Biology subjects

Weiss, A.

Publications and source records attributed to Weiss, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Differential multi-omics analysis of pulmonary arterial hypertension microvascular endothelial cells for differential drug response

Pulmonary arterial hypertension (PAH) represents a heterogeneous group of disorders that involves complex molecular dysregulations, which are not fully captured by single-omics analyses. We apply our network-based multi-omics analysis framework, DrDimont, to transcriptomic, proteomic, phosphoproteomic, and kinase screening data from lung microvascular endothelial cells of PAH patients and controls. Thereby, we extend the functionality of DrDimont to incorporate kinase-kinase interactions during the construction of condition-specific multi-omics networks. Kinase interactions are inferred from phosphorylations of screened substrates that are weighted by kinase-substrate predictions. Differential interaction scores from the network-based analysis between PAH and control uncover alterations centered on kinases, in particular top hits relating to MAPK signaling, such as MAPK13, MAP2K, or upstream IRAK1, and other MAPK/MAP2K family members. Further highly differential nodes were ACADSB, GPX7, DSE (for proteins), and AIM1, LY96, CHSY3 (for mRNAs). When prioritizing drug candidates by mapping drug targets onto the differential network, we find high scores for the drug tacrolimus (FK506) and several anti-neoplastic MAPK inhibitors (e.g., selumetinib, trametinib), as well as agents acting on general proliferation via (mitochondrial) DNA transcription (e.g., epirubicin, topotecan). Integrating kinase activity screens into our explainable multi-omics network-based analyses reveals kinase-centered alterations and therapeutic hypotheses in PAH that complement single-layer classical differential expression analyses.

bioinformatics↗

bioRxiv2026

Efficacy of RMC-6236 (Daraxonrasib) and novel combination strategies targeting resistance in RAS pathway-driven neuroblastoma

Metastatic neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, has a cure rate of <50%. DNA-sequencing studies have demonstrated rare recurrent driver mutations at diagnosis, with the most common alterations detected in ALK-RAS-MAPK pathway. Activating ALK and RAS-MAPK mutations are associated with inferior outcome and are increased at relapse, and thus, represent therapeutic vulnerabilities in NB. Previously, we identified combinations of RAS/MAPK inhibitors, including SHP2 and MEK, with efficacy in resistant MAPK-altered tumor cells, including those with the most common NB-associated RAS mutation NRAS-Q61K. However, toxicities of SHP2 inhibitors and promising results using compounds that directly target RAS suggest there may be superior strategies to target RAS/MAPK pathway in NB. Here, we have assessed the efficacy of RAS/MAPK inhibitors, including tovorafenib (pan-RAF), RMC-6236/daraxonrasib (pan-active-RAS) and avutometinib (RAF/MEK) in NB in vitro and in vivo using NB models harboring differing genomic status of RAS/MAPK pathway effectors. We demonstrate selective efficacy of RMC-6236 and avutometinib via RAS-MAPK pathway inhibition in NB cells and xenografts harboring RAS, NF1 or ALK alterations. Importantly, we demonstrate that presence of the NRAS-Q61K mutation confers drug sensitivity. Using newly generated and previously established NB cell models of acquired resistance to RMC-6236 or the ALK inhibitor lorlatinib, we identified targeted combinations, including RMC-6236 plus avutometinib, that demonstrate re-sensitization in resistant NB cell and xenograft models. Finally, transcriptomic studies of RMC-6236-resistant cells detected upregulation of RAS/MAPK signatures, as well as TNF/NF{kappa}B and IL-6/JAK/STAT3 pathway enrichment, thus informing future combinations to enhance sensitivity to RAS inhibitors. STATEMENT OF SIGNIFICANCEOur work demonstrates that newly available RAS pathway inhibitors RMC-6236/daraxonrasib and avutometinib have efficacy in neuroblastoma tumors, which have frequent alterations in the RAS/MAPK pathway. These drugs with early efficacy results in adult RAS-driven tumors provide an important option for patients with relapsed neuroblastoma alone or in combination.

Cancer Biology↗

bioRxiv2026

T-bet Expressing B cells are Key Determinants of Protective Immunity Against Norovirus Infection

The gastrointestinal tract (GI) is the largest environmental mucosal interface and is exposed to diverse commensal and pathogenic microbes. B cells are a prevalent immune component of the GI tract and its associated secondary lymphoid organs, yet we know little about the diversity and stability of distinct transcriptional programs that modulate B cell responses against different classes of pathogens or environmental perturbations. A subset of B cells defined by expression of the transcription factor T-bet, has been canonically associated with antiviral immunity through IgG production. However, the role of T-bet expressing B cells in mucosal tissues, where IgA responses predominate, is poorly understood. Here, we identify a population of intestinal T-bet+ B cells that, in the absence of overt perturbation, constitutes a minor fraction of intestinal associated B cells and undergoes continuous turnover. In contrast, during enteric viral infection with murine norovirus (MNV), T-bet B cells undergo a marked expansion, with T-bet expression stably maintained in the majority of virus-specific B cells, including IgG2c and IgA switched B cells. Moreover, virus-reactive IgG2c and IgA B cells arise independently rather than through sequential switching, and B cell intrinsic T-bet expression is required for effective germinal center responses but dispensable for IgA class switching. Moreover, T-bet expressing B cells are required for the generation of all MNV-specific circulating IgG and mucosal IgA, and for protection upon re-encounter with the virus. Together, these findings establish T-bet expressing B cells as a specialized B cell subset essential for mucosal immunity and protection against norovirus infection.

immunology↗

bioRxiv2026

iPSC modeling of pulmonary arterial hypertension to uncover pathomechanisms and unrecognized modes of action of sotatercept

Pulmonary arterial hypertension (PAH) is a potentially fatal disease characterized by obliterative remodeling of distal pulmonary arteries, commonly associated with bone morphogenetic receptor type 2 (BMPR2) gene mutations. In patients with PAH, sotatercept, an activin signaling inhibitor, improves hemodynamics and outcomes, but clinical responses vary and sometimes occur within weeks, suggesting additional mechanisms beyond its anti-proliferative, pro-apoptotic and anti-remodeling effects. Using patient-specific induced pluripotent stem cell-derived smooth muscle cells (iSMCs) with BMPR2 extracellular- or kinase-domain mutations, we were able to reproduce Activin A-driven PAH traits, including hyperproliferation, reduced apoptosis, enhanced contraction and excessive matrix production. We identified smooth muscle cell-to-myofibroblast transition as a previously unknown contributor to pulmonary vascular remodeling and demonstrate that it is blocked by sotatercept. Beyond its established effects, sotatercept rapidly reduced contractility, collagen-integrin mechanotransduction and TGF{beta} receptor expression, disrupting a pathological positive feedback loop, reflected by lower levels of circulating TGF{beta}1 in patients on sotatercept. Taken together, our patient-derived iSMC platform links mutation-dependent mechanisms of pulmonary vascular remodeling to variable drug responsiveness and reveals previously unrecognized, potentially rapid-acting modes of sotatercept in PAH. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/711267v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18d5e53org.highwire.dtl.DTLVardef@2bc80dorg.highwire.dtl.DTLVardef@5ace98org.highwire.dtl.DTLVardef@1b16171_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Acute IFN-γ elicits long-term proteomic IFN-γ signature in melanoma cells

Interferon-{gamma} (IFN-{gamma}) is a central mediator of antitumor immunity, and its downstream effects are widely assumed to require continuous cytokine exposure. Here, we applied state-of-the-art mass spectrometry (MS)-based proteomics to patient-derived melanoma cells exposed either continuously to IFN-{gamma} or to a 3-hour pulse followed by cytokine removal. A transient IFN-{gamma} exposure was sufficient to establish a stable proteomic state indistinguishable from continuous stimulation, demonstrating that IFN-{gamma}-induced remodeling is rapidly triggered and durably maintained independently of ligand persistence. Proximal JAK-STAT signaling was rapid, saturable at low cytokine concentrations, and fully reversible upon washout, yet downstream proteomic programs remained durably imprinted. In contrast, functional consequences such as cytostasis remained dose-dependent, revealing a disconnect between signaling activation and phenotypic sensitivity. Together, these findings challenge long-standing assumptions about IFN-{gamma} signaling, redefine the temporal requirements for cytokine-driven tumor cell programming, and provide a mechanistic framework for improving immunotherapies primarily working via IFN-{gamma}-signaling and physiologically relevant in vitro models of tumor-immune interactions.

biochemistry↗

bioRxiv2025

Sample-level modeling of single-cell data at scale with tinydenseR

Single-cell studies now routinely encompass hundreds of samples and millions of cells, offering unprecedented opportunities to link sample-level phenotypes with cellular and molecular states. However, current workflows often depend on cell-level inference and rigid clustering, which can distort significance and obscure subtle, continuous variation, in particular for complex experimental designs. Here, we present tinydenseR, a clustering-independent framework that enables robust, scalable, and statistically sensitive detection of differential cell state density, outperforming existing workflows in speed and memory usage. Technology-agnostic at its core, tinydenseR works seamlessly on scRNA-seq, flow, mass and spectral cytometry. Across synthetic benchmarks, a preclinical xenograft model, a publicly available COVID-19 study, two immuno-oncology trials and a multi-study atlas, tinydenseR uncovers disease and treatment history-associated effects, including subtle cell state heterogeneity, while embedding samples in a quantitative manner. Designed to accelerate discovery in clinical, preclinical, and translational research, the open-source package is available at GitHub.com/Novartis/tinydenseR.

bioinformatics↗

bioRxiv2025

Dynamical memory underlies prolonged plasmid persistence after transient antibiotic treatment

Plasmids play critical roles in spreading and maintaining antimicrobial resistance (AMR). They often exhibit prolonged persistence upon antibiotic treatment, even when they impose substantial burden on their hosts. This persistence has been primarily attributed to rapid horizontal transfer or low plasmid cost. However, these mechanisms cannot account for the slow decay of burdensome plasmids with poor mobility. Here, we show that the decoupling of time scales between slow segregation loss and fast growth competition leads to a slow-down in plasmid abundance decay at high initial plasmid abundance, reminiscent of the ghost effect from nonlinear dynamical systems. Integrating theory, simulations, and quantitative experiments across clonal populations and multi-species bacterial communities, we demonstrate that a transient antibiotic pulse can eliminate plasmid-free cells and create a ghost state that extends plasmid persistence from days to months. Our research reveals a generalizable mechanism for the prolonged ecological memory of antibiotic exposure and underscores the need for proactive strategies to curb the spread of AMR.

microbiology↗

bioRxiv2025

Frataxin depletion leads to decreased soma size and activation of AMPK metabolic pathway in dorsal root ganglia sensory neurons

IntroductionFriedreichs ataxia (FA) is an inherited neurodegenerative disorder caused by frataxin deficiency, leading to impaired iron-sulfur (Fe-S) cluster biogenesis and mitochondrial dysfunction. Proprioceptive dorsal root ganglia (DRG) neurons are among the most vulnerable cell types in FA, yet the mechanisms underlying their selective susceptibility remain unclear. MethodsWe developed a primary culture model of embryonic mouse DRG neurons with complete frataxin depletion, enabling longitudinal analysis of cellular and metabolic alterations. ResultsThis novel model reproduces several key biochemical hallmarks of FA, including Fe-S enzyme deficiency, mitochondrial dysfunction, altered iron homeostasis and oxidative stress. Despite severe mitochondrial impairment, frataxin-deficient neurons remained viable but exhibited a marked reduction in soma growth. Mechanistically, this phenotype was associated with activation of AMP-activated protein kinase (AMPK) hyperactivation and inhibition of mTOR signaling. Genetics inhibition of AMPK or re-expression of frataxin restored neuronal growth, demonstrating a functional link between metabolic stress signaling and soma size regulation. Treatment with -lipoic acid (ALA) rescued soma growth, normalize ATP levels and reduce AMPK activation. ConclusionsOur findings identify AMPK-mTOR signaling as a key pathway linking mitochondrial dysfunction to impaired neuronal growth impairment in frataxin-deficient sensory neurons. This robust, scalable cellular model provides new insights into sensory neuron vulnerability and offers a platform for therapeutic discovery.

molecular biology↗

bioRxiv2025

SONIC: A Benchmarking Paradigm for Brain-Computer Interfaces

Brain-computer interfaces (BCIs) can restore function for individuals with neuro-logical disorders and have the potential to transform the way people interact with digital systems. However, the development of advanced BCI applications, such as fluent speech synthesis, is dependent on the underlying information transfer capacity of the physical neural interface employed. A significant barrier to progress has been the lack of standardized, application-agnostic methods for benchmarking BCI system performance prior to clinical trials. Here, we introduce SONIC, a novel preclinical benchmarking paradigm designed to evaluate the information transfer rate (ITR) of a BCI system. This paradigm treats the brain and BCI as a noisy communication channel, where information is sent into the brain via precisely controlled sensory stimuli and read out by the neural interface. We implemented this paradigm in an ovine model by presenting rapid sequences of pure tones while recording neural activity from the primary auditory cortex with the Paradromics Connexus(C) BCI, a fully implanted system utilizing high-density intracortical micro-electrode arrays with wireless power and data transmission. A convolutional neural network was used to decode tones based on neural features. Our results demonstrate an achieved ITR of over 200 bits per second (bps), which is the highest reported BCI ITR to date. For reference, this rate exceeds the linguistic information content of human speech. This ITR is achieved with a total neural interface, filtering, and data aggregation delay of 56 milliseconds. Further analysis demonstrated that ITR remains high (> 100 bps) for the lowest total delay tested (11 ms), supporting the needs of latency-sensitive applications (e.g., direct speech synthesis). This work establishes a new benchmark for BCI performance and demonstrates that the Connexus BCI possesses the bandwidth necessary to support highly advanced applications. This benchmark provides a robust framework for preclinical BCI evaluation, enabling principled system design optimization to accelerate the translation of next-generation neurotechnology.

bioengineering↗

bioRxiv2025

Mapping human microglial morphological diversity via handcrafted and deep learning-derived image features

Microglia play critical roles in brain health and disease by adopting a spectrum of dynamic activation states. However, capturing this continuous heterogeneity in a scalable way remains a major challenge. To address this, we developed an imaging and data analysis framework to map the activation landscape of human iPSC-derived microglia (iMG) at single-cell resolution. We combined high-content imaging using two complementary strategies: a hypothesis-driven immunofluorescence (IF) panel targeting key activation markers (NF-{kappa}B, ASC, CD45) and a discovery-oriented, pan-morphological Cell Painting (CP) assay. Diverse phenotypes were captured through handcrafted and representation learning-based features. To classify cells, we applied Gaussian Mixture Models (GMMs) to image-derived features, enabling soft probabilistic assignments that capture transitional states between phenotypes. Compared to graph-based methods like the Leiden algorithm, GMMs provided comparable classification performance while offering a more nuanced and biologically interpretable view of microglial heterogeneity. We demonstrate that deep learning features from the targeted IF panel are most powerful, achieving high classification accuracy and strong correlation with biological states such as functional NLRP3 inflammasome activation. Our model system provides a robust and scalable platform for quantifying microglial heterogeneity, offering a new tool to identify novel disease-associated states and compounds that precisely modulate microglial phenotypes for therapeutic discovery.

neuroscience↗

bioRxiv2025

Lotka-Volterra Dynamics Facilitate Sustainable Biocontrol of Wastewater Sludge Bulking

Biological wastewater treatment is driven by complex interactions between prokaryotes and eukaryotes. Occasionally, filamentous bacteria species, first and foremost Ca. Microthrix parvicella, increase in abundance and lead to detrimental wastewater sludge bulking or floating, causing environmental harm and financial losses. Current mitigation strategies rely heavily on nonspecific chemical interventions, which present environmental risks and lack prolonged effectiveness. Here, we utilise long-term monitoring data from four German wastewater treatment plants (WWTPs) to explore sustainable biocontrol alternatives. Our findings reveal Lotka-Volterra dynamics between Ca. Microthrix parvicella and the protist Arcella spp. Visual and experimental validation demonstrate the suppression of filamentous bacterial growth by predation. We further model these interactions, predicting the biocontrol potential of Arcella spp. for both immediate and sustained efficacy in managing sludge bulking. These results highlight the potential of protists as biological control agents, providing a more sustainable and environmentally friendly alternative to chemical treatment.

ecology↗

bioRxiv2024

Morphological profiling in human dopaminergic neurons identifies mitochondrial uncoupling as a neuroprotective effect

Parkinsons disease (PD) involves multiple pathological processes in midbrain dopaminergic (mDA) neurons, including protein degradation defects, vesicular trafficking disruption, endolysosomal dysfunction, mitochondrial issues, and oxidative stress. Current PD models often lack complexity and focus on single phenotypes. We used patient-derived SNCA triplication (SNCA-4x) and isogenic control (SNCA-corr) mDA neurons, applying high-content imaging-based morphological profiling to identify and rescue multiple phenotypes. Screening 1,020 compounds, we identified top-scoring compounds that restored healthy profiles in SNCA-4x neurons, increasing Tyrosine hydroxylase (TH) and decreasing -synuclein (Syn) levels. Several hits were linked to mitochondrial biology. Tyrphostin A9, a mitochondrial uncoupler, and several of its structural analogues decreased ROS levels, normalized mitochondrial membrane potential, and increased respiration. Western blotting confirmed that Tyrphostin A9 reduces Syn levels. Our study highlights the neuroprotective potential of mild mitochondrial uncoupling in mDA neurons.

neuroscience↗

bioRxiv2024

DNA Damage Response Deficiency Enhances Neuroblastoma Progression and Sensitivity to Combination PARP and ATR Inhibition

Next generation sequencing of neuroblastoma (NB) tumors have revealed frequent somatic and germline genetic alterations in genes encoding proteins involved in DNA damage response (DDR) pathways. Despite being well-studied in many adult cancers, roles for DDR disruption in pediatric solid tumors remains poorly understood. To address this, patient-relevant loss-of-function mutations in DDR pathway components including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations were found to enhance NB formation and metastasis in vivo, and result in upregulation of proliferation, cell cycle checkpoint and DNA damage repair transcriptional signatures, revealing potential molecular vulnerabilities in DDR-deficient NB. Zebrafish DDR-deficient NB and human NB cells with DDR protein knock-down were sensitive to the poly(ADP-ribose)-polymerase (PARP) inhibitor olaparib, and this effect was further enhanced by inhibition of the ataxia telangiectasia and rad3 related (ATR) kinase. Altogether, our data supports a functional role for DDR-deficiency in NB in vivo and therapeutic potential for combination PARP + ATR inhibition in NB patients with alterations in DDR genes. SignificanceThis work provides the first in vivo evidence supporting a functional role for DDR-deficiency in NB by demonstrating that alterations in certain DDR pathway genes promote NB formation and metastasis. NGS and pre-clinical drug testing also provides rationale for PARP + ATR inhibitor therapy combinations for patients with NB and pathogenic DDR pathway alterations.

cancer biology↗

bioRxiv2024

Malnutrition drives infection susceptibility and dysregulated myelopoiesis that persists after refeeding intervention

Undernutrition is one of the largest persistent global health crises, with nearly 1 billion people facing severe food insecurity. Infectious disease represents the main underlying cause of morbidity and mortality for malnourished individuals, with infection during malnutrition representing the leading cause of childhood mortality worldwide. In the face of this complex challenge, simple refeeding protocols have remained the primary treatment strategy. Although an association between undernutrition and infection susceptibility has been appreciated for over a century, the underlying mechanisms remain poorly understood and the extent to which refeeding intervention is sufficient to reverse nutritionally acquired immunodeficiency is unclear. Here we investigate how malnutrition leads to immune dysfunction and the ability of refeeding to repair it. We find that chronic malnutrition induced through prolonged dietary restriction (40% reduction in food intake) severely impairs the ability of mice to control a sub-lethal Listeria monocytogenes infection. Malnourished mice exhibit blunted immune cell expansion, impaired immune function, and accelerated contraction prior to pathogen clearance. While this defect is global, we find that myelopoiesis is uniquely impacted, resulting in reduced neutrophil and monocyte numbers prior to and post-infection. Upon refeeding, we observe that mice recover body mass, size, cellularity across all major immune organs, the capacity to undergo normal immune cell expansion in response to infection, and a restoration in T cell responses. Despite this broad improvement, refed mice remain susceptible to Listeria infection, uncoupling global lymphoid atrophy from immunodeficiency. We find peripheral neutrophil and monocyte numbers fail to fully recover and refed mice are unable to undergo normal emergency myelopoiesis. Altogether, this work identifies dysregulated myelopoiesis as a link between prior nutritional state and immunocompetency. We believe these findings raise the possibility that exposure to food scarcity should be treated as an immunologic variable, even post-recovery, with considerations for how patient medical history and public health policy.

immunology↗

bioRxiv2024

Antibiotic-mediated microbial community restructuring is dictated by variability in antibiotic susceptibility and population interactions

It is widely known that faster-growing bacterial cells are more susceptible to antibiotics. Given this notion, it appears intuitive that antibiotic treatment would enrich slower-growing cells in a clonal population or slower-growing populations in a microbial community, which has been commonly observed. However, experimental observations also show the enrichment of faster-growing subpopulations under certain conditions. Does this apparent discrepancy suggest uniqueness about different growth environments or the role of additional confounding factors? If so, what could be the major determinant in antibiotic-mediated community restructuring? Combining modeling and quantitative measurements using a barcoded heterogeneous E. coli library, we show that the outcome of antibiotic-mediated community restructuring can be driven by two major factors. One is the variability among the clonal responses of different subpopulations to the antibiotic; the other is their interactions. Our results suggest the importance of quantitative measurements of antibiotic responses in individual clones in predicting community responses to antibiotics and addressing subpopulation interactions.

microbiology↗

bioRxiv2024

SSR42 is a Novel Regulator of Cytolytic Activity in Staphylococcus aureus

SSR42 is the longest noncoding RNA in the S. aureus cell and the second-most abundant transcript in the stationary phase transcriptome, second only to RNAIII. It is highly conserved across strains and exhibits pronounced stability in stationary phase, however the mechanism behind its regulatory role has yet to be fully elucidated. Herein, we used transcriptomic and proteomic approaches to probe the role of SSR42, revealing that it is a powerful, novel activator of the primary leukocidin LukAB. SSR42 is required for cytotoxicity towards, and escape from within, human neutrophils, and also mediates survival within human blood. We show that SSR42 wields this role via derepression by the peroxide repressor PerR in response to the presence of human neutrophils and governs lukAB induction in this niche. Importantly, this regulation is driven by direct RNA-RNA interaction, as we show binding of the 5 UTR of the lukAB transcript with the 3 end of SSR42, which ultimately modulates transcript stability as well as translational activity. Finally, we demonstrate that this behavior is absolutely required for full virulence of S. aureus in murine models of both pneumonia and sepsis. Collectively, we present SSR42 as a pleiotropic regulatory RNA that acts as a nexus between environmental sensing and the regulation of pathogenesis, responding to environmental stimuli and host immune factors to bolster cytotoxic behavior and facilitate infection in S. aureus. ImportanceS. aureus is a master pathogen due to its formidable collection of virulence factors. These are tightly controlled by a diverse group of regulators that titrate their abundance to adapt to unique infectious niches. The role of regulatory RNAs in stress adaptation and pathogenesis is becoming increasingly more relevant in S. aureus. In this study, we provide the most comprehensive global analysis to date of just such a factor, SSR42. Specifically, we uncover that SSR42 is required for mediating cytotoxicity - one of the pillars of infection - in response to phagocytosis by human neutrophils. We find that SSR42 is induced by components of the host immune system and facilitates downstream activation of cytotoxic factors via RNA-RNA interactions. This illustrates that SSR42 forms a pivotal link between sensing the external environment and mediating resistance to oxidative stress while promoting virulence, solidifying it as a major global regulator in S. aureus.

microbiology↗

bioRxiv2024

RNAi-mediated silencing of SOD1 profoundly extends survival and functional outcomes in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders. One sentence summarySilencing SOD1 with chemically optimized divalent siRNA profoundly extends the lifespan of G93A mice and prevents neurodegenerative biochemical and behavioral phenotypes.

neuroscience↗

bioRxiv2024

Intestinal tissue response to Shiga toxin exposure

Shiga toxin (Stx) is produced by some pathogenic strains of E. coli. To study the impact of Stx on the human intestine we utilized human intestinal organoids (HIOs) and human intestinal enteroids (HIEs) grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. Medium used for epithelial cells contains added growth factors, while mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in medium lacking growth factors suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to buildup in the medium over time, since daily medium changes were not as effective as medium changes performed every three days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models. ImportanceThese studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, STEC-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage, therefore they are resistant to epithelial delivery of Stx2, while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.

microbiology↗