Biology subjects

Schmidt, A.

Publications and source records attributed to Schmidt, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Computer-Assisted Mitotic Count Using a Deep Learning-based Algorithm Improves Inter-Observer Reproducibility and Accuracy in canine cutaneous mast cell tumors

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intra-observer discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying/classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, the computer-assisted review by pathologists may ensure reliability. In the present study we have compared partial (MC-ROI preselection) and full (additional visualization of MF candidate proposal and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MF and improving classification against imposters. The inter-observer consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study prove that computer assistance may lead to a more reproducible and accurate MCs in ccMCTs.

pathology↗

bioRxiv2021

Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19

The introduction of vaccines has inspired new hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against COVID-19, thus we profiled the earliest humoral signatures in a large cohort of severe and asymptomatic COVID-19 individuals. While a SARS-CoV-2-specific immune response evolved rapidly in survivors of COVID-19, non-survivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibody evolution. Given the conservation of S2 across {beta}-coronaviruses, we found the early development of SARS-CoV-2-specific immunity occurred in tandem with pre-existing common {beta}-coronavirus OC43 humoral immunity in survivors, which was selectively also expanded in individuals that develop paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.

immunology↗

bioRxiv2021

Structures of active melanocortin-4 receptor--Gs-protein complexes with NDP-α-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R- Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP--MSH and setmelanotide, with 2.9 [A] and 2.6 [A] resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP--MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor- Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

biochemistry↗

bioRxiv2021

Shared and unique properties of place cells in anterior cingulate cortex and hippocampus

Hippocampal sharp-wave ripples (SWRs) and the associated replay of neuronal sequences are well-established as the primary physiological substrates for memory consolidation. While the local transformations they induce within the hippocampus are well-characterized, their impact on the downstream cortical representations remains unknown. Here, we identify a distinct functional class of neurons in the anterior cingulate cortex "spatial context cells" that emerge through the multi-day integration of discrete hippocampal inputs. Using closed-loop disruption of SWRs and longitudinal calcium imaging, we demonstrate that formation of this global spatial framework depends on SWRs during offline periods. Whereas standard cortical place maps remain intact following SWR disruption, the emergence of spatial context cells is selectively abolished. These cells preferentially express c-Fos, identifying them as constituents of the cortical memory engram, and exhibit broad receptive fields that develop through an experience-dependent process of field convergence. Collectively, these findings establish SWR-mediated replay as a computational bridge transforming high-resolution, fragmented hippocampal spatial codes into generalized, stable cortical schemas. This work reveals a bifurcated consolidation process, identifying SWR-dependent synaptic remodeling as a fundamental mechanism for the ontogeny of remote memory.

neuroscience↗

bioRxiv2021

Discovery of lipotoxicity-sensitive transcription factors in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH.

molecular biology↗

bioRxiv2021

Proteogenomic characterization of hepatocellular carcinoma

We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-{beta}-catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs.

genomics↗

bioRxiv2020

Morc3 silences endogenous retroviruses by enabling Daxx-mediated H3.3 incorporation

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although, specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as top scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3 and increased chromatin accessibility of distinct ERV classes. We found that the GHKL ATPase domain of Morc3 is critical for ERV silencing, since mutants which cannot bind ATP, or which are defective in ATP hydrolysis cannot rescue the Morc3 ko phenotype. Proteomic analysis revealed that Morc3 mutant protein which cannot bind ATP fails to interact with the H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation, as Daxx lacking the SUMO interaction domain shows reduced association with Morc3. Notably, in Morc3 ko cells, we observed strongly reduced H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as critical regulator of Daxx-mediated H3.3 incorporation to ERV regions.

molecular biology↗

bioRxiv2020

RNA-bound PGC-1α controls gene expression in liquid-like nuclear condensates

The peroxisome-proliferator-activated receptor-{gamma} coactivator-1 (PGC-1) integrates environmental cues by controlling complex transcriptional networks in various metabolically active tissues. However, it is unclear how a transcriptional coregulator coordinates dynamic biological programs in response to multifaceted stimuli such as endurance training or fasting. Here, we discovered a central function of the poorly understood C-terminal domain (CTD) of PGC-1 to bind RNAs and assemble multi-protein complexes. Surprisingly, in addition to controlling the coupling of transcription and processing of target genes, RNA binding is indispensable for the recruitment of PGC-1 to chromatin into liquid-like nuclear condensates, which compartmentalize and regulate active transcription. These results demonstrate a hitherto unsuspected molecular mechanism by which complexity in the regulation of large transcriptional networks by PGC-1 is achieved. These findings are not only essential for the basic understanding of transcriptional coregulator-driven control of biological programs, but will also help to devise new strategies to modulate these processes in pathological contexts in which PGC-1 function is dysregulated, such as type 2 diabetes, cardiovascular diseases or skeletal muscle wasting.

molecular biology↗

bioRxiv2020

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata

17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant and potent anti-herbivore defense metabolites in Nicotiana attenuata whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 members of family-1 UGTs. Tissue-specific time-course transcriptional profiling revealed that the transcripts of three UGTs were highly correlated with two HGL-DTG key biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLSs role in HGL-DTG biosynthesis was confirmed by virus-induced gene-silencing. Silencing the UDP-rhamnosyltransferase, UGT91T1, indicated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. UGT74P3 and UGT74P5 stably silenced plants were severely developmentally deformed, suggesting a phytotoxic effect of 17-HGL. Applications of synthetic 17-HGL and silencing of these UGTs in HGL-DTG-free plants confirmed the phytotoxic effect of 17-HGL. Feeding assays with Manduca sexta larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation is a critical step that contributes to the metabolites defensive function and solves the autotoxicity problem of this potent chemical defense.

plant biology↗

bioRxiv2020

Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria at mucosal surfaces. Though several GBS adhesins have been identified, the host receptor targets of these adhesins remain unknown. We report here that surface-expressed {beta} protein from GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that the IgSF domain in {beta} represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessments revealed that this newly identified IgI3 fold is not exclusively present in GBS. Instead, the IgI3 fold is predicted to be present in adhesins from other clinically important human pathogens. We confirmed the interaction between CEACAM1 and the predicted IgI3-containing adhesin in two different streptococcal pathogens. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

microbiology↗

bioRxiv2019

Single-cell analysis of intestinal immune cells during helminth infection

Single cell isolation from helminth infected intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to isolate millions of immune cells from heavily infected tissues. Using this protocol, we validated many hallmarks of anti-parasite immunity and analyzed immune cells from the lamina propria and granulomas during helminth development, as well as acute and chronic worm infection.

immunology↗

bioRxiv2019

mTORC1 signaling is not essential for the maintenance of muscle mass and function in adult sedentary mice

BackgroundThe balance between protein synthesis and degradation (proteostasis) is a determining factor for muscle size and function. Signaling via the mammalian target of rapamycin complex 1 (mTORC1) regulates proteostasis in skeletal muscle by affecting protein synthesis and autophagosomal protein degradation. Indeed, genetic inactivation of mTORC1 in developing and growing muscle causes atrophy resulting in a lethal myopathy. However, systemic dampening of mTORC1 signaling by its allosteric inhibitor rapamycin is beneficial at the organismal level and increases lifespan. Whether the beneficial effect of rapamycin comes at the expense of muscle mass and function is yet to be established.\n\nMethodsWe conditionally ablated the gene coding for the mTORC1-essential component raptor in muscle fibers of adult mice (iRAmKO). We performed detailed phenotypic and biochemical analyses of iRAmKO mice and compared them with RAmKO mice, which lack raptor in developing muscle fibers. We also used polysome profiling and proteomics to assess protein translation and associated signaling in skeletal muscle of iRAmKO mice.\n\nResultsAnalysis at different time points reveal that, as in RAmKO mice, the proportion of oxidative fibers decreases, but slow-type fibers increase in iRAmKO mice. Nevertheless, no significant decrease in body and muscle mass, or muscle fiber area was detected up to 5 months post-raptor depletion. Similarly, ex vivo muscle force was not significantly reduced in iRAmKO mice. Despite stable muscle size and function, inducible raptor depletion significantly reduced the expression of key components of the translation machinery and overall translation rates.\n\nConclusionsRaptor depletion and hence complete inhibition of mTORC1 signaling in fully-grown muscle leads to metabolic and morphological changes without inducing muscle atrophy even after 5 months. Together, our data indicate that maintenance of muscle size does not require mTORC1 signaling, suggesting that rapamycin treatment is unlikely to negatively affect muscle mass and function.

molecular biology↗

bioRxiv2019

Emergence of the subapical domain is associated with the midblastula transition

Epithelial domains and cell polarity are determined by polarity proteins which are associated with the cell cortex in a spatially restricted pattern. Early Drosophila embryos are characterized by a stereotypic dynamic and de novo formation of cortical domains. For example, the subapical domain emerges at the transition from syncytial to cellular development during the first few minutes of interphase 14. The dynamics in cortical patterning is revealed by the subapical markers Canoe/Afadin and ELMO-Sponge, which widely distributed in interphase 13 but subapically restricted in interphase 14. The factors and mechanism determining the timing for the emergence of the subapical domain have been unknown. In this study, we show, that the restricted localization of subapical markers depends on the onset of zygotic gene expression. In contrast to cell cycle remodeling, the emergence of the subapical domain does not depend on the nucleo-cytoplasmic ratio. Thus, we define cortical dynamics and specifically the emergence of the subapical domain as a feature of the midblastula transition.\n\nAuthor summaryMidblastula transition is a paradigm of a developmental transition. Multiple processes such as cell cycle, cell mobility, onset of zygotic gene expression, degradation of maternal RNA and chromatin structure are coordinated to lead to defined changes in visible morphology. The midblastula transition in Drosophila embryos is associated with a change from fast nuclear cycles to a cell cycle mode with gap phase and slow replication, a strong increase in zygotic transcription and cellularization. The timing of the processes associated with the midblastula transition are controlled by the onset of zygotic gene expression or the nucleocytoplasmic ratio. Here we define the patterning of cortical domains, i. e. the emergence of a subapical domain as a novel feature of the midblastula transition whose appearance is controlled by the onset of zygotic transcription but not the nucleocytoplasmic ratio. Our findings will help to gain further understanding of the coordination of complex developmental processes during the midblastula transition.

developmental biology↗

bioRxiv2019

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina(R) and Ion Torrent(R) sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

microbiology↗

bioRxiv2019

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Metabolic heterogeneity between individual cells of a population harbors offers significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e. the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

synthetic biology↗

bioRxiv2019

Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris

Novel thermostable forms of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene with its localization on a new episomal expression vector pPEH in Pichia pastoris cells. Of 3000 yeast transformants screened, six new thermostable GA mutants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asp9His/Ile82Phe, Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of every single substitution in the double mutants, we have constructed an appropriate single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg mutations caused an increased thermostability. While Leu7Met and Asp9His mutations decreased the thermal stability of GA, and Gln338Leu had little effect, the synergistic effect of double mutant forms Leu7Met/His391Tyr, Asp9His/Ile82Phe and Ser8Arg/Gln338Leu revealed the significant thermostability improvement as compared to wild type GA.

molecular biology↗

bioRxiv2019

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

Higher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.

microbiology↗

bioRxiv2019

An investigation of PS-b-PEO polymersomes for the oral treatment and diagnosis of hyperammonemia

Ammonia-scavenging transmembrane pH-gradient poly(styrene)-b-poly(ethylene oxide) polymersomes were investigated for the oral treatment and diagnosis of hyperammonemia, a condition associated with serious neurologic complications in patients with liver disease as well as in infants with urea cycle disorders. While these polymersomes were highly stable in simulated intestinal fluids at extreme bile salt and osmolality conditions, they unexpectedly did not reduce plasmatic ammonia levels in cirrhotic rats after oral dosing. Incubation in dietary fiber hydrogels mimicking the colonic environment suggested that the vesicles were probably destabilized during the dehydration of the intestinal chyme. Our findings question the relevance of commonly used simulated intestinal fluids for studying vesicular stability. With the encapsulation of a pH-sensitive dye in the polymersome core, the local pH increase upon ammonia influx could be exploited to assess the ammonia concentration in the plasma of healthy and cirrhotic rats as well as in other fluids. Due to its high sensitivity and selectivity, this novel polymersome-based assay could prove useful in the monitoring of hyperammonemic patients and in other applications such as drug screening tests.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC=\"FIGDIR/small/631630v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@dd9b02org.highwire.dtl.DTLVardef@ebb129org.highwire.dtl.DTLVardef@1bc15e3org.highwire.dtl.DTLVardef@1411a28_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗