Biology subjects

Schneider, C.

Publications and source records attributed to Schneider, C..

63 records · Page 4Linked to original sources

bioRxiv2020

Biodiversity genomics of small metazoans: high quality de novo genomes from single specimens of field-collected and ethanol-preserved springtails

Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in evolutionary sciences, ecology, systematics and in biodiversity-related applied fields such as environmental management and natural product research. Advances in DNA sequencing technologies make genome sequencing feasible for many non-genetic model species. However, genome sequencing today relies on large quantities of high quality, high molecular weight (HMW) DNA which is mostly obtained from fresh tissues. This is problematic for biodiversity genomics of Metazoa as most species are small and yield minute amounts of DNA. Furthermore, briging living specimens to the lab bench not realistic for the majority of species. Here we overcome those difficulties by sequencing two species of springtails (Collembola) from single specimens preserved in ethanol. We used a newly developed, genome-wide amplification-based protocol to generate PacBio libraries for HiFi long-read sequencing. The assembled genomes were highly continuous. They can be considered complete as we recovered over 95% of BUSCOs. Genome-wide amplification does not seem to bias genome recovery. Presence of almost complete copies of the mitochondrial genome in the nuclear genome were pitfalls for automatic assemblers. The genomes fit well into an existing phylogeny of springtails. A neotype is designated for one of the species, blending genome sequencing and creation of taxonomic references. Our study shows that it is possible to obtain high quality genomes from small, field-preserved sub-millimeter metazoans, thus making their vast diversity accessible to the fields of genomics.

genomics↗

bioRxiv2020

Characterizing the diverse cells that associate with the developing commissures of the zebrafish forebrain

During embryonic development of bilateral organisms, neurons send axons across the midline at specific points to connect the two halves of the nervous system with a commissure. Little is known about the cells at the midline that facilitate this tightly regulated process. We exploit the con served process of vertebrate embryonic development in the zebrafish model system to elucidate the identity of cells at the midline that may facilitate postoptic (POC) and anterior commissure (AC) development. We have discovered that three differentgfap+ astroglial cell morphologies persist in contact with pathfinding axons throughout commissure formation. Similarly, olig2+ progenitor cells occupy delineated portions of the postoptic and anterior commissures. These early olig2+ progenitors demonstrate glial-like morphologies despite the lack of a myelination marker. Moreover, we conclude that both the gfap+ and olig2+progenitor cells give rise to neuronal populations in both the telencephalon and diencephalon. Interestingly, these varied cell populations showed significant developmental heterochrony between the telencephalon and diencephalon. Lastly, we also showed that fli1a+ mesenchymal cells migrate along the presumptive commissure regions before and during midline axon crossing. Furthermore, following commissure maturation, specific blood vessels formed at the midline of the POC and immediately ventral and parallel to the AC. This comprehensive account of the cellular populations that correlate with the timing and position of commissural axon pathfinding has supported the conceptual modeling and identification of the early forebrain architecture that may be necessary for proper commissure development.

developmental biology↗

bioRxiv2020

Primate heart regeneration via migration and fibroblast repulsion by human heart progenitors

SUMMARYHuman heart regeneration is one of the most critical unmet clinical needs at a global level1. Muscular regeneration is hampered both by the limited renewing capacity of adult cardiomyocytes2-4 and the onset of cardiac fibrosis5,6, resulting in reduced compliance of the tissue. Primate have proven to be ideal models for pluripotent stem cell strategies for heart regeneration, but unravelling specific approaches to drive cell migration to the site of injury and inhibition of subsequent fibrosis have been elusive. Herein, by combining human cardiac progenitor lineage tracing and single-cell transcriptomics in injured non-human primate heart bio-mimics, we uncover the coordinated muscular regeneration of the primate heart via directed migration of human ventricular progenitors to sites of injury, subsequent fibroblast repulsion targeting fibrosis, and ultimate functional replacement of damaged cardiac muscle by differentiation and electromechanical integration. Single-cell RNAseq captured distinct modes of action, uncovering chemoattraction mediated by CXCL12/CXCR4 signalling and fibroblast repulsion regulated by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Moreover, transplantation of human cardiac progenitors into hypo-immunogenic CAG-LEA29Y transgenic porcine hearts following injury proved their chemotactic response and their ability to generate a remuscularized scar without the risk of arrhythmogenesis in vivo. Our study demonstrates that inherent developmental programs within cardiac progenitors are sequentially activated in the context of disease, allowing the cells to sense and counteract injury. As such, they may represent an ideal bio-therapeutic for functional heart rejuvenation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Cytoskeletal tension forces mitohormesis

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programmed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of ageing. However, the physical characteristics of the extracellular matrix are also altered in cancer and in aging tissues. We demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via SLC9A1-dependent ion exchange and HSF1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/979583v3_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1ecdaceorg.highwire.dtl.DTLVardef@1112c32org.highwire.dtl.DTLVardef@6c99a1org.highwire.dtl.DTLVardef@1da1e5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

SAMHD1 is a key regulator of the lineage-specific response of acute lymphoblastic leukaemias to nelarabine

The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), has been known for decades to be effective against acute lymphoblastic leukaemias of T-cell (T-ALL), but not of B-cell (B-ALL) origin. The mechanisms underlying this lineage-specific drug sensitivity have remained elusive. Data from pharmacogenomics studies and from a panel of ALL cell lines revealed an inverse correlation of SAMHD1 expression and nelarabine sensitivity. SAMHD1 can hydrolyse and thus inactivate triphosphorylated nucleoside analogues. Transcriptomic and protein expression profiling of cell lines and patient-derived leukaemic blasts revealed lower SAMHD1 abundance in T-ALL than in B-ALL. Mechanistically, SAMHD1 promoter methylation strongly correlated with suppressed SAMHD1 expression, while T-ALL cells did not display increased global DNA methylation. Targeted SAMHD1 degradation using virus-like particles containing Vpx sensitised B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induced AraG resistance. SAMHD1 had a larger impact on cytarabine activity than on nelarabine/ AraG activity in acute myeloid leukaemia (AML) cells, but more strongly affected nelarabine/ AraG activity in ALL cells. This indicates a critical role of the cancer entity. In conclusion, lineage-specific differences in SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine. SAMHD1 is a potential biomarker for the identification of ALL patients likely to benefit from nelarabine therapy and a therapeutic target to overcome nelarabine resistance.

pharmacology and toxicology↗

bioRxiv2019

BRAIN-DERIVED EXTRACELLULAR VESICLES ARE HIGHLY ENRICHED IN THE PRION PROTEIN AND ITS C1 FRAGMENT: RELEVANCE FOR CELLULAR UPTAKE AND IMPLICATIONS IN STROKE

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischemic region. The fate of neurons in the surrounding penumbra depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24h after induction of transient ischemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs) whereas after ischemia, the main EV population originates from astrocytes. Moreover, sEVs presented high amounts of the prion protein (PrP) which were increased after stroke. Conspicuously, sEVs were particularly enriched in a truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or containing PrP (WT). We show that PrP-KO-EVs are rapidly taken up by neurons and colocalize with lysosomes. Although eventually WT-EVs are also found in lysosomes, the amount taken up by neurons is significantly higher for PrP-KO-EVs. Likewise, microglia and astrocytes were also engulfing PrP-KO-sEVs more efficiently than WT-sEVs. Our results provide information on the relative contribution of brain cell types to the sEV pool in mice and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to their cellular uptake.

neuroscience↗

bioRxiv2019

Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

Herpesvirus genome replication, capsid assembly and packaging take place in the host cell nucleus. Matured capsids leave the nucleus through a unique envelopment-de-envelopment process at the nuclear membranes called nuclear egress. How assembled and DNA-containing herpesvirus capsids reach the sites of nuclear egress is however still controversially discussed, as host chromatin that marginalizes during infection might constitute a major barrier. For alphaherpesviruses, previous work has suggested that nuclear capsids use active transport mediated by nuclear filamentous actin (F-actin). However, direct evidence for nuclear capsid motility on nuclear F-actin was missing. Our subsequent work did not detect nuclear F-actin associated with motile capsids, but instead found evidence for chromatin remodeling to facilitate passive capsid diffusion. A recent report described that human cyto-megalovirus, a betaherpesvirus, induces nuclear F-actin and that the motor protein myosin V localizes to these structures. Direct evidence of capsid recruitment to these structures and motility on them was however missing. In this study, we tested the functional role of HCMV-induced, nuclear actin assemblies for capsid transport. We did not observe transport events along nuclear F-actin. Instead, reproduction of nuclear F-actin was only possible using strong overexpression of the fluorescent marker LifeAct-mCherry-NLS. Also, two alternative fluo-rescent F-actin markers did not detect F-actin in HCMV-infected cells. Furthermore, single particle tracking of nuclear HCMV capsids showed no indication for active transport, which is in line with previous work on alphaherpesviruses.

microbiology↗

bioRxiv2018

TP53 mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

BackgroundMDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.\n\nMethods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.\n\nResultsAll MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.\n\nConclusionWe introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

pharmacology and toxicology↗

bioRxiv2018

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analogue drugs important for treating a variety of cancers, including Acute Myeloid Leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We find that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogues tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies.\n\nSignificanceNucleoside analogue drugs are widely used to treat a variety of cancers and viral infections. With an essential role in regulating the nucleotide pool in the cell by degrading cellular nucleotides, SAMHD1 has the potential to decrease the cellular concentration of frequently prescribed nucleotide analogues and thereby decrease their clinical efficacy in cancer therapy. To improve future nucleotide analogue treatments, it is important to understand SAMHD1 interactions with these drugs. Our work thoroughly examines the extent to which nucleotide analogues interact with the catalytic and allosteric sites of SAMHD1. This work contributes to the assessment of SAMHD1 as a potential therapeutic target for cancer therapy and the future design of SAMHD1 modulators that might improve the efficacy of existing therapies.

biochemistry↗