Biology subjects

Weiss, S.

Publications and source records attributed to Weiss, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Transcriptional regulators ensuring specific gene expression and decision making at high TGFβ doses

TGF{beta}-signaling regulates cancer progression by controlling cell division, migration and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision making towards specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing and morphological assays to quantitatively link signaling, gene expression and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that the majority of TGF{beta} target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGF{beta} doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGF{beta} doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGF{beta} stimulus. Taken together, we present a global view of TGF{beta}-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision making.

systems biology↗

bioRxiv2024

Exploiting a metabolic vulnerability in brain tumour stem cells using a brain-penetrant drug with safe profile

Glioblastoma (GB) remains one of the most treatment refractory and fatal tumour in humans. GB contains a population of self-renewing stem cells, the brain tumour stem cells (BTSC) that are highly resistant to therapy and are at the origin of tumour relapse. Here, we report, for the first time, that mubritinib potently impairs stemness and growth of patient-derived BTSCs harboring different oncogenic mutations. Mechanistically, by employing bioenergetic assays and rescue experiments, we provide compelling evidence that mubritinib acts on complex I of the electron transport chain to impair BTSC stemness pathways, self-renewal and proliferation. Global gene expression profiling revealed that mubritinib alters the proliferative, neural-progenitor-like, and the cell-cycling state signatures. We employed in vivo pharmacokinetic assays to establish that mubritinib crosses the blood-brain barrier. Using preclinical models of patient-derived and syngeneic murine orthotopic xenografts, we demonstrated that mubritinib delays GB tumourigenesis, and expands lifespan of animals. Interestingly, its combination with radiotherapy offers survival advantage to animals. Strikingly, thorough toxicological and behavioral studies in mice revealed that mubritinib does not induce any damage to normal cells and has a well-tolerated and safe profile. Our work warrants further exploration of this drug in in-human clinical trials for better management of GB tumours.

cancer biology↗

bioRxiv2023

Doxycycline Levofloxacin or Moxifloxacin are superior to ciprofloxacin in treating anthrax meningitis in rabbits

Efficient treatment of anthrax related meningitis in patients poses a significant therapeutic challenge. Previously we demonstrated in our anthrax meningitis rabbit model that ciprofloxacin treatment in ineffective with most of the treated animals succumbing to the infection. Herein we tested the efficacy of Doxycycline in our rabbit model and found it highly effective. To test whether the low efficacy of Ciprofloxacin is an example of low efficacy of all fluoroquinolones or only this substance, we treated rabbits that were inoculated intra cisterna magna (ICM) with Levofloxacin or Moxifloxacin. We found that in contrast to Ciprofloxacin, Levofloxacin and Moxifloxacin were highly efficient in treating lethal anthrax related meningitis in rabbits. We demonstrated (in naive rabbits) that this deference probably results from variances in blood brain barrier (BBB) penetration of the different fluoroquinolones. The combined treatment of doxycycline and any one of the tested fluoroquinolones was highly effective in the rabbit CNS infection model. The combined treatment of doxycycline and levofloxacin was effective in inhalation rabbit model, as good as the doxycycline monotherapy. These findings imply that while Ciprofloxacin is highly effective as a post exposure prophylactic drug, using this drug to treat symptomatic patients should be reconsidered.

microbiology↗

bioRxiv2023

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca2+ channel modulate its β-adrenergic regulation

The adrenergic nervous system augments cardiac contraction by increasing the activity of L-type voltage-gated CaV1.2 channels. Dysregulation of this process is linked to severe cardiac dysfunctions. The signaling cascade involves activation of {beta}-adrenergic receptors, elevation of cAMP levels, separation of protein kinase A (PKA) regulatory subunit (PKAR) from catalytic subunit (PKAC), and phosphorylation of the inhibitory protein Rad leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2 (1C) exists in two forms: full-length (FL) or proteolytically processed (truncated), lacking the distal C-terminus (dCT). Specificity and efficiency in the cascade are believed to emanate from unique protein-protein interactions, such as anchoring PKA (via PKAR) to 1C by A-kinase anchoring proteins (AKAPs). However, most AKAPs do not interact with the truncated 1C, and their role in {beta}AR regulation of cardiac CaV1.2 remains unclear. Here we show that PKAC, independently of PKAR or AKAPs, directly interacts with 1C at two domains in 1C-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Furthermore, we find that DCRD competes with PCRD and reduces its interaction with PKAC. The physiological consequences of these complex interactions are incompletely understood; our data suggest that they may fine-tune the {beta}AR regulation of CaV1.2. We propose that the newly discovered interactions take part in governing colocalization of regulatory proteins within the {beta}AR-CaV1.2 multimolecular signaling complexes in cardiomyocytes.

biochemistry↗

bioRxiv2023

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 A using a free-electron laser

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

biophysics↗

bioRxiv2023

Ultra-high density electrodes improve detection, yield, and cell type specificity of brain recordings

To understand the neural basis of behavior, it is essential to sensitively and accurately measure neural activity at single neuron and single spike resolution. Extracellular electrophysiology delivers this, but it has biases in the neurons it detects and it imperfectly resolves their action potentials. To minimize these limitations, we developed a silicon probe with much smaller and denser recording sites than previous designs, called Neuropixels Ultra (NP Ultra). This device samples neuronal activity at ultra-high spatial density ([~]10 times higher than previous probes) with low noise levels, while trading off recording span. NP Ultra is effectively an implantable voltage-sensing camera that captures a planar image of a neurons electrical field. We use a spike sorting algorithm optimized for these probes to demonstrate that the yield of visually-responsive neurons in recordings from mouse visual cortex improves up to [~]3-fold. We show that NP Ultra can record from small neuronal structures including axons and dendrites. Recordings across multiple brain regions and four species revealed a subset of extracellular action potentials with unexpectedly small spatial spread and axon-like features. We share a large-scale dataset of these brain-wide recordings in mice as a resource for studies of neuronal biophysics. Finally, using ground-truth identification of three major inhibitory cortical cell types, we found that these cell types were discriminable with approximately 75% success, a significant improvement over lower-resolution recordings. NP Ultra improves spike sorting performance, detection of subcellular compartments, and cell type classification to enable more powerful dissection of neural circuit activity during behavior.

neuroscience↗

bioRxiv2023

Autophagy in maternal tissues contributes to Arabidopsis thaliana seed development.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development. One degradation mechanism is autophagy, facilitating cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy was shown to affect nutrient remobilization from mother plants and function in the embryo. Yet, these studies examined autophagy-knockout (atg mutant) plants, making it impossible to distinguish between the contribution of autophagy in the source (i.e., the mother plant) and the sink tissue (i.e., the embryo). To address this, we employed a novel approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between WT and atg mutant Arabidopsis thaliana plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from different seed coat development. Our study emphasizes the significance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It sheds light on the tissue-specific functions of autophagy, offering potential for new research into the underlying mechanisms governing seed development and crop yield.

plant biology↗

bioRxiv2023

Adult thymus-derived cMaf+ RORγt+ γδ T cells lack Scart2 chromatin accessibility and do not reach periphery

T cell receptor (TCR) V{gamma}4+ expressing {gamma}{delta} T cells can be divided into IFN-{gamma} and IL-17-producing effector T cell subsets. A bias towards {gamma}{delta}17 effector fate decisions is observed during early ontogeny. In contrast, the existence of V{gamma}4+ {gamma}{delta}17 cells derived from adult thymus is still controversial. In the present work, we used a mouse model where T cells are exclusive generated within an adult thymus. Additionally, we employed single-cell chromatin state analysis from thymocytes of normal mice. A small, but considerable population of immature Cd24+ Gzma+ V{gamma}4 cells was found that exhibit molecular programs of {gamma}{delta}17 cells. These adult thymus-derived immature Cd24a+ cMaf+ V{gamma}4 cells secrete small amounts of IL-17A and IL-17F. Interestingly, do not reach the periphery under steady-state conditions. Furthermore, de novo generated {gamma}{delta}17-like cells from adult thymus lack transcriptional activity of the Scart2 encoding gene, suggesting that Scart2 is a distinct trait of fetal {gamma}{delta} T cell precursors. Together, this study provides valuable insights into developmental traits of V{gamma}4 cells during adulthood and raises the question on signals suppressing the full maturation and/or thymic export of {gamma}{delta}17-like cells within the adult thymus. HighlightsO_LITranscriptional and epigenetic profiling identifies developmental plasticity of Gzma+ Cd24a+ V{gamma}4 cells in adult thymus. C_LIO_LIThymic c-Maf+ and ROR{gamma}t+ V{gamma}4 T cells can be generated during adulthood, but do not reach the periphery under steady-state conditions. C_LIO_LIInnate CD44highCD45RBneg {gamma}{delta}17 cells are completely absent upon induction of T cell development during adulthood. C_LIO_LIScart2 expression might be a key molecule to track developmental traits of fetal-derived {gamma}{delta}17 cell precursors. C_LI

immunology↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

Monkeypox DNA correlates with virus infectivity in clinical samples

In light of the alarming global spread of Monkeypox and the lack of data regarding virus infectivity in clinical specimens, we examined virus infectivity and its correlation with PCR results, the currently available diagnostic tool. Due to the high risk of Monkeypox virus infection, only a limited number of approved BSL-3 laboratories employed with vaccinated staff, including ourselves, are capable of determining Monkeypox virus infectivity. We show strong correlation between viral DNA content and virus infectivity in clinical specimens. Moreover, we define a PCR threshold value which correspond to non-infectious virus, and suggest that our data can be translated into informed decision-making regarding risk assessment, protective measures and guidelines for Monkeypox patients.

microbiology↗

bioRxiv2022

Lateral Axonal Modulation is Required for Stimulus-Specific Olfactory Conditioning in Drosophila

Effective and stimulus-specific learning is essential for animals survival. Two major mechanisms are known to aid stimulus-specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is limited effective temporal window for the reinforcing signals to induce neuromodulation only after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. Here, we report a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. We find that KCs have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, we show that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation. HighlightsO_LILateral KC axo-axonic connections are mediated by muscarinic type-B receptor C_LIO_LIKC connections suppress odor-evoked calcium responses and dopamine-evoked cAMP C_LIO_LIknockdown of the muscarinic type-B receptor impairs olfactory learning C_LIO_LIImpaired learning is due to changes to the valence of the unconditioned odor C_LI

neuroscience↗

bioRxiv2022

Ecological Dynamics Imposes Fundamental Challenges in Microbial Source Tracking

Quantifying the contributions of possible environmental sources ("sources") to a specific microbial community ("sink") is a classical problem in microbiology known as microbial source tracking (MST). Solving the MST problem will not only help us understand how microbial communities were formed, but also have far-reaching applications in pollution control, public health, and forensics. Numerous computational methods, referred to as MST solvers hereafter, have been developed in the past and applied to various real datasets to demonstrate their utility across different contexts. Yet, those MST solvers do not consider microbial interactions and priority effects in microbial communities. Here, we revisit the performance of several representative MST solvers. We show compelling evidence that solving the MST problem using existing MST solvers is impractical when ecological dynamics plays a role in community assembly. In particular, we clearly demonstrate that the presence of either microbial interactions or priority effects will render the MST problem mathematically unsolvable for any MST solver. We further analyze data from fecal microbiota transplantation studies, finding that the state-of-the-art MST solvers fail to identify donors for most of the recipients. Finally, we perform community coalescence experiments to demonstrate that the state-of-the-art MST solvers fail to identify the sources for most of the sinks. Our findings suggest that ecological dynamics imposes fundamental challenges in solving the MST problem using computational approaches.

microbiology↗

bioRxiv2022

Parsing the role of NSP1 in SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

microbiology↗

bioRxiv2022

Alternate response of Bacillus anthracis atxA and acpA to serum, HCO3- and CO2

Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is independently mediated by two regulators; acpA and acpB. In addition, it was demonstrated that acpA has at least two promotors, one of them shared with atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works utilizing NBY-HCO3- medium under CO2 enriched conditions, we used a sDMEM-based medium. Thus, toxin and capsule production can be induced in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA in an atxA-independent manner, with little to no toxin (protective antigen PA) production. atxA is activated in response to serum independently of CO2, inducing toxin and capsule production in an acpA or acpB dependent manner. HCO3- was also found to activate atxA, but in non-physiological concentrations. Our findings may help explain the first stages of inhalational infection, in which spores germinating in dendritic cells require protection (by encapsulation) without affecting cell migration to the draining lymph-node by toxin secretion.

microbiology↗

bioRxiv2021

In vitro and in vivo NIR Fluorescence Lifetime Imaging with a time-gated SPAD camera

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche photodiode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Forster Resonant Energy Transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated-ICCD camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

bioengineering↗

bioRxiv2021

Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

biophysics↗

bioRxiv2021

PySOFI: an open source Python package for SOFI

Super-resolution optical fluctuation imaging (SOFI) is a highly democratizable technique that provides optical super-resolution (SR) without requirement of sophisticated imaging instruments. An open source package for SOFI algorithm is needed to support not only the utilization of SOFI, but also the community adoption and participation for further development of SOFI. In this work, we developed PySOFI, an open source python package for SOFI analysis that offers the flexibility to inspect, test, modify, improve and extend the algorithm. We provide a complete documentation for the package and a collection of Jupyter Notebooks to demonstrate the usage of the package. We discuss the architecture of PySOFI, illustrate how to use each functional module, and demonstrate how to extend the PySOFI package with additional modules. We expect PySOFI to facilitate efficient adoption, testing, modification, dissemination and prototyping of new SOFI-relevant algorithms.

biophysics↗

bioRxiv2021

Long chain lipids facilitate insertion of large nanoparticles into membranes of small unilamellar vesicles

Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into the hydrophobic membrane core in between the two lipid leaflets. Incorporation of nanoparticles above this size limit requires development of challenging surface engineering methodologies. In principle, increasing membrane thickness should facilitate incorporation of larger nanoparticles. Here, we explore the effect of incorporating very long phospholipids (C24:1) into small unilamellar vesicles on the membrane insertion efficiency of hydrophobic nanoparticles that are 5-13 nm in diameter. To this end, we improved an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and to evaluate the insertion efficiency of membrane-inserted nanoparticles. We also perform classical, coarse-grained molecular dynamics simulations to identify changes in lipid membrane structural properties that may increase insertion efficiency. Our results indicate that long-chain lipids increase the insertion efficiency by preferentially accumulating near membrane-inserted nanoparticles to reduce the thermodynamically unfavorable disruption of the membrane.

biophysics↗