Biology subjects

Muller, K.

Publications and source records attributed to Muller, K..

26 records · Page 2Linked to original sources

bioRxiv2022

Combination drug screen targeting glioblastoma core vulnerabilities reveals pharmacological synergisms

Synergistic drug combinations are an attractive anticancer strategy but prove challenging to identify. Here we present a stepwise approach consisting in revealing core cancer vulnerabilities and exploiting them through drug combination screen to uncover synergistic treatments for glioblastoma patients. MethodsWe established an innovative method, based on high-throughput screening, target deconvolution and functional genomics, to reveal core vulnerabilities in glioblastoma. Combination drug screen targeting these vulnerabilities was then designed to unveil synergistic associations. The therapeutic potential of the top drug combination was validated in two different clinically-relevant models: an organotypic ex vivo model and a syngeneic orthotopic mouse model of glioblastoma. ResultsLarge-scale monotherapy drug screening identified 83 potent anti-glioblastoma compounds. Target deconvolution using public chemoinformatic databases uncovered 1,100 targets and interactors of the hit compounds. Screening of a focused siRNA library targeting the top 292 drug interactors revealed 22 targetable vulnerabilities, 9 of which were confirmed as core glioblastoma vulnerabilities by mining the CRISPR screen cohort data from the online Cancer Dependency Map portal. Six selective inhibitors of the core vulnerabilities were then screened in combination with a custom-made library of 88 compounds and synergies amongst the 528 tested pairwise combinations were predicted. The combinations of CHK1 / MEK and AURKA / BET inhibitors were highlighted and validated in 3D tumor spheroids. Using an organotypic ex vivo model and a syngeneic orthotopic mouse model, we definitively ascertained the efficacy of dual AURKA / BET inhibition in glioblastoma. ConclusionsCollectively, we uncovered that dual inhibition of BET proteins and aurora kinase A is highly synergistic against GBM. Moreover, our study indicates that our approach to exploit drug poly-pharmacology for the rational design of drug combination screens represent a valuable strategy to discover synergistic treatments against refractory cancers.

pharmacology and toxicology↗

bioRxiv2022

Fusion-negative Rhabdomyosarcoma 3D-organoids as an innovative model to predict resistance to cell death inducers

Rhabdomyosarcoma (RMS) is the main form of soft-tissue sarcoma in children and adolescents. For 20 years, and despite international clinical trials, its cure rate has not really improved, and remains stuck at 20% in case of relapse. The definition of new effective therapeutic combinations is hampered by the lack of reliable models, which complicate the transposition of promising results obtained in pre-clinical studies into efficient solutions for young patients. Inter-patient heterogeneity, particularly in the so-called fusion-negative group (FNRMS), adds an additional level of difficulty in optimizing the clinical management of children and adolescents with RMS. Here, we describe an original 3D-organoid model derived from relapsed FNRMS and show that it finely mimics the characteristics of the original tumor, including inter- and intra-tumoral heterogeneity. Moreover, we have established the proof-of-concept of their preclinical potential by re-evaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on the exploitation of bulk and single-cell omics data.

cancer biology↗

bioRxiv2022

RaPiD-chamber: Easy to self-assemble live-imaging chamber with adjustable LEDs allows to track small differences in dynamic plant movement adaptation on tissue level

Plants rely on fine-tuning organ movement to ensure their survival and productivity. Even subtle loss of directional growth orchestration can result in a huge impact when the plant is impaired to adapt to an ever-changing environment, where it is exposed to manifold exogenous stimuli simultaneously. We present a newly designed chamber to obtain live images to track organ growth and movement differences, called RaspberyPi Dark Chambers (RaPiD-chamber). The RaPiD-chamber is easy to self-assemble and cost-efficient and allows to monitor the continuous growth of etiolated seedlings, as well as their response to light of different wavelengths and from chosen positions. We tested the advice by comparing hypocotyl elongation rate and response to unilateral white and blue light exposure of Arabidopsis thaliana Col0. Additionally, we compared the elongation rate of etiolated hypocotyls between Col0 and kin10, a mutant lacking the catalytic subunit of the cellular signaling hub SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1). kin10 is known for its diminished ability to control hypocotyl elongation. As a case study, we compared the growth dynamics of etiolated Col0 versus kin10. Without further energy source supplementation to the growth medium, the mutant cannot keep up with hypocotyl elongation. Additionally, continuous observation of the dark-grown seedlings allowed us to determine a shift in the dynamics of apical hook angle formation for the mutant.

plant biology↗

bioRxiv2021

Total number and ratio of GABAergic neuron types in the mouse lateral and basal amygdala

GABAergic neurons are key circuit elements in cortical networks. In spite of growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types have been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22 %) is significantly higher than in the LA (16 %) in both male and female mice. No difference was observed between the right and left hemispheres in either sexes. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5-6 %), basket cells expressing parvalbumin (17-20 %) or cholecystokinin (7-9 %), dendrite-targeting inhibitory cells expressing somatostatin (10-16 %), NPY-containing neurogliaform cells (14-15 %), VIP and/or calretinin-expressing interneuron-selective interneurons (29-38 %) and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (nNOS, 5.5-8 %). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathological conditions, and to model functioning amygdalar networks in health and disease. Significance statementGABAergic cells in cortical structures, like in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyper-excitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions including anxiety, posttraumatic stress disorder, schizophrenia and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathological brain states.

neuroscience↗

bioRxiv2020

A role of DAO1 in oxidation of IAA amino acid conjugates revealed through metabolite, high throughput transcript and protein level analysis.

Auxin metabolism is, together with auxin transport, a key determinant of auxin signalling output in plant cells, yet details on the underlying mechanisms and factors involved are still largely unknown. Processes involved in the auxin metabolism are subject to regulation based on numerous signals, including auxin concentration itself. Altered auxin availability and the subsequent changes of auxin metabolite profiles can therefore elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. After analysing auxin metabolism in auxin dependent tobacco BY-2 cell line grown in presence or absence of synthetic auxin 2,4-D we found that both conditions were similarly characterized by very low levels of endogenous indole-3-acetic acid (IAA) and its metabolites. However, metabolic profiling after exogenous application of IAA uncovered that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundantly formed auxin metabolite in the control culture, dramatically decreased in auxin-starved conditions. To describe the molecular mechanism behind this regulation, we analysed transcriptome and proteome changes caused by auxin starvation. While no changes in the expression of auxin biosynthetic machinery were observed, many genes related to auxin conjugation and degradation showed differential expression. Selected putative auxin glycosylating enzymes as well as members of the Gretchen Hagen 3 gene family involved in auxin amino acid conjugation showed both up- and down-regulation. Contrarily to that, all tobacco homologs of Arabidopsis thaliana DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), known to be responsible for the formation of oxIAA from IAA, showed significant downregulation at both transcript and protein levels. To validate the role of DAO1 in auxin metabolism, we performed auxin metabolite profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis thaliana plants. Both mutants showed not only expectedly lower levels of oxIAA, but also significantly lower abundance of oxidated amino acid conjugates of IAA (oxIAA-Asp). Our results thus represent the first direct evidence on DAO1 activity on IAA amino acid conjugates. Statement of significanceHere we present an analysis of auxin metabolism on metabolite, transcript and protein levels in tobacco BY-2 cell line, collectively identifying oxidation of IAA amino acid conjugates as a new role of DIOXYGENASE FOR AUXIN OXIDATION 1 within an auxin-level-responsive metabolic system.

plant biology↗

bioRxiv2020

Formation of flavone-based wooly fibres by glandular trichomes of Dionysia tapetodes.

Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "wooly" farina. This contrasts with some related Primula which instead possess a powdery farina. Using a combination of cell biology and analytical chemical techniques, we provide a detailed insight of wooly farina formation by glandular trichomes that produce a mixture of flavone and substituted flavone derivatives, including hydroxyflavones. Conversely, our analysis show that the powdery form consist almost entirely of flavone. The wooly farina in D. tapetodes is extruded through specific sites at the surface of the glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle. The data is consistent with formation and thread elongation occurring from within the cell. The putative mechanism of wool thread formation and its stability is discussed.

plant biology↗

bioRxiv2018

Homologous recombination changes the context of Cytochrome b transcription in the mitochondrial genome of Silene vulgaris KRA

BackgroundSilene vulgaris (bladder campion) is a gynodioecious species existing as two genders - male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity.\n\nResultsWe assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression the CMS gene by the fertility restorer genes. In addition, by analyzing RNA editing, we found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes despite completely identical gene sequences.\n\nConclusionsFrequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.

genomics↗

bioRxiv2017

Comprehensive skin microbiome analysis reveals the uniqueness of human-associated microbial communities among the class Mammalia

Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. The back, torso, and inner thigh regions of 177 non-human mammals were collected to include representatives from 38 species and 10 mammalian orders. Animals were collected from local farms, zoos, households, and the wild. All samples were amplified using the V3-V4 16S rRNA gene region and sequenced using a MiSeq (Illumina). For reference, previously published skin microbiome data from 20 human participants, sampled using an identical protocol to the non-human mammals, were included in the analysis. Human skin was significantly less diverse than all other mammalian orders and the factor most strongly associated with community variation for all samples was whether the host was a human. Within non-human samples, host taxonomic order was the most significant factor influencing the skin community, followed by the geographic location of the habitat. By comparing the congruence between known host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing first evidence of phylosymbiosis between skin communities and their hosts.\n\nSignificanceSkin forms a critical protective barrier between a mammal and its external environment. Baseline data on the mammalian skin microbiome is crucial for making informed decisions related to veterinary research and biodiversity conservation strategies, in addition to providing insight into mammalian evolutionary history. To our knowledge, this study represents the largest mammalian skin microbiota project to date. These findings demonstrate that human skin is distinct, not only from other Primates, but from all 10 mammalian orders sampled. Using phylosymbiosis analysis, we provide the first evidence that co-evolution may be occurring between skin communities and their mammalian hosts, which warrants more in-depth future studies of the relationships between mammals and their skin microbiota.

microbiology↗