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Roth, S.

Publications and source records attributed to Roth, S..

29 records · Page 2Linked to original sources
bioRxiv2020

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

Chinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

genomics↗
bioRxiv2020

Microbiota-derived short chain fatty acids promote Aβ plaque deposition

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimers disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD, including the microbial mediators and their cellular targets in the brain, were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as key metabolites along the gut-brain axis in AD. Germ-free (GF) AD mice exhibit a substantially reduced A{beta} plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice was sufficient to increase the A{beta} plaque load to levels of conventionally colonized animals. While A{beta} generation was only mildly affected, we observed strong microglial activation and upregulation of ApoE upon the SCFA supplementation. Taken together, our results demonstrate that microbiota-derived SCFA are the key mediators along the gut-brain axis resulting in increased microglial activation, ApoE upregulation and A{beta} deposition.

neuroscience↗
bioRxiv2019

Fibrillar Aβ triggers microglial proteome alterations and dysfunction in Alzheimer mouse models

Microglial dysfunction is a key pathological feature of Alzheime[r]s disease (AD), but little is known about proteome-wide changes in microglia during the course of AD pathogenesis and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid {beta} (A{beta}) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial A{beta} Response Proteins (MARPs) that reflect a heterogeneity of microglial alterations during early, middle and advanced stages of A{beta} deposition. Although both mouse models display severe microglial alterations at late stages of amyloid pathology, MARP signatures occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar A{beta}, rather than dystrophic neurites, suggesting that fibrillar A{beta} aggregates are the main drivers of the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy.

neuroscience↗
bioRxiv2019

Targeting endogenous K-RAS for degradation through the affinity-directed protein missile system

For over three decades, K-RAS has been known as the holy grail of cancer targets, one of the most frequently mutated oncogenes in cancer. Because the development of conventional small molecule K-RAS inhibitors has been extremely challenging, K-RAS has been dubbed as an undruggable target, and only recently a mutation specific inhibitor has reached clinical trials. Targeted protein degradation has emerged as a new modality in drug discovery to tackle undruggable targets. However, no degrader for K-RAS has been described thus far. Our laboratory has developed an Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis of endogenous proteins through the ubiquitin proteasome system. Here, we show that we can achieve degradation of endogenous K-RAS and H-RAS in different cell lines in a targeted manner using our AdPROM system. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, thereby offering tantalising possibilities for an alternative therapeutic approach to these so-called undruggable targets in cancer.

molecular biology↗
bioRxiv2019

Dynamic perineuronal net expression during learned maternal behavior is cortical hemisphere-specific and dependent on methyl-CpG-binding protein 2

Cortical neuronal circuits along the sensorimotor pathways are shaped by experience during critical periods of heightened plasticity in early postnatal development. After closure of critical periods, measured histologically by the formation and maintenance of extracellular matrix structures called perineuronal nets (PNNs), the adult mouse brain exhibits restricted plasticity and maturity. Mature PNNs are typically considered to be stable structures that restrict synaptic plasticity on cortical parvalbumin+ GABAergic neurons. Changes in environment (i.e. novel behavioral training) or social contexts (i.e. motherhood) are known to elicit synaptic plasticity in relevant neural circuitry. However, little is known about concomitant changes in the PNNs surrounding the cortical parvalbumin+ GABAergic neurons. Here, we show novel changes in PNN density in the primary somatosensory cortex (SS1) of adult female mice after maternal experience, using systematic microscopy analysis of a whole brain region. On average, PNNs were increased in the right barrel field and decreased in the left forelimb regions. Individual mice had left hemisphere dominance in PNN density. Using adult female mice deficient in methyl-CpG-binding protein 2 (MECP2), an epigenetic regulator involved in regulating experience-dependent plasticity, we found that MECP2 is critical for this precise and dynamic expression of PNN. Adult naive Mecp2-heterozygous females (Het) had increased PNN density in specific subregions in both hemispheres before maternal experience. The laterality in PNN expression seen in naive Het was lost after maternal experience, suggesting possible intact mechanisms for plasticity. Together, our results identify subregion and hemisphere-specific alterations in PNN expression in adult females, suggesting extracellular matrix plasticity as a possible neurobiological mechanism for adult behaviors in rodents.

neuroscience↗
bioRxiv2019

Reconstitution of basic mitotic spindles in cell-like confinement

Bipolar organization of the mitotic spindle is the result of forces generated by dynamic microtubules and associated proteins in interaction with chromosomes and the cell boundary1-4. Biophysical experiments on isolated spindle components have provided important insights into the force-generating properties of different components5-8, but a quantitative understanding of the force balance that results from their concerted action is lacking. Here we present an experimental platform based on water-in-oil emulsion droplets that allows for the bottom-up reconstitution of basic spindles. We find a typical metaphase organization, where two microtubule asters position symmetrically at moderate distance from the mid-zone, is readily obtained even in the absence of chromosomes. Consistent with simulations, we observe an intrinsic repulsive force between two asters that can be counterbalanced alternatively by cortical pulling forces, anti-parallel microtubule crosslinking, or adjustment of microtubule dynamics, emphasizing the robustness of the system. Adding motor proteins that slide anti-parallel microtubules apart drives the asters to maximum separation, as observed in cells during anaphase9,10. Our platform offers a valuable complementary approach to in vivo experiments where essential mitotic components are typically removed, instead of added, one by one.

cell biology↗
bioRxiv2019

Fog signaling has diverse roles in epithelial morphogenesis in insects

The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, here we show in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fogs role during cellularisation is widely conserved and therefore might represent the ancestral function of the pathway.

developmental biology↗
bioRxiv2018

In vivo widefield calcium imaging of the mouse cortex for analysis of network connectivity in health and brain disease

The organization of brain areas in functionally connected networks, their dynamic changes, and perturbations in disease states are subject of extensive investigations. Research on functional networks in humans predominantly uses functional magnetic resonance imaging (fMRI). However, adopting fMRI and other functional imaging methods to mice, the most widely used model to study brain physiology and disease, poses major technical challenges and faces important limitations. Hence, there is great demand for alternative imaging modalities for network characterization. Here, we present a refined protocol for in vivo widefield calcium imaging of both cerebral hemispheres in mice expressing a calcium sensor in excitatory neurons. We implemented a stringent protocol for minimizing anesthesia and excluding movement artifacts which both imposed problems in previous approaches. We further adopted a method for unbiased identification of functional cortical areas using independent component analysis (ICA) on resting-state imaging data. Biological relevance of identified components was confirmed using stimulus-dependent cortical activation. To explore this novel approach in a model of focal brain injury, we induced photothrombotic lesions of the motor cortex, determined changes in inter- and intrahemispheric connectivity at multiple time points up to 56 days post-stroke and correlated them with behavioral deficits. We observed a severe loss in interhemispheric connectivity after stroke, which was partially restored in the chronic phase and associated with corresponding behavioral motor deficits. Taken together, we present an improved widefield calcium imaging tool accounting for anesthesia and movement artifacts, adopting an advanced analysis pipeline based on human fMRI algorithms and with superior sensitivity to recovery mechanisms in mouse models compared to behavioral tests. This tool will enable new studies on interhemispheric connectivity in murine models with comparability to human imaging studies for a wide spectrum of neuroscience applications in health and disease.

neuroscience↗
bioRxiv2018

A molecular phylogeny of bedbugs elucidates the evolution of host associations and sex-reversal of reproductive trait diversification

All 100+ bedbug species (Cimicidae) are obligate blood-sucking parasites and well-known for their habit of traumatic insemination but the evolutionary trajectory of these characters is unknown. Our new, fossil-dated, molecular phylogeny estimates that ancestral Cimicidae evolved ca. 115MYA as hematophagous specialists on an unidentified host, 50MY before bats, switching to bats and birds thereafter. Humans were independently colonized three times and our phylogeny rejects the idea that the divergence of the two current urban pests (Cimex lectularius and C. hemipterus) 47MYA was associated with the divergence of Homo sapiens and H. erectus (1.6MYA). The females functional reproductive tract is unusually diverse and heterotopic, despite the unusual and strong morphological stasis of the male genitalia. This sex-reversal in genital co-variation is incompatible with current models of genital evolution. The evolutionary trait diversification in cimicids allowed us to uncover fascinating biology and link it to human pre-history and current activity.

ecology↗
bioRxiv2017

A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos.\n\nIn spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders.\n\nDespite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression.\n\nTo address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate.

developmental biology↗