Biology subjects

Krawetz, R.

Publications and source records attributed to Krawetz, R..

7 recordsLinked to original sources

bioRxiv2025

Elevated GLUT4 Levels in Human Skeletal Muscle Microtissues is Accompanied by Functional Insulin Dependence

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended [~]3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were [~]12-fold higher (p < 0.0001), leading to a [~]60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach.

cell biology↗

bioRxiv2025

Evaluating gene representation in spatial transcriptomics across pre-designed panels

Spatial transcriptomics (ST) has revolutionized our understanding of gene expression within tissues by preserving spatial context. Over the past few years, this technology has led to a number of paradigm-shifting discoveries that have enabled a more comprehensive understanding of cellular functions and interactions in normal and diseased states. However, ST technologies still face challenges related to resolution, sensitivity, and technical variability. In this study, we evaluate the read coverage of commercialized pre-designed panels using publicly available ST datasets generated from the 10X Genomics and Nanostring platforms. We introduce the Coverage Index (CI) as a quantitative metric to assess the representation of established gene signatures across multiple ST datasets. Our findings reveal that cancer-related gene lists exhibit the highest CI values, while genes encoding for ligands and receptors tend to have low coverage. Additionally, CI analysis can help highlight intrinsic biases in gene panel design, influencing the detection capacity and thereby downstream comprehension of certain biological pathways. The insights gained from this study provide a framework for assessing ST panel performance and optimizing gene panels for future spatial transcriptomic applications.

bioinformatics↗

bioRxiv2024

Cell and Transcriptomic Diversity of Infrapatellar Fat Pad during Knee Osteoarthritis

ObjectivesIn this study, we employ a multi-omic approach to identify major cell types and subsets, and their transcriptomic profiles within the infrapatellar fat pad (IFP), and to determine differences in the IFP based on knee osteoarthritis (KOA), sex, and obesity status. MethodsSingle-nucleus RNA sequencing of 82,924 nuclei from 21 IFPs (n=6 healthy control and n=15 KOA donors), spatial transcriptomics and bioinformatic analysis were used to identify contributions of the IFP to KOA. We mapped cell subclusters from other white adipose tissues using publicly available literature. The diversity of fibroblasts within the IFP was investigated by bioinformatic analyses, comparing by KOA, sex, and obesity status. Metabolomics was used to further explore differences in fibroblasts by obesity status. ResultsWe identified multiple subclusters of fibroblasts, macrophages, adipocytes, and endothelial cells with unique transcriptomic profiles. Using spatial transcriptomics, we resolved distributions of cell types and their transcriptomic profiles, and computationally identified putative cell-cell communication networks. Furthermore, we identified transcriptomic differences in fibroblasts from KOA versus healthy control donor IFPs, female versus male KOA-IFPs, and obese versus normal body mass index (BMI) KOA-IFPs. Finally, using metabolomics, we defined differences in metabolite levels in supernatants of naive, profibrotic- and proinflammatory stimuli-treated fibroblasts from obese compared to normal BMI KOA-IFP. ConclusionsOverall, by employing a multi-omic approach, this study provides the first comprehensive map of cellular and transcriptomic diversity of human IFP and identifies IFP fibroblasts as a key cell type contributing to transcriptomic and metabolic differences related to KOA disease, sex, or obesity.

cell biology↗

bioRxiv2021

The level of synovial human VEGFA, IL-8 and MIP-1α correlate with truncation of lubricin glycans in osteoarthritis

Osteoarthrithis (OA) is an endemic disease due to the increase of the worlds elderly population. Previously thought to be a consequence of an imbalance between cartilage degradation and biosynthesis, it is now recognized as a disease also involving inflammation, hence influencing the level of inflammatory cytokines, growth factors and chemokines. Lubricin is a mucin type molecule where its OA induced glycosylation truncation propels a deteriorating lubrication of the articular cartilage. The objective of this study was to explore the OA driven truncation of O-linked glycosylation of synovial lubricin and its cross talk with systemic and local (synovial fluid, SF) inflammation. We compared the systemic level of cytokines/chemokine in OA patients and controls plasma with their local level in SF using a 44 plex screen. The level of 27 cytokines and chemokines was consistently measured in both plasma and SF. The data showed that the levels of cytokines and chemokines in OA plasma display limited correlation to their counterpart in SF. The level of synovial IL-8 and MIP-1 and VEGFA in OA patients, but not their plasma level, where the only cytokines that displayed a significant correlation to the observed lubricin O-linked glycosylation truncation. These cytokines were also shown to be upregulated exposing fibroblast like synoviocytes from healthy and OA patients to recombinant lubricin with truncated glycans mainly consisting of Tn-antigens, while lubricin with sialylated and non-sialylated T anigens did not have any effect. The data suggest that truncated glycans of lubricin, as found in OA, promotes the synovial cytokine production and exerebate the local synovial inflammation.

pathology↗

bioRxiv2019

Core-2 O-glycans are required for Galectin-3 interaction with the osteoarthritis related protein lubricin

Synovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of the mass) biological lubricant involved in osteoarthritis (OA) development. Lubricin has been reported to be cross-linked by synovial galectin-3 on the lubricating articular surface. Here, we confirm that binding to galectin-3 depended on core-2 O-linked glycans, where surface plasmon resonance of a recombinant lubricin (rhPRG4) devoid of core-2 structures lacked binding capacity to recombinant galectin-3. Both galectin-3 levels and interactions with synovial lubricin were found to be decreased in late-stage OA patients coinciding with an increase of truncated and less sialylated core 1 O-glycans. These data suggest a defect cross-linking of surface active molecules in OA and provides novel insights into OA molecular pathology.

biochemistry↗

bioRxiv2019

Cathepsin G Degrades Synovial Fluid Lubricin: Relevance For Osteoarthritis Pathogenesis

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff in gels, and with proteomics. Full length lubricin ([~]300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain ([~]250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in different domains of lubricin. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the lubrication of OA joints.

pathology↗

bioRxiv2018

Unbiased whole genomes from mammalian feces using fluorescence-activated cell sorting

Ecological flexibility, extended lifespans, and large brains, have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have the largest relative brain size of any monkey and a lifespan that exceeds 50 years, despite their small (3-5 kg) body size. We assemble a de novo reference genome for Cebus imitator and provide the first genome annotation of a capuchin monkey. Through high-depth sequencing of DNA derived from blood, various tissues and feces via fluorescence activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of non-invasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides new insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity. SIGNIFICANCESurviving challenging environments, living long lives, and engaging in complex cognitive processes are hallmark characteristics of human evolution. Similar traits have evolved in parallel in capuchin monkeys, but their genetic underpinnings remain unexplored. We developed and annotated a reference assembly for white-faced capuchin monkeys to explore the evolution of these phenotypes. By comparing populations of capuchins inhabiting rainforest versus dry forests with seasonal droughts, we detected selection in genes associated with kidney function, muscular wasting, and metabolism, suggesting adaptation to periodic resource scarcity. When comparing capuchins to other mammals, we identified evidence of selection in multiple genes implicated in longevity and brain development. Our research was facilitated by our new method to generate high- and low-coverage genomes from non-invasive biomaterials.

genomics↗