Mid-term surgical success after transscleral ab interno glaucoma gel stent implantation.

PURPOSE: To investigate the surgical success and efficacy of XEN45 implantation (XEN45 µm, AbbVie Inc., USA) with and without combined cataract surgery up to the first 5 years. METHODS: In a prospective observational monocentric trial, 192 eyes of 157 patients with open-angle glaucoma received either XEN45 implants only (solo surgery group) or combined surgery/cataract surgeries (combined surgery group). Surgical success (qualified and full success; IOP-limit: ≤12, 15, 18, 21 mmHg), time to secondary IOP-lowering procedure, IOP and number of IOP-lowering medications were analysed for 1, 2, 3, 4 and 5 years. RESULTS: Compared to baseline, IOP (24.1 ± 8.1 to 12.6 ± 2.8 mmHg, -48%, p < 0.001) and the number of IOP-lowering medications (3.0 ± 1.0 to 1.5 ± 1.2, -50%, p < 0.001) decreased significantly at 5 years. Although no differences between IOP and the number of IOP-lowering medication courses between the groups were detected at 5 years (p > 0.11), the combined procedure (63%, 37%) showed better success rates compared to the solo procedure (36%, 13%) in the definition IOP ≤18 and ≤12 mmHg (p = 0.035, 0.028). Solo XEN45 procedures had a higher rate of secondary IOP-lowering procedures compared to combined XEN45 cataract procedures (hazard ratio: 2.02, 95%CI: 1.03-3.97, p = 0.04). Twenty per cent of the eyes, including both procedures, required a secondary IOP-lowering procedure within 5 years. CONCLUSIONS: The XEN45 implant is effective in lowering IOP and the number of IOP-lowering medications in patients with open-angle glaucoma in the mid-term. Comparing XEN45 implant results with the results of trabeculectomy available in current literature, we speculate that there might be a higher surgical success rate without medications in favour of trabeculectomy.

Breeding for durable resistance against biotrophic fungal pathogens using transgenes from wheat.

Breeding for resistant crops is a sustainable way to control disease and relies on the introduction of novel resistance genes. Here, we tested three strategies on how to use transgenes from wheat to achieve durable resistance against fungal pathogens in the field. First, we tested the highly effective, overexpressed single transgene Pm3e in the background of spring wheat cultivar Bobwhite in a long-term field trial over many years. Together with previous results, this revealed that transgenic wheat line Pm3e#2 conferred complete powdery mildew resistance during a total of nine field seasons without a negative impact on yield. Furthermore, overexpressed Pm3e provided resistance to powdery mildew isolates from our worldwide collection when crossed into the elite wheat cultivar Fiorina. Second, we pyramided the four overexpressed transgenes Pm3a, Pm3b, Pm3d, and Pm3f in the background of cultivar Bobwhite and showed that the pyramided line Pm3a,b,d,f was completely resistant to powdery mildew in five field seasons. Third, we performed field trials with three barley lines expressing adult plant resistance gene Lr34 from wheat during three field seasons. Line GLP8 expressed Lr34 under control of the pathogen-inducible Hv-Ger4c promoter and provided partial barley powdery mildew and leaf rust resistance in the field with small, negative effects on yield components which might need compensatory breeding. Overall, our study demonstrates and discusses three successful strategies for achieving fungal disease resistance of wheat and barley in the field using transgenes from wheat. These strategies might confer long-term resistance if applied in a sustainable way. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01451-2.

Pyramiding of transgenic immune receptors from primary and tertiary wheat gene pools improves powdery mildew resistance in the field.

Introgression of resistance genes from wild or related species is a common strategy to improve disease resistance of wheat cultivars. Pm17 is a gene that confers powdery mildew resistance in wheat. It encodes an NLR type of immune receptor and was introgressed from rye to wheat as part of the 1RS chromosome arm translocation several decades ago. So far it has not been possible to separate Pm17 from its co-introgressed rye genes due to suppressed recombination. Here we tested in the field transgenic Bobwhite wheat overexpressing Pm17 without any other rye genes. Four transgenic events showed high levels of PM17 protein accumulation, strong powdery mildew resistance, and no pleiotropic effects during three field seasons. We used a combined approach of transgene insertion and cross-breeding to generate lines co-expressing Pm17 and Pm3, or Pm17 and Pm8. Blumeria graminis f. sp. tritici infection tests confirmed additive, race-specific resistance of the two pyramided transgenes in lines Pm17+Pm3b and Pm17+Pm8. Furthermore, pyramided lines showed strong powdery mildew resistance during three field seasons. We conclude that the combination of overexpressed NLR genes from the extended gene pool broadens and diversifies wheat disease resistance.

Inhibition of CysLTR1 reduces the levels of aggregated proteins in retinal pigment epithelial cells.

The endosomal-lysosomal system (ELS), which carries out cellular processes such as cellular waste degradation via autophagy, is essential for cell homeostasis. ELS inefficiency leads to augmented levels of damaged organelles and intracellular deposits. Consequently, the modulation of autophagic flux has been recognized as target to remove damaging cell waste. Recently, we showed that cysteinyl leukotriene receptor 1 (CysLTR1) antagonist application increases the autophagic flux in the retinal pigment epithelial cell line ARPE-19. Consequently, we investigated the effect of CysLTR1 inhibition-driven autophagy induction on aggregated proteins in ARPE-19 cells using flow cytometry analysis. A subset of ARPE-19 cells expressed CysLTR1 on the surface (SE+); these cells showed increased levels of autophagosomes, late endosomes/lysosomes, aggregated proteins, and autophagy as well as decreased reactive oxygen species (ROS) formation. Furthermore, CysLTR1 inhibition for 24 h using the antagonist zafirlukast decreased the quantities of autophagosomes, late endosomes/lysosomes, aggregated proteins and ROS in CysLTR1 SE- and SE+ cells. We concluded that high levels of plasma membrane-localized CysLTR1 indicate an increased amount of aggregated protein, which raises the rate of autophagic flux. Furthermore, CysLTR1 antagonist application potentially mimics the physiological conditions observed in CysLTR1 SE+ cells and can be considered as strategy to dampen cellular aging.

Prehabilitation in older patients prior to elective cardiac procedures (PRECOVERY): study protocol of a multicenter randomized controlled trial.

BACKGROUND: Previous studies have demonstrated the efficacy of rehabilitation after a cardiovascular procedure. Especially older and multimorbid patients benefit from rehabilitation after a cardiac procedure. Prehabilitation prior to cardiac procedures may also have positive effects on patients' pre- and postoperative outcomes. Results of a current meta-analysis show that prehabilitation prior to cardiac procedures can improve perioperative outcomes and alleviate adverse effects. Germany currently lacks a structured cardiac prehabilitation program for older patients, which is coordinated across healthcare sectors. METHODS: In a randomized, controlled, two-arm parallel group, assessor-blinded multicenter intervention trial (PRECOVERY), we will randomize 422 patients aged 75 years or older scheduled for an elective cardiac procedure (e.g., coronary artery bypass graft surgery or transcatheter aortic valve replacement). In PRECOVERY, patients randomized to the intervention group participate in a 2-week multimodal prehabilitation intervention conducted in selected cardiac-specific rehabilitation facilities. The multimodal prehabilitation includes seven modules: exercise therapy, occupational therapy, cognitive training, psychosocial intervention, disease-specific education, education with relatives, and nutritional intervention. Participants in the control group receive standard medical care. The co-primary outcomes are quality of life (QoL) and mortality after 12 months. QoL will be measured by the EuroQol 5-dimensional questionnaire (EQ-5D-5L). A health economic evaluation using health insurance data will measure cost-effectiveness. A mixed-methods process evaluation will accompany the randomized, controlled trial to evaluate dose, reach, fidelity and adaptions of the intervention. DISCUSSION: In this study, we investigate whether a tailored prehabilitation program can improve long-term survival, QoL and functional capacity. Additionally, we will analyze whether the intervention is cost-effective. This is the largest cardiac prehabilitation trial targeting the wide implementation of a new form of care for geriatric cardiac patients. TRIAL REGISTRATION: German Clinical Trials Register (DRKS; http://www.drks.de ; DRKS00030526). Registered on 30 January 2023.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Pericyte-derived cells participate in optic nerve scar formation.

Introduction: Pericytes (PCs) are specialized cells located abluminal of endothelial cells on capillaries, fulfilling numerous important functions. Their potential involvement in wound healing and scar formation is achieving increasing attention since years. Thus, many studies investigated the participation of PCs following brain and spinal cord (SC) injury, however, lacking in-depth analysis of lesioned optic nerve (ON) tissue. Further, due to the lack of a unique PC marker and uniform definition of PCs, contradicting results are published. Methods: In the present study the inducible PDGFRß-P2A-CreERT2-tdTomato lineage tracing reporter mouse was used to investigate the participation and trans-differentiation of endogenous PC-derived cells in an ON crush (ONC) injury model, analyzing five different post lesion time points up to 8 weeks post lesion. Results: PC-specific labeling of the reporter was evaluated and confirmed in the unlesioned ON of the reporter mouse. After ONC, we detected PC-derived tdTomato+ cells in the lesion, whereof the majority is not associated with vascular structures. The number of PC-derived tdTomato+ cells within the lesion increased over time, accounting for 60-90% of all PDGFRß+ cells in the lesion. The presence of PDGFRß+tdTomato- cells in the ON scar suggests the existence of fibrotic cell subpopulations of different origins. Discussion: Our results clearly demonstrate the presence of non-vascular associated tdTomato+ cells in the lesion core, indicating the participation of PC-derived cells in fibrotic scar formation following ONC. Thus, these PC-derived cells represent promising target cells for therapeutic treatment strategies to modulate fibrotic scar formation to improve axonal regeneration.

The Role of Traffic Volume on Sound Pressure Level Reduction before and during COVID-19 Lockdown Measures-A Case Study in Bochum, Germany.

During the SARS-CoV-2 pandemic, sound pressure levels (SPL) decreased because of lockdown measures all over the world. This study aims to describe SPL changes over varying lockdown measure timeframes and estimate the role of traffic on SPL variations. To account for different COVID-19 lockdown measures, the timeframe during the pandemic was segmented into four phases. To analyze the association between a-weighted decibels (dB(A)) and lockdown phases relative to the pre-lockdown timeframe, we calculated a linear mixed model, using 36,710 h of recording time. Regression coefficients depicting SPL changes were compared, while the model was subsequently adjusted for wind speed, rainfall, and traffic volume. The relative adjusted reduction of during pandemic phases to pre-pandemic levels ranged from -0.99 dB(A) (CI: -1.45; -0.53) to -0.25 dB(A) (CI: -0.96; 0.46). After controlling for traffic volume, we observed little to no reduction (-0.16 dB(A) (CI: -0.77; 0.45)) and even an increase of 0.75 dB(A) (CI: 0.18; 1.31) during the different lockdown phases. These results showcase the major role of traffic regarding the observed reduction. The findings can be useful in assessing measures to decrease noise pollution for necessary future population-based prevention.

Topical Diacerein Decreases Skin and Splenic CD11c+ Dendritic Cells in Psoriasis.

Psoriasis is an inflammatory skin disease characterized by increased neo-vascularization, keratinocyte hyperproliferation, a pro-inflammatory cytokine milieu and immune cell infiltration. Diacerein is an anti-inflammatory drug, modulating immune cell functions, including expression and production of cytokines, in different inflammatory conditions. Therefore, we hypothesized that topical diacerein has beneficial effects on the course of psoriasis. The current study aimed to evaluate the effect of topical diacerein on imiquimod (IMQ)-induced psoriasis in C57BL/6 mice. Topical diacerein was observed to be safe without any adverse side effects in healthy or psoriatic animals. Our results demonstrated that diacerein significantly alleviated the psoriasiform-like skin inflammation over a 7-day period. Furthermore, diacerein significantly diminished the psoriasis-associated splenomegaly, indicating a systemic effect of the drug. Remarkably, we observed significantly reduced infiltration of CD11c+ dendritic cells (DCs) into the skin and spleen of psoriatic mice with diacerein treatment. As CD11c+ DCs play a pivotal role in psoriasis pathology, we consider diacerein to be a promising novel therapeutic candidate for psoriasis.

Ageing is associated with a reduction in markers of mitochondrial energy metabolism in the human epidermis.

The decline of mitochondrial function throughout the lifespan is directly linked to the development of ageing phenotypes of the skin. Here, we assessed alterations in markers of epidermal mitochondrial energy metabolism as a function of skin age. Human skin samples from distinct anatomical regions were obtained during routine dermatological surgery from 21 young (27.6 ± 1.71 year) and 22 old (76.2 ± 1.73 year) donors. Sections of skin samples were analysed by immunohistochemistry for mitochondrial subunits of each electron transport chain complex (I-V)/oxidative phosphorylation (OXPHOS), as well as proteins serving as a marker of mitochondrial mass (VDAC1) and the regulation of DNA transcription (TFAM). Staining intensities of ATP5F1A (comprising complex V) and TFAM in the epidermis of older subjects were significantly decreased compared with younger donors. Moreover, these effects were independent of UV exposure of the stained skin section. Overall, we demonstrate that ageing is associated with reduced protein levels of complex V of the mitochondrial respiratory chain and TFAM. These alterations may impair essential mitochondrial functions, exacerbating the cutaneous ageing process.

Cysteinyl leukotriene receptor 1 is a potent regulator of the endosomal-lysosomal system in the ARPE-19 retinal pigment epithelial cell line.

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Navigating chemical reaction space - application to DNA-encoded chemistry.

Databases contain millions of reactions for compound synthesis, rendering selection of reactions for forward synthetic design of small molecule screening libraries, such as DNA-encoded libraries (DELs), a big data challenge. To support reaction space navigation, we developed the computational workflow Reaction Navigator. Reaction files from a large chemistry database were processed using the open-source KNIME Analytics Platform. Initial processing steps included a customizable filtering cascade that removed reactions with a high probability to be incompatible with DEL, as they would e.g. damage the genetic barcode, to arrive at a comprehensive list of transformations for DEL design with applicability potential. These reactions were displayed and clustered by user-defined molecular reaction descriptors which are independent of reaction core substitution patterns. Thanks to clustering, these can be searched manually to identify reactions for DEL synthesis according to desired reaction criteria, such as ring formation or sp3 content. The workflow was initially applied for mapping chemical reaction space for aromatic aldehydes as an exemplary functional group often used in DEL synthesis. Exemplary reactions have been successfully translated to DNA-tagged substrates and can be applied to library synthesis. The versatility of the Reaction Navigator was then shown by mapping reaction space for different reaction conditions, for amines as a second set of starting materials, and for data from a second database.

Minimally Invasive Bimanual Fetal Surgery-A Review.

BACKGROUND: The aim of this review is to discuss experimental and clinical techniques and interventions of fetal surgery which have been performed minimally invasively by the means of a three-port approach for the fetoscope and instruments for the left and right hand of the surgeon (bimanual minimally invasive fetal surgery). METHODS: a print and electronic literature search was performed; the titles and abstracts were screened and included reports were reviewed in a two-step approach. First, reports other than minimally invasive fetal surgery were excluded, then a full text review and analysis of the reported data was performed. RESULTS: 17 reports were included. The heterogeneity of the included reports was high. Although reports on human fetoscopic surgical procedures can be found, most of them do not pick out bimanual fetal surgery as a central theme but rather address interventions applying a fetoscope with a working channel for a laser fiber, needle or flexible instrument. Most reports were on experimentation in animal models, the human application of minimally invasive fetoscopic bimanual surgery is rare and has at best been explored for the prenatal treatment of spina bifida. Some reported bimanual fetoscopic procedures were performed on the exteriorized uterus via a maternal laparotomy and can therefore not be classified as being truly minimally invasive. DISCUSSION: our results demonstrate that minimally invasive fetoscopic bimanual surgery is rare, even in animal models, excluding many other techniques and procedures that are loosely termed 'minimally invasive fetal surgery' which we suggest to better label as 'interventions'. Thus, more research on percutaneous minimally invasive bimanual fetoscopic surgery is warranted, with the aim to reduce the maternal, uterine and fetal trauma for correction of congenital malformations.

Fetal Surgery for Gastroschisis-A Review with Emphasis on Minimally Invasive Procedures.

(1) Background: The morbidity of gastroschisis is defined by exposure of unprotected intestines to the amniotic fluid leading to inflammatory damage and consecutive intestinal dysmotility, the viscero-abdominal disproportion which results in an abdomen too small to incorporate the herniated and often swollen intestine, and by associated pathologies, such as in complex gastroschisis. To prevent intestinal damage and to provide for growth of the abdominal cavity, fetal interventions such as amnio exchange, gastroschisis repair or covering have been evaluated in several animal models and human trials. This review aims to evaluate the reported techniques for the fetal treatment of gastroschisis by focusing on minimally invasive procedures. (2) Methods: We conducted a systematic database search, quality assessment and analyzed relevant articles which evaluate or describe surgical techniques for the prenatal surgical management of gastroschisis in animal models or human application. (3) Results: Of 96 identified reports, 42 eligible studies were included. Fetal interventions for gastroschisis in humans are only reported for EXIT procedures and amnio exchange. In animal models, particularly in the fetal sheep model, several techniques of open or minimally invasive repair of gastroschisis or covering the intestine have been described, with fetoscopic covering being the most encouraging. (4) Discussion: Although some promising minimally invasive techniques have been demonstrated in human application and animal models, most of them are still associated with relevant fetal morbidity and mortality and barely appear to be currently applicable in humans. Further research on specific procedures, instruments and materials is needed before any human application.

Single Point Insulin Sensitivity Estimator in Pediatric Non-Alcoholic Fatty Liver Disease.

Background: Attenuated insulin-sensitivity (IS) is a central feature of pediatric non-alcoholic fatty liver disease (NAFLD). We recently developed a new index, single point insulin sensitivity estimator (SPISE), based on triglycerides, high-density-lipoprotein and body-mass-index (BMI), and validated by euglycemic-hyperinsulinemic clamp-test (EHCT) in adolescents. This study aims to assess the performance of SPISE as an estimation of hepatic insulin (in-)sensitivity. Our results introduce SPISE as a novel and inexpensive index of hepatic insulin resistance, superior to established indices in children and adolescents with obesity. Materials and Methods: Ninety-nine pubertal subjects with obesity (13.5 ± 2.0 years, 59.6% males, overall mean BMI-SDS + 2.8 ± 0.6) were stratified by MRI (magnetic resonance imaging) into a NAFLD (>5% liver-fat-content; male n=41, female n=16) and non-NAFLD (≤5%; male n=18, female n=24) group. Obesity was defined according to WHO criteria (> 2 BMI-SDS). EHCT were used to determine IS in a subgroup (n=17). Receiver-operating-characteristic (ROC)-curve was performed for diagnostic ability of SPISE, HOMA-IR (homeostatic model assessment for insulin resistance), and HIRI (hepatic insulin resistance index), assuming null hypothesis of no difference in area-under-the-curve (AUC) at 0.5. Results: SPISE was lower in NAFLD (male: 4.8 ± 1.2, female: 4.5 ± 1.1) than in non-NAFLD group (male 6.0 ± 1.6, female 5.6 ± 1.5; P< 0.05 {95% confidence interval [CI]: male NAFLD 4.5, 5.2; male non-NAFLD 5.2, 6.8; female NAFLD 4.0, 5.1, female non-NAFLD 5.0, 6.2}). In males, ROC-AUC was 0.71 for SPISE (P=0.006, 95% CI: 0.54, 0.87), 0.68 for HOMA-IR (P=0.038, 95% CI: 0.48, 0.88), and 0.50 for HIRI (P=0.543, 95% CI: 0.27, 0.74). In females, ROC-AUC was 0.74 for SPISE (P=0.006), 0.59 for HOMA-IR (P=0.214), and 0.68 for HIRI (P=0.072). The optimal cutoff-level for SPISE between NAFLD and non-NAFLD patients was 5.18 overall (Youden-index: 0.35; sensitivity 0.68%, specificity 0.67%). Conclusion: SPISE is significantly lower in juvenile patients with obesity-associated NAFLD. Our results suggest that SPISE indicates hepatic IR in pediatric NAFLD patients with sensitivity and specificity superior to established indices of hepatic IR.

Expression of Oxidative Phosphorylation Complexes and Mitochondrial Mass in Pediatric and Adult Inflammatory Bowel Disease.

INTRODUCTION: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a multifactorial intestinal disorder but its precise etiology remains elusive. As the cells of the intestinal mucosa have high energy demands, mitochondria may play a role in IBD pathogenesis. The present study is aimed at evaluating the expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes in IBD. Material and Methods. 286 intestinal biopsy samples from the terminal ileum, ascending colon, and rectum from 124 probands (34 CD, 33 UC, and 57 controls) were stained immunohistochemically for all five OXPHOS complexes and the voltage-dependent anion-selective channel 1 protein (VDAC1 or porin). Expression levels were compared in multivariate models including disease stage (CD and UC compared to controls) and age (pediatric/adult). RESULTS: Analysis of the terminal ileum of CD patients revealed a significant reduction of complex II compared to controls, and a trend to lower levels was evident for VDAC1 and the other OXPHOS complexes except complex III. A similar pattern was found in the rectum of UC patients: VDAC1, complex I, complex II, and complex IV were all significantly reduced, and complex III and V showed a trend to lower levels. Reductions were more prominent in older patients compared to pediatric patients and more marked in UC than CD. CONCLUSION: A reduced mitochondrial mass is present in UC and CD compared to controls. This is potentially a result of alterations of mitochondrial biogenesis or mitophagy. Reductions were more pronounced in older patients compared to pediatric patients, and more prominent in UC than CD. Complex I and II are more severely compromised than the other OXPHOS complexes. This has potential therapeutic implications, since treatments boosting biogenesis or influencing mitophagy could be beneficial for IBD treatment. Additionally, substances specifically stimulating complex I activity should be tested in IBD treatment.

Deviating glucose results in an international dual-center study. A root cause investigation.

During a dual-center study on obese and normal weight children and adolescents, focusing on glucose metabolism, we observed a marked difference in glucose results (N = 16,840) between the two sites, Salzburg, Austria and Uppsala, Sweden (P < 0.001). After excluding differences in patient characteristics between the two populations as cause of this finding, we investigated other preanalytic influences. Finally, only the tubes used for blood collection at the two sites were left to evaluate. While the Vacuette FC-Mix tube (Greiner Bio-One, Kremsmünster, Austria) was used in Uppsala, in Salzburg blood collections were performed with a lithium heparin tube (LH-Monovette, Sarstedt, Germany). To prove our hypothesis, we collected two blood samples in either of these tubes from 51 children (Salzburg N = 27, Uppsala N = 24) and compared the measured glucose results. Indeed, we found the suspected bias and calculated a correction formula, which significantly diminished the differences of glucose results between the two sites (P = 0.023). Our finding is in line with those of other studies and although this issue should be widely known, we feel that it is widely neglected, especially when comparing glucose concentrations across Europe, using large databases without any information on preanalytic sample handling.

Characterization of the Two Inducible Cre Recombinase-Based Mouse Models NG2-CreER™ and PDGFRb-P2A-CreERT2 for Pericyte Labeling in the Retina.

PURPOSE: Pericytes (PCs), located abluminal of endothelial cells on capillaries, are essential for vascular development and stability. They display a heterogeneous morphology depending on organ localization, differentiation state, and function. Consequently, PCs show a diverse gene expression profile, impeding the usage of a unique PC marker and therefore the distinct identification of PCs. Inducible reporter mouse models represent an important tool for investigating the fate of PCs under physiological and pathophysiological conditions. PC-specific expression efficiency of the fluorescence reporter tdTomato following tamoxifen induction was analyzed and compared in two inducible Cre recombinase-expressing mouse models under control of the NG2 and PDGFRb promotor. METHODS: The NG2-CreER™-tdTomato and the PDGFRb-P2A-CreERT2-tdTomato mice were treated with tamoxifen at three defining time points of retinal vascular development: post-natal days (P)5, P10/11/12, and P48/49/50/51. TdTomato reporter induction efficiency was determined by analyzing retinal whole mounts utilizing confocal microscopy, using the antibodies Anti-neural/glial antigen 2 (PCs), Anti-Collagen IV (basement membrane), and Anti-Glutamine Synthetase (Müller glial cells). RESULTS: Tamoxifen induction at the three different time points resulted in PC-specific expression of tdTomato in both reporter models. In the NG2-CreER™-tdTomato mouse, the induction efficiency ranged from 21.9 to 35.5%. In the PDGFRb-P2A-CreERT2-tdTomato mouse, an induction efficiency between 78.9 and 94.1% was achieved. TdTomato expression in the retina was restricted to PCs and vascular smooth muscle cells in the NG2-CreER™-tdTomato mouse, however, in the PDGFRb-P2A-CreERT2-tdTomato mouse, tdTomato was also expressed in Müller glial cells. CONCLUSION: Both reporter mouse models represent promising tools for fate-mapping studies of PCs. While the NG2-CreER™-tdTomato mouse reveals very specific labeling of PCs in the retina, its induction efficiency is lower compared to the PDGFRb-P2A-CreERT2-tdTomato mouse. Although the latter revealed a high percentage of tdTomato-positive PCs in the retina, additional labeling of Müller cells potentially hampers analysis of reporter-positive PCs.

Intranasal Delivery of a Methyllanthionine-Stabilized Galanin Receptor-2-Selective Agonist Reduces Acute Food Intake.

The regulatory (neuro)peptide galanin is widely distributed in the central and peripheral nervous systems, where it mediates its effects via three G protein-coupled receptors (GAL1-3R). Galanin has a vast diversity of biological functions, including modulation of feeding behavior. However, the clinical application of natural galanin is not practicable due to its rapid in vivo breakdown by peptidases and lack of receptor subtype specificity. Much effort has been put into the development of receptor-selective agonists and antagonists, and while receptor selectivity has been attained to some degree, most ligands show overlapping affinity. Therefore, we aimed to develop a novel ligand with specificity to a single galanin receptor subtype and increased stability. To achieve this, a lanthionine amino acid was enzymatically introduced into a galanin-related peptide. The residue's subsequent cyclization created a conformational constraint which increased the peptide's receptor specificity and proteolytic resistance. Further exchange of certain other amino acids resulted in a novel methyllanthionine-stabilized galanin receptor agonist, a G1pE-T3N-S6A-G12A-methyllanthionine[13-16]-galanin-(1-17) variant, termed M89b. M89b has exclusive specificity for GAL2R and a prolonged half-life in serum. Intranasal application of M89b to unfasted rats significantly reduced acute 24 h food intake inducing a drop in body weight. Combined administration of M89b and M871, a selective GAL2R antagonist, abolished the anorexigenic effect of M89b, indicating that the effect of M89b on food intake is indeed mediated by GAL2R. This is the first demonstration of in vivo activity of an intranasally administered lanthipeptide. Consequently, M89b is a promising candidate for clinical application as a galanin-related peptide-based therapeutic.

Chronobiological activity of cysteinyl leukotriene receptor 1 during basal and induced autophagy in the ARPE-19 retinal pigment epithelial cell line.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line. Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy. CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.