Biology subjects

Lange, T.

Publications and source records attributed to Lange, T..

5 recordsLinked to original sources

bioRxiv2022

GAS2 encodes a 2-oxoglutarate dependent dioxygenase involved in ABA catabolism

Liu et al.1 recently reported the characterization of Arabidopsis thaliana GAS2 (Gain of Function in ABA-modulated Seed Germination 2), which was described as an enzyme that catalyzes the stereospecific hydration of GA12 to produce GA12 16, 17-dihydro-16-ol (DHGA12). However, as previously reported2, we did not find conversion of [17-14C]-labeled or [1-,7-,12-,18-14C4]-labeled GA12 by GAS2. Furthermore, the authors1 state the isolation of endogenous DHGA12 from dry Arabidopsis seeds, which we cannot confirm by our attempts to isolate this compound from 0.5 g dry Arabidopsis Col-0 seeds (data not shown). Instead, we present here data showing that the recombinant GAS2 enzyme is able to catabolize abscisic acid (ABA) to phaseic acid (PA) and further to a second product, putative 8-carboxy-ABA (Fig. 1a). O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/516706v1_fig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18fe917org.highwire.dtl.DTLVardef@11006a1org.highwire.dtl.DTLVardef@1235c94org.highwire.dtl.DTLVardef@930b98_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO GAS2 is an ABA catabolizing oxidase. (a) The proposed ABA catabolic pathway catalyzed by GAS2. (b) Metabolism of d6-ABA incubated with 70 l cell-lysate of recombinant ATGA2ox1 and cofactors (negative control, upper lane). Metabolism of d6-ABA incubated with different volumes of cell-lysates containing recombinant GAS2 and cofactors as described in Methods. Chromatograms of characteristic single ions are shown in the first row for ABA (194 Da), in the middle row for PA (125 DA), and in the right row for compound A (194 Da). Products that were identified on the basis of their mass spectra of the methyl ester derivatives are labelled in red. (c) Top lane: Representative mass spectra of the d6-ABA substrate (KRI = 2087), and of its incubation products, PA (KRI = 2141) and compound A (KRI = 2197), by recombinant GAS2 enzyme. Bottom lane: Mass spectra of unlabeled PA substrate (KRI = 2146), and of the incubation product, compound A (KRI = 2201), by recombinant GAS2 enzyme. C_FIG

plant biology↗

bioRxiv2022

Integrated workflow of down-scaled Smart-seq2 and absolute quantitative, single-cell digital PCR validates lack of representability of low-abundant transcripts

Single-cell RNA sequencing (scRNA-seq) can unmask transcriptional heterogeneity facilitating the detection of rare subpopulations at unprecedented resolution. In response to challenges related to coverage and quantity of transcriptome analysis, the lack of unbiased and absolutely quantitative validation methods hampers further improvements. Digital PCR (dPCR) represents such a method as we could show that the inherent partitioning enhances molecular detections by increasing effective mRNA concentrations. We developed a scRT-ddPCR method and validated it using two breast cancer cell lines, MCF7 and BT-474, and bulk methods. ErbB2, a low-abundant transcript in MCF7 cells, suffers from dropouts in scRNA-seq and thus calculated fold changes are biased. Using our scRT-ddPCR, we could improve the detection of ErbB2 and based on the absolute counts obtained we could validate the scRNA-seq fold change. We think this workflow is a valuable addition to the single-cell transcriptomic research toolbox and could even become a new standard in fold change validation because of its reliability, ease of use and increased sensitivity.

molecular biology↗

bioRxiv2021

The distinct role of ALDH1A1 and ALDH1A3 in the regulation of prostate cancer metastases

Cancer stem cells (CSC) are characterized by high self-renewal capacity, tumor-initiating potential, and therapy resistance. Aldehyde dehydrogenase (ALDH)+ cell population serves as an indicator of prostate CSCs with increased therapy resistance, enhanced DNA double-strand break repair, and activated epithelial-mesenchymal transition (EMT) and migration. Numerous ALDH genes contribute to ALDH enzymatic activity; however, only some of them showed clinical relevance. We found that ALDH1A1 and ALDH1A3 genes functionally regulate CSC properties and radiation sensitivity of PCa. We revealed a negative correlation between ALDH1A1 and ALDH1A3 expression in publicly available prostate cancer (PCa) datasets and demonstrated that ALDH1A1 and ALDH1A3 have opposing predictive value for biochemical recurrence-free survival. Our data suggest an association of ALDH1A1 with the metastatic burden, elucidating the role of ALDH genes in the metastatic spread and homing to the bone, which can be, at least partially, attributed to regulating the transforming growth factor beta 1 (TGFB1) and matrix metalloproteinases (MMPs). ALDH genes play a diverse role in PCa development under AR and {beta}-catenin-dependent regulation, with ALDH1A1 becoming dominant in later stages of tumor development when PCa cells gain androgen independence. Taken together, our results indicate that ALDH1A1 and ALDH1A3 modulate PCa radiosensitivity, regulate CSCs phenotype, and spread of PCa cells to the bone, therefore having clinical implication for identifying patients at high risk for progression to metastatic disease.

cancer biology↗

bioRxiv2021

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.

molecular biology↗

bioRxiv2021

Association of stress-related limbic activity and baseline interleukin-6 plasma levels in healthy adults

Several studies suggest a link between acute changes in inflammatory parameters due to an endotoxin or (psychological) stressor and the brains stress response. The extent to which basal circulating levels of inflammatory markers are associated with the brains stress response has been hardly investigated so far. In the present study, baseline plasma levels of the cytokine interleukin (IL)-6 were obtained and linked to neural markers of psychosocial stress using a modified version of the Montreal Imaging Stress Task in a sample of N=65 healthy subjects (N=39 female). Of three a-priori defined regions of interest - the amygdala, anterior insula, and anterior cingulate cortex - baseline IL-6 was significantly and negatively associated with stress-related neural activation in the right amygdala and left anterior insula. Our results suggest that baseline cytokines might be related to differences in the neural stress response and that this relationship could be inverse to that previously reported for induced acute changes in inflammation markers.

neuroscience↗