathogenesis of hematologic diseases. The present study aimed to establish an isolation method and culture system for chicken bone marrow (BM)-derived HSPCs and test their proliferation and differentiation abilities. Mononuclear cells were collected from chicken BM, and CD34+ HSPCs were isolated. Then, the cells were cultured in media with different cytokine compositions, and the growth status, cell phenotype, and morphological appearance of the cells were analyzed at different time points. Our results showed that Iscove’s Modified Dulbecco’s Medium supplemented with 50 ng/mL stem cell factor, 30 ng/mL Flt-3 ligand, 10 μg/mL interleukin 3, 50 ng/mL interleukin 6%, and 10% chicken serum supported chicken CD34+ HSPC survival ex vivo for approximately 10 d. Further, 80 ng/mL granulocyte-colony stimulating factor and 30 ng/mL granulocyte macrophage-colony stimulating factor were added into the above culture system to form a myeloid cell differentiation induction culture system. After culturing in this system for 72 h, approximately 66% of chicken CD34+ HSPCs exhibited a CD11b+ phenotype, indicating that HSPCs differentiated into myeloid cells. In conclusion, chicken BM-derived CD34+ cells possess HSPC characteristics that can self-renew and differentiate into myeloid cells in a culture medium containing growth factors.

Pancreatic Stellate Cells and the Targeted Therapeutic Strategies in Chronic Pancreatitis.

Chronic pancreatitis (CP) is a disease characterized by inflammatory recurrence that accompanies the development of pancreatic fibrosis. As the mystery of CP pathogenesis is gradually revealed, accumulating evidence suggests that the activation of pancreatic stellate cells (PSCs) and the appearance of a myofibroblast-like phenotype are the key gatekeepers in the development of CP. Targeting PSCs to prevent their activation and conversion to a myofibroblast-like phenotype, as well as increasing antioxidant capacity to counteract ongoing oxidative stress, are effective strategies for preventing or treating CP. Therefore, we reviewed the crosstalk between CP and pancreatic fibrosis, summarized the activation mechanisms of PSCs, and investigated potential CP therapeutic strategies targeting PSCs, including, but not limited to, anti-fibrosis therapy, antioxidant therapy, and gene therapy. Meanwhile, the above therapeutic strategies are selected in order to update the available phytopharmaceuticals as novel complementary or alternative approaches for the prevention and treatment of CP to clarify their potential mechanisms of action and their relevant molecular targets, aiming to provide the most comprehensive therapeutic treatment direction for CP and to bring new hope to CP patients.

Acetic acid positively modulates proline metabolism for mitigating PEG-mediated drought stress in Maize and Arabidopsis.

Osmotic imbalance is one of the major consequences of drought stress, negatively affecting plant growth and productivity. Acetic acid has modulatory roles in osmotic balance in plants; however, the mechanistic insights into acetic acid-mediated osmotic adjustment under drought stress remains largely unknown. Here, we investigated how seed priming and seedling root treatment with acetic acid enabled maize plants overcoming polyethylene glycol (PEG)-induced drought effects. Maize seeds primed with acetic acid showed better growth performance when compared with unprimed seeds under PEG application. This growth performance was mainly attributed to improved growth traits, such as fresh weight, dry weight, length of shoots and roots, and several leaf spectral indices, including normalized difference vegetation index (NDVI) and chlorophyll absorption in reflectance index (MCARI). The levels of oxidative stress indicators hydrogen peroxide (H2O2) and malondialdehyde (MDA) did not alter significantly among the treatments, but proline content as well as the expression of proline biosynthetic gene, Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) was significantly elevated in plants receiving acetic acid under PEG-treatments. On the other hand, treating the seedlings root with acetic acid led to a significant recovery of maize plants from drought-induced wilting. Although growth traits remained unchanged among the treatments, the enhancement of leaf water content, photosynthetic rate, proline level, expression of P5CS1, and antioxidant enzyme activities along with reduced level of H2O2 and MDA in acetic acid-supplemented drought plants indicated a positive regulatory role of acetic acid in maize tolerance to drought. Moreover, the high expression of P5CS1 and the subsequent elevation of proline level upon acetic acid application were further validated using wild type and proline biosynthetic mutant p5cs1 of Arabidopsis. Results showed that acetic acid application enabled wild type plants to maintain better phenotypic appearance and recovery from drought stress than p5cs1 plants, suggesting a crosstalk between acetic acid and proline metabolism in plants under drought stress. Our results highlight the molecular and intrinsic mechanisms of acetic acid conferring plant tolerance to drought stress.

Three-dimensional neuroimmune co-culture system for modeling Parkinson’s disease microenvironments in vitro

Parkinson’s disease (PD) is a complex and multifaceted neurodegenerative disorder that results from multiple environmental factors and multicellular interactions. Although several PD neuropathologies have been identified and described, the thorough understanding of PD pathophysiology and research has been largely limited by the absence of reliable in vitro models that truly recapitulate PD microenvironments. Here, we propose a neuroimmune co-culture system that models PD neuropathologies by combining relevant multicellular interactions with environments that mimic the brain. This system is composed of: (i) 3D bioprinted cultures of mature human dopaminergic (DA) neurons grown on extracellular matrix (ECM)-derived scaffolds doped with electroconductive nanostructures, and (ii) a direct co-culture of human astrocytes and differentiated monocytes that models neuroinflammatory responses. When co-cultured in a transwell format, these two compartments recreate relevant multicellular environments that model PD pathologies after exposure to the neurotoxin A53T α-synuclein. With immunofluorescent staining and gene expression analyses, we show that functional and mature DA 3D networks are generated within our ECM-derived scaffolds with superior performance to standard 2D cultures. Moreover, by analyzing cytokine secretion, cell surface markers, and gene expression, we define a human monocyte differentiation scheme that allows the appearance of both monocyte-derived macrophages and dendritic cell phenotypes, as well as their optimal co-culture ratios with human astrocytes to recreate synergistic neuroinflammatory responses. We show that the combined response of both compartments to A53T α-synuclein stimulates the formation of intracellular α-synuclein aggregates, induces progressive mitochondrial dysfunction and reactive oxygen species production, downregulates the expression of synaptic, DA, and mitophagy-related genes, and promotes the initiation of apoptotic processes within the DA networks. Most importantly, these intracellular pathologies were comparable or superior to those generated with a rotenone-stimulated 2D control that represents the current standard for in vitro PD models and showed increased resilience towards these neurotoxic insults, allowing the study of disease progression over longer time periods than current models. Taken together, these results position the proposed model as a superior alternative to current 2D models for generating PD-related pathologies in vitro.

Mild TSC phenotype and non-penetrance associated with a frameshift variant in TSC2 prompts caution in evaluating pathogenicity of frameshift variants

IntroductionTechnological advances in genetic testing, particularly the adoption of noninvasive prenatal screening (NIPS) for single gene disorders such as tuberous sclerosis complex (TSC, OMIM# 613254), mean that putative/possible pathogenetic DNA variants can be identified prior to the appearance of a disease phenotype. Without a phenotype, accurate prediction of variant pathogenicity is crucial. Here, we report a TSC2 frameshift variant, NM_000548.5(TSC2):c.4255_4256delCA, predicted to result in nonsense-mediated mRNA decay (NMD) and cessation of TSC2 protein production and thus pathogenic according to ACMG criteria, identified by NIPS and subsequently detected in family members with few or no symptoms of TSC. Due to the lack of TSC-associated features in the family, we hypothesized that the deletion created a non-canonical 5′ donor site resulting in cryptic splicing and a transcript encoding active TSC2 protein. Verifying the predicted effect of the variant was key to designating pathogenicity in this case and should be considered for other frameshift variants in other genetic disorders. MethodsPhenotypic information on the family members was collected via review of the medical records and patient reports. RNA studies were performed using proband mRNA isolated from blood lymphocytes for RT-PCR and Sanger sequencing. Functional studies were performed by transient expression of the TSC2 variant proteins in cultured cells, followed by immunoblotting. ResultsNo family members harboring the variant met any major clinical diagnostic criteria for TSC, though a few minor features non-specific to TSC were present. RNA studies supported the hypothesis that the variant caused cryptic splicing, resulting in an mRNA transcript with an in-frame deletion of 93 base pairs r.[4255_4256del, 4251_4343del], p.[(Gln1419Valfs*104), (Gln1419_Ser1449del)]. Expression studies demonstrated that the canonical function of the resulting truncated TSC2 p.Gln1419_Ser1449del protein product was maintained and similar to wildtype. ConclusionAlthough most frameshift variants are likely to result in NMD, the NM_000548.5(TSC2):c.4255_4256delCA variant creates a cryptic 5′ splice donor site, resulting in an in-frame deletion that retains TSC2 function, explaining why carriers of the variant do not have typical features of TSC. The information is important for this family and others with the same variant. Equally important is the lesson that predictions can be inaccurate, and that caution should be used when designating frameshift variants as pathogenic, especially when phenotypic information to corroborate testing results is unavailable. Our work demonstrates that functional RNA- and protein-based confirmation of the effects of DNA variants improves molecular genetic diagnostics.

ʟ-glutamic acid modulates antioxidant defense systems and nutrient homeostasis in lentil (Lens culinaris Medik.) under copper toxicity.

Copper (Cu), an essential micronutrient, can generate reactive oxygen species (ROS) at its supra-optimal level in living cells as a transition metal, thus producing oxidative stress in plants. Therefore, protecting plants from Cu-induced oxidative stress via the exogenous application of chemical substances, particularly L-glutamic acid (L-Glu), could be a viable strategy for mitigating the toxicity of Cu. The aim of our present study was to investigate how ʟ-Glu protects lentil seedlings from oxidative stress produced by toxic Cu and allows them to survive under Cu toxicity. The results exhibited that when lentil seedlings were exposed to excessive Cu, their growth was inhibited and their biomass decreased due to an increase in Cu accumulation and translocation to the root, shoot, and leaves. Exposure to toxic Cu also depleted photosynthetic pigments, imbalanced water content, and other essential nutrients, increased oxidative stress, and reduced enzymatic and non-enzymatic antioxidants. However, pre-treatment of ʟ-Glu improved the phenotypic appearance of lentil seedlings, which was distinctly evidenced by higher biomass production, maintenance of water balance, and an increase in photosynthetic pigments when exposed to toxic Cu. ʟ-Glu also protected the seedlings from Cu-induced oxidative stress by reducing the oxidative stress marker, specifically by the efficient action of enzymatic and non-enzymatic antioxidants, particularly ascorbate, catalase, monodehydroascorbate, and glutathione peroxidase and maintaining redox balance. Furthermore, ʟ-Glu assisted in maintaining the homeostasis of Cu and other nutrient in the roots, shoots, and leaves of lentil. Collectively, our results provide evidence of the mechanism of ʟ-Glu-mediated protective role in lentil against Cu toxicity, thus proposed as a potential chemical for managing Cu toxicity not only in lentil but also other plants.

WS07.03 A long-term study of Pseudomonas aeruginosa early patient isolate adaptation to the hypoxic environment

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which causes chronic lung infections in people with cystic fibrosis (CF) and is a major contributor to morbidity and mortality. Despite this, the mechanism driving its adaptation towards chronic colonisation in the CF lung is not yet fully understood. This work focuses on the adaptations of P. aeruginosa to hypoxia, one of the important environmental pressures present in the lung. Long term hypoxia has yet to be examined as a driver of adaptation towards the persistence of this bacterium in CF patients. We have studied the effect of long term adaptation to 6% oxygen for up to 28 days. Interestingly, distinctive colony morphotypes developed throughout the experiments, including small colony variants (SCV). One of the observed morphotypes appeared exclusively under low-oxygen pressure. Importantly, the colonies with visibly different morphologies were more common in hypoxia adapted cultures, comprising up to 98% of the population, while they never exceeded 35% in normoxia adapted cultures. Proteomic analysis showed changes in the abundance of over 700 proteins within 5 days of adaptation, including those involved in respiration and carbon metabolism, quorum sensing and phenazine biosynthesis. Interestingly, comparable changes in several pathways were consistent across both conditions while phenotype differences in motility, pyocyanin production, and protease production between the different colony morphotypes in samples taken over time were observed. Both the SCV and the normal colony variants (NCV) from the hypoxia adapted strains showed decreases in motility, pyocyanin production and protease production. A Congo red assay showed increased exopolysaccharide production in both hypoxia adapted SCV and NCVs. Overall, these results confirm that hypoxia indeed promotes the appearance of many phenotypes in P. aeruginosa that are associated with persistence in the lung and with poor patient outcomes.

CDH12 and the development of the neuronal subtype in MIBC.

e16579 Background: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. Methods: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from > 2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. Results:Enforced expression of CDH12 led to enhanced tumor growth and the appearance of the neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. Conclusions: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process.

Cell surface composition, released polysaccharides, and ionic strength mediate fast sedimentation in the cyanobacterium Synechococcus elongatus PCC 7942.

Cyanobacteria are photosynthetic prokaryotes of high ecological and biotechnological relevance that have been cultivated in laboratories around the world for more than 70 years. Prolonged laboratory culturing has led to multiple microevolutionary events and the appearance of a large number of 'domesticated' substrains among model cyanobacteria. Despite its widespread occurrence, strain domestication is still largely ignored. In this work we describe Synechococcus elongatus PCC 7942-KU, a novel domesticated substrain of the model cyanobacterium S. elongatus PCC 7942, which presents a fast-sedimenting phenotype. Under higher ionic strengths the sedimentation rate increased leading to complete sedimentation in just 12 h. Through whole genome sequencing and gene deletion, we demonstrated that the Group 3 alternative sigma factor F plays a key role in cell sedimentation. Further analysis showed that significant changes in cell surface structures and a three-fold increase in released polysaccharides lead to the appearance of a fast-sedimenting phenotype. This work sheds light on the determinants of the planktonic to benthic transitions and provides genetic targets to generate fast-sedimenting strains that could unlock cost-effective cyanobacterial harvesting at scale.

New insights into the correlation between genotype and cardiac phenotype in laminopathies

Abstract Funding Acknowledgements Type of funding sources: None. Background Genotype-phenotype correlation and risk stratification for heart failure progression in patients with cardiolaminopathies are poorly defined. Purpose The primary aim of the present study is to characterize and correlate phenotype with genotype and sex. The secondary aim is to test the two main existing prognostic scores (the Arbustini and the Whabi score), both developed to predict ventricular arrhythmic events in cardiolaminopathies, for both major ventricular arrhythmias (MVA) and end-stage heart failure (ESHF). Methods This is a retrospective, single-center study, including patients with lamin (LMNA) and/or emerin (EMD) genetic variants. Data were collected from the first medical contact (FMC) to the last follow-up (FU). Results 28 patients (57% male, 82% probands, 68% with a family history of sudden death) were included: 96% harbored LMNA heterozygous variants, 15% had non-missense variants, 11% had variant previously never described. Functionally, 50% were variant of undetermined significance (VUS, C3), the rest were either pathogenetic or likely pathogenic (C4/C5) variants. Median age at FMC was 41 years (IQR 30-55 years), median FU was 11 years (IQR 8-18 years). At FMC, all patients had cardiac involvement, 54% had muscle involvement. By the last FU, 54% developed atrial fibrillation, 43% atrioventricular block (AVB) > I degree, 50% carried an implantable cardioverter defibrillator (21% with resynchronization), 50% had suffered at least one episode of MVA, 25% developed ESHF: 11% died due to refractory HF, 14% underwent heart transplantation (mean age 43.5 ± 9 years). The age of onset of the different types of cardiac phenotype was not influenced by sex. The penetrance of atrioventricular (AV) conduction defects (Figure 1 Panel A) and of atrial tachyarrhythmias (Figure 1 Panel B) was higher and earlier in subjects with non-missense variants. Subjects with VUS, compared to those with pathogenetic/likely pathogenetic variants, showed a significantly lower and later penetrance of MVA (Figure 2 Panel A) and a later penetrance of non- sustained ventricular tachycardia (NSVT, Figure 2, Panel B), but not of AV conduction disturbances, atrial arrhythmias, or contractile dysfunction. Arbustini score at FMC proved to be a predictor of both MVA and ESHF, while Whabi score only predicted MVA. Conclusions In our series, the prevalence and the age of appearance of the different cardiac phenotypes of cardiolaminopathies were not influenced by sex, while the presence of VUS correlated with a lower risk of ventricular arrhythmias, but not of the other cardiac manifestations including contractile disfunction and heart failure. Our data suggest the importance of a close cardiac monitoring and of larger prospective studies even in patients with VUS. All patients developed a sever cardiac phenotype by the age of 60.

Cell-free DNA screening for trisomy 21 in twin pregnancy: a large multicenter cohort study

Analysis of cell-free DNA from maternal blood provides effective screening for trisomy 21 in singleton pregnancies. Data on cell-free DNA screening in twin gestations are promising although limited. In previous twin studies, cell-free DNA screening was primarily performed in the second trimester and many studies did not report chorionicity. This study aimed to evaluate the screening performance of cell-free DNA for trisomy 21 in twin pregnancies in a large, diverse cohort. A secondary aim was to evaluate screening performance for trisomy 18 and trisomy13. This was a retrospective cohort study of twin pregnancies from 17 centers for which cell-free DNA screening was performed from December 2011 to February 2020 by one laboratory using massively parallel sequencing technology. Medical record review was conducted for all newborns and data on the birth outcome, the presence of any congenital abnormalities, phenotypic appearance at birth, and any chromosomal testing that was undertaken in the antenatal or postnatal period were extracted. Cases with a possible fetal chromosomal abnormality with no genetic test results were reviewed by a committee of maternal-fetal medicine geneticists. Cases with a vanishing twin and inadequate follow-up information were excluded. A minimum of 35 confirmed cases of trisomy 21 was required to capture a sensitivity of at least 90% with a prevalence of at least 1.9% with 80% power. Test characteristics were calculated for each outcome. A total of 1764 samples were sent for twin cell-free DNA screening. Of those, 78 cases with a vanishing twin and 239 cases with inadequate follow-up were excluded, leaving a total of 1447 cases for inclusion in the analysis. The median maternal age was 35 years and the median gestational age at cell-free DNA testing was 12.3 weeks. In total, 81% of the twins were dichorionic. The median fetal fraction was 12.4%. Trisomy 21 was detected in 41 of 42 pregnancies, yielding a detection rate of 97.6% (95% confidence interval, 83.8-99.7). There was 1 false negative and no false positive cases. Trisomy 21 was detected in 38 out of 39 dichorionic twin pregnancies, yielding a detection rate of 97.4% (95% confidence interval, 82.6-99.7). Trisomy 18 was detected in 10 of the 10 affected pregnancies. There was 1 false positive case. Trisomy 13 was detected in 4 of the 5 cases, yielding a detection rate of 80% (95% confidence interval, 11.1-99.2). There was one false negative and no false positive cases. The nonreportable rate was low at 3.9 %. Cell-free DNA testing is effective in screening for trisomy 21 in twin gestations from the first trimester of pregnancy. Detection of trisomy 21 was high in dichorionic and monochorionic twins, and the nonreportable result rates were low. This study included high numbers of cases of trisomy 18 and 13 when compared with the current literature. Although screening for these conditions in twins seems to be promising, the numbers were too small to make definitive conclusions regarding the screening efficacy for these conditions. It is possible that cell-free DNA testing performance may differ among laboratories and vary with screening methodologies.

LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC

You have accessJournal of UrologyCME1 Apr 2023LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC Xingyu Chen, Mukta Basu, Simon Knott, and Dan Theodorescu Xingyu ChenXingyu Chen More articles by this author , Mukta BasuMukta Basu More articles by this author , Simon KnottSimon Knott More articles by this author , and Dan TheodorescuDan Theodorescu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003426.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. METHODS: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from >2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. RESULTS: Enforced expression of CDH12 led to enhanced tumor growth and the appearance of a neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. CONCLUSIONS: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process. Source of Funding: CA274375 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1195 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xingyu Chen More articles by this author Mukta Basu More articles by this author Simon Knott More articles by this author Dan Theodorescu More articles by this author Expand All Advertisement PDF downloadLoading ...

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation

Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.

Molecular Mechanisms in the Etiology of Polycystic Ovary Syndrome (PCOS): A Multifaceted Hypothesis Towards the Disease with Potential Therapeutics.

Among the premenopausal women, Polycystic Ovary Syndrome (PCOS) is the most prevalent endocrinopathy affecting the reproductive system and metabolic rhythms leading to disrupted menstrual cycle. Being heterogeneous in nature it is characterized by complex symptomology of oligomennorhoea, excess of androgens triggering masculine phenotypic appearance and/or multiple follicular ovaries. The etiology of this complex disorder remains somewhat doubtful and the researchers hypothesize multisystem links in the pathogenesis of this disease. In this review, we attempt to present several hypotheses that tend to contribute to the etiology of PCOS. Metabolic inflexibility, aberrant pattern of gonadotropin signaling along with the evolutionary, genetic and environmental factors have been discussed. Considered a lifelong endocrinological implication, no universal treatment is available for PCOS so far however; multiple drug therapy is often advised along with simple life style intervention is mainly advised to manage its cardinal symptoms. Here we aimed to present a summarized view of pathophysiological links of PCOS with potential therapeutic strategies.

Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

Human populations are not biologically and genetically discrete.

Human populations are not biologically and genetically discrete.