Biology subjects

Schlueter, R.

Publications and source records attributed to Schlueter, R..

7 recordsLinked to original sources

bioRxiv2026

Exosome-mediated LNA anti-miR delivery achieves potent miR-21 inhibition and PTEN restoration in podocytes.

BackgroundPodocyte injury and loss are central drivers of chronic kidney disease (CKD) progression and are involved in the majority of glomerular diseases. Among the molecular pathways implicated in podocyte dysfunction, microRNA-21 (miR-21) is consistently upregulated during glomerular injury and suppresses protective target genes including PTEN. Although anti-miR-21 approaches have shown high efficacy in preclinical models for Alport Syndrome and diabetic nephropathy, clinical translation of free anti-miR-21 oligonucleotides remains challenging, potentially reflecting limited glomerular and podocyte target engagement. Here, we show that an exosome-mediated delivery of an LNA-miR-21 inhibitor results in a higher intracellular uptake, leading to functional miR-21 inhibition and PTEN restoration in podocytes. MethodsExosomes were isolated from immortalized murine podocytes SVI and were directly loaded with fluorescently-labeled LNA anti-miR-21 (LNA-21) and anti-miR-Ctrl. Exosome integrity was assessed by transmission electron microscopy, dynamic light scattering and Western blot analysis of CD9 and TSG101. Uptake efficiency and intracellular delivery were analyzed by confocal laser-scanning microscopy, time-series imaging (2-48 h) and (imaging) flow cytometry. Functional efficacy was evaluated by TaqMan RT-qPCR and PTEN protein expression in differentiated SVI podocytes, primary murine podocytes and puromycin aminonucleoside (PAN)-injured primary glomeruli. ResultsExosome number, morphology and marker expression remained unchanged after direct LNA-miR-21 loading. Time-series imaging demonstrated progressive intracellular accumulation of exosome-delivered LNA-21 cargo from 2 to 48 h, whereas free LNA-21 showed no detectable uptake. Flow cytometry and imaging flow cytometry confirmed an efficient (76.6%) intracellular delivery exclusively via exosomes. Exosome-mediated LNA-21 delivery induced a robust miR-21 suppression, resulting in an 99% reduction in immortalized podocytes, a 97% reduction in primary podocytes and a 97% reduction in PAN-injured glomeruli (all p < 0.05). In contrast, free LNA-21 did not achieve significant suppression in all investigated models. To evaluate the functional consequences of miR-21 inhibition, we examined PTEN expression, as PTEN is a known direct target of miR-21. Notably, restoration of PTEN protein expression was observed only following exosome-mediated LNA delivery which was confirmed by Western blot and immunofluorescence, suggesting efficient functional inhibition of miR-21 in podocytes. Transfection of scrambled controls confirmed sequence specificity throughout. ConclusionThese findings demonstrate that exosome-mediated LNA delivery overcomes the limited uptake of free inhibitor oligonucleotides in podocytes and enables functional inhibition of miR-21 across multiple experimental systems. These data provide mechanistic proof-of-principle for exosome-based miR inhibitor delivery as a potential strategy to improve podocyte-targeted therapies in CKD.

cell biology↗

bioRxiv2025

The global proteome of Streptococcus pneumoniae EF3030 under nutrient-defined in vitro conditions

Streptococcus pneumoniae is a pathobiont that colonises the upper respiratory tract of humans without causing symptoms but can cause a range of life-threatening diseases including pneumonia, sepsis, and meningitis. It also causes less severe, non-invasive infections such as otitis media and sinusitis. This bacterium thrives in the nasopharynx, where nutrient availability is limited, and has adapted to this environment by developing mechanisms to survive host stress and regulate protein abundance. To study the molecular biology of S. pneumoniae under in vitro and infection-related conditions, a suitable cultivation medium is essential for reproducible experiments. In this study, we optimized a chemically-defined minimal medium that mimics the in vivo nutrient-limited condition and used it for proteome analysis. This optimized medium not only shortened the lag phase but also improved the growth of S. pneumoniae clinical isolates and other streptococcal species. We applied this medium to analyse the global proteome of the pneumococcal colonising strain EF3030, focusing on the transition from the early to late log phase. Our proteomic analysis revealed distinct patterns of protein abundance in different functional categories including metabolism, amino acid synthesis, natural competence, RNA synthesis, cell wall synthesis, protein degradation, and stress responses. Notably, choline-binding protein CbpD, competence factors ComGA and ComEA as well as proteins involved in processing internalized single-stranded DNA (ssDNA) such as Dpr and DprA were higher in abundance in the late log phase. This proteomic profiling provides valuable insights into the pathophysiology of strain S. pneumoniae EF3030 under defined nutrient conditions.

microbiology↗

bioRxiv2024

Efficient delivery of small RNAs to podocytes in vitro by direct exosome transfection.

Podocytes are a crucial component of the glomerular filtration barrier and changes in their 3D structure contribute to over 80% of chronic kidney disease (CKD) cases. Exosomal small RNAs play a key role in cell-cell communication in CKD and may serve as nanocarriers for delivering small RNAs into podocytes. However, the uptake of exosomal cargo by podocytes remains poorly understood. This study explores the use of isolated exosomes, directly transfected with fluorescently labeled small RNAs, for tracking and delivering small RNAs to cultured podocytes. Exosomes were isolated from immortalized murine podocytes and transfected with Cy3-labeled siRNA and miRNA controls using the ExoFect siRNA/miRNA Transfection Kit. We characterized the transfected exosomes via transmission electron microscopy (TEM) and Western blot for exosomal markers CD9 and TSG101. Subsequently, we co-cultured these exosomes with podocytes and used confocal laser-scanning microscopy (cLSM) and structured illumination microscopy (SIM) to visualize cargo uptake. The isolated exosomes were also transfected with pre-miR-21 and filamin A (FlnA)-siRNAs before being co-cultured with podocytes. We confirmed the efficiency of transfection and knockdown using RT-qPCR, Western blotting, and immunofluorescence staining. TEM revealed that the exosomes maintained a consistent shape and size of approximately 20 nm post-transfection and exhibited a stable expression of CD9 and TSG101. Our imaging showed that podocytes take up Cy3-labeled exosomal miRNAs and siRNAs, utilizing various mechanisms, including encapsulation within vesicular structures, endocytosis and free distribution within the cells. Transfection of exosomes with FlnA-siRNAs resulted in a significant 2.8-fold reduction of FlnA expression in co-cultured podocytes, while pre-miR-21-transfected exosomes led to a remarkable 338-fold increase in mature miR-21 levels. These findings demonstrate that direct exosome transfection with fluorescently labeled small RNAs is an effective tool for exosomal cargo tracking in podocytes. This study is the first to show that directly transfected exosomes can deliver small RNAs to podocytes in vitro, suggesting their potential as RNA carriers for therapeutic strategies in more complex settings.

cell biology↗

bioRxiv2020

Metabolic differences between symbiont subpopulations in the deep-sea tubeworm Riftia pachyptila

The hydrothermal vent tube worm Riftia pachyptila lives in intimate symbiosis with intracellular sulfur-oxidizing gammaproteobacteria. Although the symbiont population consists of a single 16S rRNA phylotype, bacteria in the same host animal exhibit a remarkable degree of metabolic diversity: They simultaneously utilize two carbon fixation pathways and various energy sources and electron acceptors. Whether these multiple metabolic routes are employed in the same symbiont cells, or rather in distinct symbiont subpopulations, was unclear. As Riftia symbionts vary considerably in cell size and shape, we enriched individual symbiont cell sizes by density gradient centrifugation in order to test whether symbiont cells of different sizes show different metabolic profiles. Metaproteomic analysis and statistical evaluation using clustering and random forests, supported by microscopy and flow cytometry, strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. Large symbionts, on the other hand, apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis in large symbionts indicates that in this late differentiation stage the symbionts metabolism is efficiently geared towards the production of organic material. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

microbiology↗

bioRxiv2019

Placenta microbiome diversity is associated with maternal pre-pregnancy obesity and placenta biogeography

Recently there has been considerable debate in the scientific community regarding the placenta as the host of a unique microbiome. No studies have addressed the associations of clinical conditions such as maternal obesity, or localizations on the placental microbiome. We examined the placental microbiome in a multi-ethnic maternal pre-pregnant obesity cohort using controls for environmental contaminants and an optimized microbiome protocol to enrich low bacterial biomass samples. We confirmed that a distinct placenta microbiome does exist, as compared to the environmental background. The placenta microbiome consists predominantly of Lactobacillus, Enterococcus and Chryseobacterium. Moreover, the microbiome in the placentas of obese pre-pregnant mothers are less diverse when compared to those of mothers of normal pre-pregnancy weight. Lastly, microbiome richness also decreases from the maternal side to fetal side. In summary, our study reveals associations of placental microbiome with placenta biogeography and with maternal pre-pregnant obesity.

microbiology↗

bioRxiv2019

Host-microbe interactions in the chemosynthetic Riftia pachyptila symbiosis

The deep-sea tubeworm Riftia pachyptila lacks a digestive system, but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which enabled comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. We demonstrate that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion, and substrate transfer to the symbiont by abundant host proteins. Our analysis further suggests that Riftia maintains its symbiont by protecting the bacteria from oxidative damage, while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.

systems biology↗

bioRxiv2018

Metabolomics profile of umbilical cord blood is associated with maternal pre-pregnant obesity in a prospective multi-ethnic cohort displaying health disparities

Maternal obesity has become a growing global health concern that may predispose the offspring to medical conditions later in life. However, the metabolic link between maternal pre-pregnant obesity and healthy offspring has not yet been fully elucidated. In this study, we conducted a case-control study using coupled untargeted and targeted metabolomics approach, from the newborn cord blood metabolomes associated with a matched maternal pre-pregnant obesity cohort of 28 cases and 29 controls. The subjects were recruited from multi-ethnic populations in Hawaii, including rarely reported Native Hawaiian and other Pacific Islanders (NHPI). We found that maternal obesity was the most important factor contributing to differences in cord blood metabolomics. Using elastic net regularization based logistic regression model, we identified 29 metabolites as potential early-life biomarkers manifesting intrauterine effect of maternal obesity, with accuracy as high as 0.947 after adjusting for clinical confounding (maternal and paternal age and ethnicity, parity and gravidity). We validated the model results in a subsequent set of samples (N=30) with an accuracy of 0.822. Among the metabolites, six metabolites (galactonic acid, butenylcarnitine, 2-hydroxy-3-methylbutyric acid, phosphatidylcholine diacyl C40:3, 1,5-anhydrosorbitol, and phosphatidylcholine acyl-alkyl 40:3) were individually and significantly different between the maternal obese vs. norm-weight groups. Interestingly, Hydroxy-3-methylbutyric acid showed significnatly higher levels in cord blood from the NHPI group, compared to asian and caucasian groups. In summary, significant associations were observed between maternal pre-pregnant obesity and offspring metabolomics alternation at birth, revealing the inter-generational impact of maternal obesity.

bioinformatics↗