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Background: 46,XX testicular disorder/difference of sex development (46,XX DSD) is a rare congenital condition, characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of individuals with 46,XX DSD, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment and the genome-wide effects remain elusive., Methods: We performed long-read DNA sequencing, RNA sequencing and DNA methylation analyses on blood samples from 46,XX DSD (n = 11), male controls (46,XY; variable cohort sizes) and female controls (46,XX; variable cohort sizes), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measurements on all 46,XX DSD and a subset of 46,XY (n = 10)., Results: We identified variation in the translocated Y chromosome segments, enabling subcategorization into 46,XX DSD (1) lacking Y chromosome material (n = 1), (2) with short Yp arms (breakpoint at 2.7-2.8 Mb, n = 2), (3) with medium Yp arms (breakpoint at 7.3 Mb, n = 1), and (4) with long Yp arms (n = 7), including deletions of AMELY, TBLY1 and in some cases PRKY. We also identified variable expression of the X-Y homologues PRKY and PRKX. The Y-chromosomal transcriptome and methylome reflected the Y chromosome segment lengths, while changes to autosomal and X-chromosomal regions indicated global effects. Furthermore, transcriptional changes tentatively correlated with phenotypic traits of 46,XX DSD, including reduced height, lean mass and testicular size., Conclusion: This study refines our understanding of the genetic composition in 46,XX DSD, describing the translocated Y chromosome segment in more detail than previously and linking variability herein to genome-wide changes in the transcriptome and methylome., (© 2024. The Author(s).)

Background: Sex chromosome aneuploidies (SCAs) give rise to a broad range of phenotypic traits and diseases. Previous studies based on peripheral blood samples have suggested the presence of ripple effects, caused by altered X chromosome number, affecting the methylome and transcriptome. Whether these alterations can be connected to disease-specific tissues, and thereby having clinical implication for the phenotype, remains to be elucidated., Methods: We performed a comprehensive analysis of X chromosome number on the transcriptome and methylome in blood, fat, and muscle tissue from individuals with 45,X, 46,XX, 46,XY, and 47,XXY., Results: X chromosome number affected the transcriptome and methylome globally across all chromosomes in a tissue-specific manner. Furthermore, 45,X and 47,XXY demonstrated a divergent pattern of gene expression and methylation, with overall gene downregulation and hypomethylation in 45,X and gene upregulation and hypermethylation in 47,XXY. In fat and muscle, a pronounced effect of sex was observed. We identified X chromosomal genes with an expression pattern different from what would be expected based on the number of X and Y chromosomes. Our data also indicate a regulatory function of Y chromosomal genes on X chromosomal genes. Fourteen X chromosomal genes were downregulated in 45,X and upregulated in 47,XXY, respectively, in all three tissues (AKAP17A, CD99, DHRSX, EIF2S3, GTPBP6, JPX, KDM6A, PP2R3B, PUDP, SLC25A6, TSIX, XIST, ZBED1, ZFX). These genes may be central in the epigenetic and genomic regulation of sex chromosome aneuploidies., Conclusion: We highlight a tissue-specific and complex effect of X chromosome number on the transcriptome and methylome, elucidating both shared and non-shared gene-regulatory mechanism between SCAs., (© 2023. The Author(s).)

Background: Offspring born to women with pregestational type 1 diabetes (T1DM) are exposed to an intrauterine hyperglycemic milieu and has an increased risk of metabolic disease later in life. In this present study, we hypothesize that in utero exposure to T1DM alters offspring DNA methylation and gene expression, thereby altering their risk of future disease., Methods: Follow-up study using data from the Epigenetic, Genetic and Environmental Effects on Growth, Metabolism and Cognitive Functions in Offspring of Women with Type 1 Diabetes (EPICOM) collected between 2012 and 2013., Setting: Exploratory sub-study using data from the nationwide EPICOM study., Participants: Adolescent offspring born to women with T1DM (n=20) and controls (n=20) matched on age, sex, and postal code., Main Outcome Measures: This study investigates DNA methylation using the 450K-Illumina Infinium assay and RNA expression (RNA sequencing) of leucocytes from peripheral blood samples., Results: We identified 9 hypomethylated and 5 hypermethylated positions (p < 0.005, |ΔM-value| > 1) and 38 up- and 1 downregulated genes (p < 0.005, log2FC ≥ 0.3) in adolescent offspring born to women with T1DM compared to controls. None of these findings remained significant after correction for multiple testing. However, we identified differences in gene co-expression networks, which could be of biological significance, using weighted gene correlation network analysis. Interestingly, one of these modules was significantly associated with offspring born to women with T1DM. Functional enrichment analysis, using the identified changes in methylation and gene expression as input, revealed enrichment in disease ontologies related to diabetes, carbohydrate and glucose metabolism, pathways including MAPK1/MAPK3 and MAPK family signaling, and genes related to T1DM, obesity, atherosclerosis, and vascular pathologies. Lastly, by integrating the DNA methylation and RNA expression data, we identified six genes where relevant methylation changes corresponded with RNA expression (CIITA, TPM1, PXN, ST8SIA1, LIPA, DAXX)., Conclusions: These findings suggest the possibility for intrauterine exposure to maternal T1DM to impact later in life methylation and gene expression in the offspring, a profile that may be linked to the increased risk of vascular and metabolic disease later in life., (© 2022. The Author(s).)

Background: Quality of life and patient self-determination are key elements in successful palliative care. To achieve these goals, a robust prediction of the remaining survival time is useful as it can provide patients and their relatives with information for individual goal setting including appropriate priorities. The Aim of our study was to assess factors that influence survival after enrollment into ambulatory palliative care., Methods: In this cross-sectional, multicenter study (n = 14 study centers) clinical records of all palliative care patients who were treated in 2017 were extracted and underwent statistical analysis. The main outcome criterion was the association of survival time with clinical characteristics such as age, type of disease, symptoms and performance status., Results: A total of 6282 cases were evaluated. Median time of survival was 26 days (95 % CI: 25-27 days). The strongest association for an increased hazard ratio was found for the following characteristics: moderate/severe weakness (aHR: 1.91; 95 % CI: 1.27-2.86) Karnofsky score 10-30 (aHR: 1.80; 95 % CI: 1.67-1.95), and age > 85 (aHR: 1.50; 95 % CI: 1.37-1.64). Surprisingly, type of disease (cancer vs. non-cancer) was not associated with a change in survival time (aHR: 1.03; 95 % CI: 0.96-1.10)., Conclusions: In this cross-sectional study, the most relevant predictor for a short survival time in specialized ambulatory palliative care was the performance status while type of disease was irrelevant to survival.

Background: Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. Deciphering the specific transcriptomic response of both pollen and stigmatic cells during their interaction constitutes an important challenge to better our understanding of this cell recognition event., Results: Here we describe a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. This strategy allowed us to distinguish 80% of transcripts according to their parental origins. We also developed a tool which predicts male/female specific expression for genes without SNP. We report an unanticipated transcriptional activity triggered in stigma upon incompatible pollination and show that following compatible interaction, components of the pattern-triggered immunity (PTI) pathway are induced on the female side., Conclusions: Our work unveils the molecular signatures of compatible and incompatible pollinations both at the male and female side. We provide invaluable resource and tools to identify potential new molecular players involved in pollen-stigma interaction.

Background: Combating the COVID-19 pandemic is a major challenge for health systems, citizens and policy makers worldwide. Early detection of affected patients within the large and heterogeneous group of patients with common cold symptoms is an important element of this effort, but often hindered by limited testing resources, false-negative test results and the lack of pathognomonic symptoms in COVID-19. Therefore, we aimed to identify anamnestic items with an increased/decreased odds ratio for a positive SARS-CoV-2 PCR (CovPCR) result in a primary care setting., Methods: We performed a multi-center cross-sectional cohort study on predictive clinical characteristics for a positive CovPCR over a period of 4 weeks in primary care patients in Germany., Results: In total, 374 patients in 14 primary care centers received CovPCR and were included in this analysis. The median age was 44.0 (IQR: 31.0-59.0) and a fraction of 10.7% (n = 40) tested positive for COVID-19. Patients who reported anosmia had a higher odds ratio (OR: 4.54; 95%-CI: 1.51-13.67) for a positive test result while patients with a sore throat had a lower OR (OR: 0.33; 95%-CI: 0.11-0.97). Furthermore, patients who had a first grade contact with an infected persons and showed symptoms themselves also had an increased OR for positive testing (OR: 5.16; 95% CI: 1.72-15.51). This correlation was also present when they themselves were still asymptomatic (OR: 12.55; 95% CI: 3.97-39.67)., Conclusions: Several anamnestic criteria may be helpful to assess pre-test probability of COVID-19 in patients with common cold symptoms.

Background: Metachromatic Leukodystrophy (MLD) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in the ARSA gene. While interventional trials often use untreated siblings as controls, the genotype-phenotype correlation is only partly understood, and the variability of the clinical course between siblings is unclear with some evidence for a discrepant clinical course in juvenile patients. The aim of this study was to systematically investigate the phenotypic variation in MLD siblings in comparison to the variability in a larger MLD cohort and to case reports published in literature., Results: Detailed clinical information was available from 12 sibling-pairs (3 late-infantile, 9 juvenile) and 61 single patients (29 late-infantile, 32 juvenile). Variability of age at onset was similar between the siblings and randomly chosen pairs of the remaining cohort (no statistically different Euclidean distances). However, in children with juvenile MLD both the type of first symptoms and the dynamic of the disease were less variable between siblings compared to the general cohort. In late-infantile patients, type of first symptoms and dynamic of disease were similarly homogeneous between siblings and the whole MLD cohort. Thirteen published case reports of families with affected siblings with MLD are presented with similar findings., Conclusions: In a systematic analysis of phenotypic variation in families with MLD, siblings with the late-infantile form showed a similar variability as unrelated pairs of children with late-infantile MLD, whereas siblings with juvenile MLD showed a more homogeneous phenotype regarding type of first symptoms and disease evolution in comparison to unrelated children with juvenile MLD, but not regarding their age at onset. These results are highly relevant with respect to the evaluation of treatment effects and for counseling of families with affected siblings.

Background: Diagnosing dementia, a syndrome affecting 35.6 million people worldwide, can be challenging, especially in patients with a migrant background. Language barriers and language-based diagnostic tools, cultural differences in the perception of the syndrome as well as restricted access to healthcare can influence medical care. For the first time in Germany, this study investigates whether German general practitioners (GPs) feel prepared to meet the diagnostic needs of these patient groups and whether there are challenges and support needs., Methods: A cross-sectional study among a random sample of 982 general practitioners in Germany was conducted from October 2017 to January 2018 (response rate: 34.5%). A self-developed, written, standardised questionnaire was used. Descriptive statistics as well as multiple logistic regression analyses were performed using data of 326 GPs., Results: Ninety-six percent of GPs reported having experienced barriers at least once. Uncertainties in diagnosing dementia in patients with a migrant background were indicated by 70.9%. There was no significant association between uncertainties in diagnosing dementia and GPs' sociodemographic characteristics. The most frequently reported barriers were language barriers that affected or prevented diagnostics (89.3%) and information deficits in patients with a migrant background (59.2%). Shameful interaction or lack of acceptance of the syndrome was also common (55.5%). A demand for more information about the topic was expressed by 70.6% of GPs., Conclusions: Public health measures supporting GPs in their interaction with patients with a migrant background as well as information and services for dementia patients are needed. Efforts to facilitate access to interpreting services and to focus on people with a migrant background in healthcare are necessary., Trial Registration: German Clinical Trials Register: DRKS00012503 , date of registration: 05/09/2017 (German Institute of Medical Documentation and Information. German Clinical Trials Register (DRKS) 2017). Clinical register of the study coordination office of the University hospital of Bonn: ID530, date of registration: 05/09/2017 (Universitätsklinikum Bonn. Studienzentrum. UKB-Studienregister 2017).

Background: Considering the targeted general practitioner-centred healthcare in Germany, general practitioners (GPs) are in the best possible position to increase awareness of all sorts of dementia, an age-related syndrome with rising relevance in the future. In Germany, a doubling of the number of cases from 1.55 million up to 3 million in 2050 is predicted. Diagnostics can be challenging, especially among patients with a migration background. Complicating factors include: Language-based diagnostic tools, cultural differences in handling the syndrome and its underlying diseases as well as a differing use of the healthcare system. Because of missing research in this field in Germany, the type, frequency and intensity of barriers as well as the way GPs cope with them is unknown. That is why it's crucial to focus research on diagnostics in total and especially among this population group., Methods: A cross-sectional study among a random sample of 1000 general practitioners in Germany is conducted in October 2017. A self-administered standardized questionnaire was developed, evaluated and send to the GP practices. A response rate of 30% is expected with one reminder letter. Descriptive statistics as well as, depending on the results, multivariable analyses will be executed. Based on these results and the stated needs, a cluster-randomized intervention study will be constructed to improve healthcare., Discussion: This study is the first in Germany focusing on how dementia diagnostics in general practice is performed, what problems occur, especially because of a migration background of patients, and how GPs cope with them. Depending on the results, it should emphasize the necessity of dementia diagnostics to be adjusted to the needs of the rising amount of people with a migration background (22.5% in Germany, 2016) like concluded from international studies., Trial Registration: German Clinical Trials Register: DRKS00012503 , date of registration: 05.09.2017. Clinical register of the study coordination office of the University hospital of Bonn: ID530 , date of registration: 05.09.2017.

Objective: High-resolution nerve ultrasound (HRUS) is a painless tool to quickly evaluate peripheral nerve morphology in vivo. This study set out to characterize peripheral nerve involvement in X-linked adrenomyeloneuropathy (AMN) by HRUS., Methods: Thirteen adults with genetically proven AMN were examined using the Ultrasound pattern sum score (UPSS) to evaluate morphological abnormalities of peripheral nerves, vagal nerves, as well as cervical nerve roots. Ultrasound results were correlated with clinical findings and nerve conduction studies., Results: UPSS was increased in six out of 13 patients. Nerve enlargement was mostly inhomogeneous and regional. The median, ulnar, and vagal nerves presented with more prominent alterations than nerves of the lower limbs. The proximal-to-distal ratio was significantly enlarged for the median nerve. HRUS findings matched nerve conduction studies, but identified one patient with enlarged nerves and yet normal conduction velocities. Sonographic findings did not correlate with disease duration or disease severity as assessed by the spastic paraplegia rating scale., Conclusion: HRUS reveals significant multifocal regional nerve swellings with reduced echo intensity as the morphological equivalent of electrophysiological peripheral nerve affection in AMN patients. Ultrasound and NCS characteristics in AMN seem to differ from other demyelinating neuropathies like CIDP or CMT1a., Trial Registration: German clinical-trial-register (DRKS) ( DRKS-ID 00005253 ) Registered 15 October 2013.

Background: DNA methylation has been found to associate with disease, aging and environmental exposure, but it is unknown how genome, environment and disease influence DNA methylation dynamics in childhood., Results: By analysing 538 paired DNA blood samples from children at birth and at 4-5 years old and 726 paired samples from children at 4 and 8 years old from four European birth cohorts using the Illumina Infinium Human Methylation 450 k chip, we have identified 14,150 consistent age-differential methylation sites (a-DMSs) at epigenome-wide significance of p < 1.14 × 10 -7 . Genes with an increase in age-differential methylation were enriched in pathways related to 'development', and were more often located in bivalent transcription start site (TSS) regions, which can silence or activate expression of developmental genes. Genes with a decrease in age-differential methylation were involved in cell signalling, and enriched on H3K27ac, which can predict developmental state. Maternal smoking tended to decrease methylation levels at the identified da-DMSs. We also found 101 a-DMSs (0.71%) that were regulated by genetic variants using cis-differential Methylation Quantitative Trait Locus (cis-dMeQTL) mapping. Moreover, a-DMS-associated genes during early development were significantly more likely to be linked with disease., Conclusion: Our study provides new insights into the dynamic epigenetic landscape of the first 8 years of life.

Background: Bitter Melon (BM) has been used as a functional food in traditional Chinese and Indian medicine for many generations and has gained a great deal of attention due to its apparent benefits in moderating some of the pathogenic processes in a variety of inflammatory conditions. BM extract (BME) has been shown to possess strong anti-oxidant properties. In addition, it can ameliorate oxidative stress and potentially ER stress. There is increasing evidence that oxidative and ER stress are major contributors for intestinal secretory cell dysfunction which leads to local inflammation and disease pathogenesis that are hallmarks of inflammatory bowel diseases (IBD). Hence, the search for potential therapeutics against ER stress and oxidative stress in intestinal epithelial secretory cells may provide valuable resources for the management of IBD. The aim of the present study was to investigate the effects of BME in ameliorating ER stress in colonic epithelial cells., Methods: Human colonic adenocarcinoma LS174T cells were used for the assessment of BME effects on colonic epithelial cells in vitro. Cell viability was assessed using trypan blue exclusion and the effect of BME in ameliorating tunicamycin (TM)-induced ER stress was determined by analysing the mRNA expression of the common ER stress markers; ATF6, XBP1, GRP78, CHOP and PERK by quantitative RT-PCR and GRP78 and CHOP by western blot., Results: In the absence of ER stress, BME exhibited no cell toxicity up to 2.0% w/v and no significant effect on the basal mRNA expression of ER stress markers in LS174T cells. In contrast, pre-treatment of LS174T cells with BME followed by induction of ER stress resulted in a significant decrease in mRNA expression of ATF6, XBP1, GRP78, CHOP and PERK and protein expression of GRP78 and CHOP. Co-treatment during induction of ER stress and post- treatment following induction of ER Stress in LS174T cells resulted in a lower but still significant reduction in mRNA expression levels of most ER stress markers., Conclusions: This is one of the first studies demonstrating the efficacy of BME in reducing expression of ER stress markers in colonic epithelial cells suggesting the potential of BME as a dietary intervention in ameliorating ER stress and oxidation in IBD. Interestingly, while the most significant effect was seen with pre-treatment of cells with BME there was a reduced but still significant effect when co-treated or even post-treated. This suggests that BME may even be effective in modulating ER stress in the face of an existing cell stress environment.

Background: In recent years the generation of antibodies by recombinant methods, such as phage display technology, has increased the speed by which antibodies can be obtained. However, in some cases when recombinant antibodies have to be validated, expression in E. coli can be problematic. This primarily occurs when codon usage or protein folding of specific antibody fragments is incompatible with the E. coli translation and folding machinery, for instance when recombinant antibody formats that include the Fc-region are needed. In such cases other expression systems can be used, including the protozoan parasite Leishmania tarentolae (L. tarentolae). This novel host for recombinant protein expression has recently shown promising properties for the expression of single-chain antibody fragments. We have utilised the L. tarentolae T7-TR system to achieve expression and secretion of two scFvs fused to the Fc-region of rabbit immunoglobulin G (IgG)., Results: Based on the commercial vector pLEXSY&#95;IE-blecherry4 (Jena Bioscience; Cat. No. EGE-255), we generated a vector containing the Fragment Crystallisable (Fc) region of rabbit IgG allowing insertions of single chain antibody fragments (scFvs) in frame via Ncol/Notl cloning (pMJ&#95;LEXSY-rFc). For the expression of rabbit Fc-fusion scFvs (scFv-rFc) we cloned two scFvs binding to human vimentin (LOB7 scFv) and murine laminin (A10 scFv) respectively, into the modified vector. The LOB7-rFc and A10-rFc fusions expressed at levels up to 2.95 mg/L in L. tarentolae T7-TR. Both scFv-rFcs were purified from the culture supernatants using protein A affinity chromatography. Additionally, we expressed three different scFvs without the rFc regions using a similar expression cassette, obtaining yields up to 1.00 mg/L., Conclusions: To our knowledge, this is the first time that antibody fragments with intact Fc-region of immunoglobulin have been produced in L. tarentolae. Using the plasmid pMJ&#95;LEXSY-rFc, L. tarentolae T7-TR can be applied as an efficient tool for expression of rFc fusion antibody fragments, allowing easy purification from the growth medium. This system provides an alternative in cases where antibody constructs express poorly in standard prokaryotic systems. Furthermore, in cases where bivalent Fc-fused antibody constructs are needed, using L. tarentolae for expression provides an efficient alternative to mammalian expression.