Student posters:
Inference of Environmental Factors Across Biomes using Bacterial Community-Wide DNA Composition David Zeevi Microbial communities exhibit rapid and extensive responses to environmental changes and their taxa or gene abundances can thus be used as environmental reporters. However, these microbial features are often biome-specific, impeding the discovery of global determinants of environmental changes. In this context, we analyzed large-scale metagenomic data coupled with environmental information from multiple marine and soil datasets. We showed that temperature could be accurately inferred across biomes using metagenomic DNA 4-mer (tetranucleotide) frequencies. We accurately inferred other, biome-specific, environmental parameters such as pH and nitrate concentrations, within each biome. Additionally, we observed specific patterns in tetranucleotide and codon frequencies that are generalizable across biomes. We also show that similarity between the inferred and actual temperature of single-cell isolate genomes associates with their relative abundance, suggesting that the inference of environmental parameters may be used to determine the success of a microbial strain in a given environment.
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Cancer paradigms ameliorates cardiac dysfunction and fibrosis Medicine Ami Aronheim Heart failure and cancer are the most deadly diseases worldwide. Murine models for cardiac remodeling and heart failure demonstrate that cardiac dysfunction promotes cancer progression and metastasis spread. Yet, no information is available on whether and how tumor progression affects cardiac remodeling. Here, we examined cardiac remodeling following transverse aortic constriction (TAC) in the presence or absence of proliferating cancer cells. We show that tumor-bearing mice, of two different cancer cell lines, display reduced cardiac hypertrophy, lower fibrosis and improved cardiac contractile function following pressure-overload induced by TAC surgery. Integrative analysis of qRT-PCR, flow cytometry and immunofluorescence identified tumor-dependent M1-to-M2 polarization in the cardiac macrophage population as a mediator of the beneficial tumor effect on the heart. Importantly, tumor-bearing mice lacking functional macrophages fail to improve cardiac function and display sustained fibrosis.
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Extending geometric mechanics beyond kinematic isotropic systems Yizhar Or Geometric mechanics analysis of locomotion has provided valuable insights into how biological and robotic systems use changes in shape to move by exploiting mechanical interaction with the environment. However, these analyses currently work only in isotropic environments on kinematic systems. In this research, we aim to extend the classic geometric mechanics method to work in non-isotropic environments on systems with dynamic effects. We will use numerical and analytical methods to evaluate the performance of the extended method and refine it as necessary. Our approach involves planning and conducting experiments with multiple robots in different environments. Then, analyzing data from the experiments using mathematical and statistical methods, and using data-driven techniques to fit models to the experimental data. By doing so, we aim to obtain accurate and reliable models that can be used to dramatically extend the range of applicability of geometric mechanics for robotic locomotion in practical scenarios.
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Complete human day 14 post-implantation embryo models from naive ES cells Molecular Genetics Jacob Hanna Studying human post-implantation development is challenging due to ethical and technical constraints. Currently, models replicating the spatial organization and structure of all embryonic and extra-embryonic tissues of the post-implantation human conceptus are lacking. Recent research showed that mouse naive embryonic stem cells can form structured stem-cell-based embryo models (SEMs) mimicking post-gastrulation development. Here, we demonstrate that genetically unmodified human naive embryonic stem cells, cultured in enhanced naive stem cell medium, can self-assemble into SEMs. These human SEMs replicate nearly all known lineages and compartments of post-implantation embryos, including epiblast, hypoblast, extra-embryonic mesoderm, and trophoblast. They exhibit key developmental features up to 13-14 days post-fertilization, such as embryonic and bilaminar disc formation, epiblast lumenogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, and development of the yolk sac, chorionic cavity, and trophoblast compartments. This platform will likely enable experimental study of early post-implantation to peri-gastrulation human development.
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Fibroblast diversification is an embryonic process dependent on muscle contraction Genetics and Developmental Biology Peleg Hasson Fibroblasts are the most common cell type found in connective tissues, known to play major roles in development, homeostasis, regeneration and disease. Recent advances in single cell (SC) technologies have demonstrated that fibroblasts are not a homogenous group of cells but rather are composed of discrete subpopulations expressing distinct transcriptomes. Although specific fibroblast subpopulations were associated with different biological processes, the mechanisms and unique activities underlying their diversity has not been thoroughly examined. Turning to skeletal muscle development, we set to dissect the variation of muscle-resident fibroblasts. Our results demonstrate fibroblasts diversify following the transition from embryonic to fetal myogenesis prior to birth. We find fibroblast segregate into two major subpopulations occupying distinct niches, with interstitial fibroblasts residing between the muscle fibers, and delineating fibroblasts sheathing individual muscles. We further show these subpopulations entail distinct cellular dynamics and transcriptomes. Notably, we find fibroblast subpopulations exert distinct regulatory roles on myoblast proliferation and differentiation. Finally, we demonstrate this diversification depends on muscle contraction. Altogether, these findings establish muscle-resident fibroblast diversify in a spatio-temporal embryonic process into distinct cell types, entailing different characteristics and roles.
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Discovery of novel small-molecule hits targeting SERCA2a for the treatment of heart failure Int Med-Cardiology Michel Espinoza Fonseca Sarco(endo)plasmic reticulum calcium ATPase (SERCA) is a transmembrane pump critical for calcium transport from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Dysregulation of this essential component in the calcium transport machinery is a hallmark of heart failure. Consequently, the development of SERCA-activating drugs has emerged as a promising approach for the pharmacological treatment of this condition. We recently developed a machine-learning model that uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, allowing screening with high precision for SERCA inhibitors. We retrained this model to identify chemical features linked to SERCA activation and used it to screen for SERCA activators in a dataset of natural compounds. The best small-molecule candidates were synthesized and experimentally tested in situ for SERCA activity and in vitro for efficacy. We found that the hit molecules activate the calcium pump and stimulate intracellular calcium transport in cardiac cells. These results demonstrate that our artificial intelligence-based approach to drug design accelerates the discovery of novel molecules for the pharmacological treatment of heart failure.
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Multi-omics analyses of the acute and long-term effects of DT109 on the molecular profile of the liver to understand DT109’s therapeutic mechanism of action in metabolic dysfunction associated steatotic liver disease (MASLD)PharmacologyCharles BurantMetabolic dysfunction associated steatotic liver disease (MASLD) is characterized by liver steatosis alongside metabolic dysfunction and can lead to serious health outcomes, such as liver failure, cancer, or stroke. Only one recently FDA-approved treatment for MASLD is available, leaving significant need for MASLD drug discovery research. DT109 (Gly-Gly-L-Leu) is a tripeptide that has shown promising therapeutic effects in preclinical mouse and non-human primate models of MASLD. However, the precise mechanism underlying DT109's efficacy remains undetermined. To gain insights into DT109’s mechanism of action, we have characterized both acute and long-term effects of DT109 treatment (450 mg/kg/day, p.o.) on liver gene expression and the liver metabolome in diet-induced mouse models of MASLD. Pathway analysis of differential hepatic genes induced by both short and long-term DT109 treatment reveal an upregulation of pathways involved in metabolism, including pathways of one carbon metabolism and glycine metabolism, and a downregulation of pathways involved in inflammation and immune function. Pathway analysis of differential annotated metabolites induced by DT109 reveal enrichment of pathways involved in one carbon metabolism and glycine metabolism. Furthermore, using quantitative LC-MS we have found that the majority of DT109 is hydrolyzed to its constituent amino acids, glycine and leucine, prior to reaching the portal circulation in mice. Future work will investigate whether our observed effects of DT109 on the molecular profile of the liver are due to the intact tripeptide or its constituent amino acids and will shed light on DT109’s site of therapeutic action to alleviate MASLD steatosis.
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The very short 5’UTR of histone genes contains a specialized enhancer regulated by the ribosome mRNA channel Biomolecular Science Rivka Dikstein Studies from our lab indicate that mRNA binding by the 40S small ribosomal subunit promotes specialized translation. RPS26, a 40S ribosomal protein , binds AUG upstream nucleotides through its C-terminal tail. Using a C-terminal truncated RPS26 mutant cells (Rps26dC), we investigated the role of RPS26 mRNA binding in translation. We employed TCP-seq, a method that enables the isolation of mRNAs engaged with the 48S complex to study scanning and AUG recognition. The data analysis showed queuing of 48S complexes along the 5'UTR up to the start codon and provide evidence for a slow movement of the early elongating 80S complex. Additionally, in the mutant cells, 48S footprints were shorter, indicating reduced mRNA-ribosome contacts. Ribo-seq analysis corroborated these findings and also showed inefficient 80S formation at initiation sites. The results also highlight the importance of Rps26 in enhancing ribosome speed during scanning and early elongation. Differential translation efficiency analysis identified the replication-dependent histone genes (H1, H2A, H2B, H3), as a major downregulated set. These genes are uniquely characterized by very short 5’UTRs and weak AUG context but are nevertheless efficiently translated. Using a reporter assay with an authentic 22 nt-long H2BC4 5’UTR revealed a strong translational activity that surpasses mRNA with optimal 5’UTR and AUG context, indicating a translation enhancer. The activity of the H2B enhancer is dependent on the RPS26 C-terminus and is driven by nucleotides positioned between -9 to -16, the site of the C-terminus binding. Likewise, the enhancer is dependent on the mRNA binding at the entry channel via RPS3. Remarkably, while in Rps26dC mutant cells general leaky scanning is decreased it is increased in the context of H2B 5’UTR. Cell cycle analysis showed a defect in G1 to S phase transition. Consistently, the H2B translational enhancer is particularly important during the S phase in WT cells. These findings underscore the specialized role of the ribosome mRNA binding channel in histone gene translation.
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Towards a systemic approach for ALS treatment Internal Medicine/Cardiology Minerva Garcia-Barrio Introduction: The cGAS/STING pathway is emerging as a common driver of pathology in neuro- and systemic inflammation and its inhibition is protective in genetic models of ALS. Our collaborators recently developed a novel STING inhibitor, CP-36, with highly favorable pharmacological properties. We hypothesize that cGAS/STING inhibition could provide benefits for ALS, beyond neurons and microglia, by targeting the peripheral immune system (with a focus on NK cells), and the blood-brain (BBB) and blood-cerebrospinal (BCSFB) barrier (through the microvascular endothelium), both which are dysregulated in ALS. Methods and Results: Using a team approach, we gathered data indicating that a) in vitro, CP-36 inhibit cGAS/STING activation in SAVI fibroblasts, with high specificity and affinity; b) in vivo, CP-36 is readily bioavailable in C57BL/6J WT mice and remains at steady levels in plasma up to 6h post-treatment (150-200nM; considered pharmacological for this family); and c) CP-36 inhibits the pathway in different tissues. Meanwhile, the lead compound in this family a) did not show toxicity in iNeurons derived from iPSCs from ALS or non-ALS individuals; b) reduced cytokine production by NK cells; and c) is anti-inflammatory in endothelial cells. Regarding NK cells, in vitro CP-36 inhibited the ability of IL-15-activated NK92 to kill K562-luc cells. Currently, CP-36 potential protection of BBB is being assessed in vitro using brain microvascular endothelial cells and immunocytochemistry. These findings suggest a protective effect of CP-36. Significance: Complex diseases like ALS present a unique challenge to drug development since they would ideally require that one drug targets different systemic compartments contributing to the disease. Additionally, current genetic mouse models are intrinsically limiting, likely not recapitulating potential, yet unknown, systemic defects unique to sporadic ALS. By focusing on cGAS/STING-driven dysfunction of NK and endothelial BBB and BCSFB, our findings may accelerate CP-36 translation to both sporadic and genetic ALS.
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Designing thermostable variants of E. coli chaperone dnaK Biochemistry Sarel Fleishman The role of thermodynamic stability in protein function and evolution is still unclear. Evolution has created an enormous array of proteins with potential therapeutic applications, however most of these proteins are inaccessible due to our inability to efficiently express them recombinantly in E. coli. The inability to express proteins in E. coli is usually due to their inherent instability outside of their host organism. There are many competing theories for why most naturally occurring proteins are only "metastable". Computational protein design allows us to create proteins that would be highly unlikely to appear in nature, due to the stepwise process of evolution, but which are very stable while maintaining function. The goal of this project was to try and clarify the role of stability in protein function and evolution. Through the use of computational tools developed in the lab we designed more-stable variants of essential E. coli chaperone protein dnaK and tested them for thermal stability, enzymatic activity, protein-protein interaction, and function. We are now working to reinsert and test the designed variants impact on the phenotype in a dnaK KO variant strain of E. coli. Finally, we want to try and understand how these designed proteins might impact the fitness of the host following reinsertion.
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Novel Z-disc localized DUF4585 containing proteins Physiology Izhak Kehat Introduction: Cardiomyocyte sarcomeres contain localized ribosomes, mRNAs and translation at the Z-disc, but the factors responsible for their localization and maintenance are unknown. Using proximity labeling, we identified two proteins containing DUF4585 (Domain of unknown function 4585) at the Z-disc of the sarcomere. Aims: We aim to study the localization of the DUF4585 containing proteins. To understand their role, we will induce loss of function of the DUF4585 containing proteins in culture and in-vivo. We will study its effects on sarcomere organization and function. Methods: We used a CRISPR/Cas9-based homology-independent targeted integration to identify the location of DUF4585 proteins. We used siRNA to target these proteins in culture and gRNAs to target these proteins in-vivo. We employed immunofluorescent imaging to determine morphological changes and to investigate the effects of the reduced expression on sarcomere organization. We used smFISH and OPP essays to determine the influence of the siRNA knockdown on the localization of mRNAs to the z-disc and on the localized translation. We used truncated protein overexpression via Adeno viral vectors to detect the localization elements. Results: We show that the DUF4585 proteins are localized to the Z-disc. We verified the knockdown using siRNA in cultured cardiomyocytes via RT-qPCR. We identified differences in cell size and in sarcomere organization post siRNA knockdown. smFISH and OPP essays showed changes to mRNA localization and altered translation localization. In-vivo knockout resulted in dilated cardiomyopathic phenotype. Using overexpression of C- and N-terminal protein fragments we showed sarcomere localization is dependent on the C-terminal fragment. Conclusion: We identified the DUF4585 proteins as novel z-disc localized proteins. We demonstrated the importance of the DUF4585 containing proteins to the cardiomyocytes morphology, to the sarcomere organization, the mRNA localization and the localized translation in culture. We show that the localization of the DUF4585 proteins is regulated by elements at the C-terminus of the mRNA transcript.
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Reovirus evasion of T cell cytotoxicity inhibits clearance of tumor cells Immunology Yotam Bar-On Reovirus is an oncolytic virus currently being tested as a possible cancer treatment. It was shown that reovirus can preferentially infect and replicate in tumor cells, leading to selective lysis of cancer cells, a process known as oncolysis. While the concept of reovirus is very promising, results from clinical trials are highly variable, and even contradictory. It was therefore suggested that the immune response to the virus plays a big factor in the treatment outcome. The consensus today is that reovirus enhances the immune response in the tumor microenvironment, with numerous studies demonstrating the immunomodulatory effects of reovirus. However, most of those studies were done on a macro-level, where the immune phenotype was studied with little relevance to the expression of immune-modulatory ligands on the surface of tumor cells, which are key to immune activation against transformed cells. In our previous work, we demonstrated that reovirus infection inhibits the expression of NKG2D-ligands, thus evading direct NK cell cytotoxicity. In this work, we show that reovirus infection lowers the expression of MHC class I on the surface of tumor cells, thus also evading direct cytotoxic T cells. This novel finding may provide insights on the varying response rate to reovirus-based treatments. Here, we investigate the in-depth mechanism by which MHC-I is downregulated and its effects on cytotoxic T cells, with the aim to develop a recombinant reovirus which does not alter T cell cytotoxicity. We propose that this virus might provide enhanced treatment with stronger therapeutic outcomes.
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Ultrastructural analysis of gene edited human induced pluripotent stem cell derived cardiomyocytes to understand mechanisms of Hypertrophic Cardiomyopathy Mechanical Engineering Leeya Engel Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease and a leading cause of sudden cardiac death in young adults. This condition is characterized by altered tissue mechanics (hypercontractility) and structure (hypertrophy and myofibril disarray) and mutations in sarcomeric proteins involved in contraction. In addition, recent studies have highlighted the links between primary changes in molecular activity and secondary consequences for cell metabolism and adaptation. However, the mechanistic links between specific mutations, alterations in cardiomyocyte (CM) architecture, and disease progression remain poorly understood. We use cryo-electron tomography (cryo-ET) for in situ visualization of contraction machinery (thick and thin filaments) in CMs. Combining engineering of cardiac stem cells, micropatterning, cryoET, and subtomogram averaging will enable us to compare architectural differences in normal CMs and those bearing disease-causing mutations in molecular detail. This would constitute an important advance in cardiac biology and shed light on the mechanistic underpinnings of this prevalent class of genetic disorders.
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LAT2 is a master regulator of neuronal amino acid metabolism Biochemistry/Medicine Prof. Herman Wolosker L-Serine is essential for the synthesis of the neuromodulator D-serine and its metabolism is regulated by glutamine. Both L-serine and glutamine are taken up by system A transporters in neurons, but they account for only a small fraction of the neuronal transport of these amino acids. The identity of the major serine/glutamine transport system in neurons remains elusive. We have obtained preliminary evidence implicating LAT2 as the major serine/glutamine uptake system in the brain. LAT2 mutations cause deafness and cataracts, but how they affect neuronal biology is unknown. We have established both constitutive (LAT2-KO) and neuronal selective LAT2 knockout (LAT2-cKO) mouse models. Using in vivo microdialysis, slices and isolated nerve terminals, we found that LAT2 is the main serine and glutamine transporter in the brain and also regulates extracellular D-serine levels. Metabolic profile show that LAT2-cKO mice also exhibit a decrease in several essential amino acid substrates and impaired energy metabolism (e.g., decrease in ATP) in the hippocampus. Our data indicate that LAT2 is the key regulator of neuronal amino acid and energy metabolism. We are currently conducting experiments to elucidate the mechanisms underlying the metabolic changes observed in LAT2-cKO mice.
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Soluble Factors Associated with Inflammation Interact with Advanced Glycation End Products to Modulate Vascular Smooth Muscle Cell Calcification Internal Medicine - Cardiology Minerva Garcia-Barrio Introduction: Cardiovascular disease (CVD) is the leading cause of death worldwide and is exacerbated by diabetes. Vascular smooth muscle cells (VSMCs) play a crucial role in vascular calcification (VC), a key predictor of CVD severity. Soluble urokinase plasminogen activator receptor (suPAR) and glycoprotein non-metastatic protein B (GPNMB) are inflammatory biomarkers implicated in atherogenesis, calcification, and diabetes. Advanced glycation end products (AGE) mediate the adverse effects of diabetes. We hypothesize these factors synergistically enhance VSMC calcification. Methods: In a well-established in vitro model using rat aortic embryonic VSMC line, A7r5 cells were treated with suPAR (2, 5, 10 ng/mL) or GPNMB (3, 6, 12 ng/mL) combined with AGE-BSA (50 and 100 µg/mL) for 7, 10, and 14 days. Calcium deposition was visualized with Alizarin red staining. RUNX2 activity, a transcription factor driving calcification genes, was assessed using a RUNX2-specific luciferase reporter plasmid, p6OSE2-Luc, and compared to a mutant construct. Apoptosis was determined by Annexin V staining. Results: Alizarin red staining showed increased calcium deposition with 10 ng/mL suPAR and 50 µg/mL AGE-BSA, suggesting synergistic effects. By day 10, RUNX2 activity was significantly increased with 10 ng/mL suPAR or 100 µg/mL AGE in calcification medium. Additionally, 10 ng/mL suPAR combined with 100 µg/mL AGE increased apoptosis in A7r5 cells. On the other hand, GPNMB effects appear to be dose and context dependent. Conclusions: These data indicate that suPAR and AGE synergistically increase VSMC calcification through elevated RUNX2 activity and apoptosis, which correlates with increased necrotic cores in advanced atherosclerosis in suPAR transgenic mice. Preliminary findings suggest that GPNMB has a dose- and context-dependent effect on VC, warranting further studies. Understanding calcification mechanisms is crucial for developing new treatments for diabetes-associated atherosclerosis.
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Conjugation mechanophore in organisms: Localized force sense in tendon Material Science Joshua M. Grolman Tendons connect muscles and bones, transferring forces to create motion.The question of how excessive tendon loading and stimulation causes tendon aging and tearing in elderly and mature athletes arouse a lot of interest. However, the in situ environmental conditions in the tendon are still not replicated by research. Mechanochemistry provides a unique approach for situ studying macroscopic material deformation and failure. We successfully incorporating the mechanophore into the tendon fiber using click chemistry. Their functional qualities are determined using FTIR and microscope. This research can help us better understand how the tendon functions and moves during forec. Also provides a mechanophore for deeper biological applications.
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Restoring Simultaneous Wrist and Finger Movements with Brain-Machine Interfaces and Functional Electrical Stimulation Biomedical Engineering Cynthia Chestek Brain-machine interface (BMI) controlled functional electrical stimulation (FES) is a promising treatment for restoring hand movements to people with spinal cord injury. This method uses a BMI to infer desired control from the brain and FES to reanimate the paralyzed hand. One challenge with restoring hand movements is that wrist and finger movements are closely linked. Multiple finger-related muscles are located in the forearm with tendons that cross the wrist. It is currently unclear how well we can use BMI and FES to continuously control two correlated degrees-of-freedom (DOF). To investigate this, one rhesus macaque (Monkey N) was implanted with microelectrode arrays in primary motor cortex. We trained the monkey to use a 2-DOF manipulandum, flexing their fingers grouped together or their wrist, to acquire virtual targets. This monkey and one other (Monkey R) were also implanted with intramuscular electrodes in forearm muscles controlling the fingers and wrist. Temporary arm paralysis was induced using a lidocaine nerve block at the elbow and stimulation was delivered through the intramuscular electrodes. Using the virtual hand task, Monkey N was able to use a ReFIT Kalman filter model to control the virtual hand in a BMI task, achieving an average 93.9% success rate. In addition, Monkey N was able to use the BMI after nerve block, although retraining was necessary to maintain performance. Additionally, using FES without a BMI, stimulation was able to move the monkey’s own nerve blocked hand to perform the 2-DOF virtual hand task. This strategy achieved an average 85.8% success in a limited range of targets. Movement range was impacted by muscle fatigue and stimulation selectivity. Ultimately, this approach achieved a high degree of success in restoring a functional range of movements and the BMI and FES could be paired to restore hand movements after spinal cord injury.
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Cracking the design principles governing damaged purine repair: a high-throughput study of 3-methyladenine DNA glycosylase Department of Chemical and Structural Biology, Weizmann Institute of Science Ariel Afek DNA damages frequently occur across the genome, with 10,000-100,000 damages occurring per day in healthy mammalian cells. These damages, if unrepaired, are considered to be a major contributing factor to mutagenesis, carcinogenesis, and ageing. Human alkyladenine DNA glycosylase (AAG) is a base-excision repair (BER) enzyme that repairs several lesions including 1,N6-ethenoA, 1,N2-ethenoG, and hypoxanthine. However, our understanding of the specificity of lesion recognition by AAG and its role in mutagenesis remains extremely limited since we still lack a global knowledge of AAG substrate discrimination across the whole genomic context and a general understanding of the mechanistic principles that govern the observed specificities of AAG for lesions in different contexts. We report the development of a microarray assay capable of measuring the repair preferences of AAG for hypoxanthine amongst thousands of sequence contexts including native base pairs, mismatched base pairs and bulges. Using this approach, we aim to significantly deepen our understanding of DNA damage signatures and mutagenesis. Our overall goal is to obtain a deep and predictive understanding of mutation formation and gene expression changes in response to cellular DNA damage.
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Effects of pathogenic variation in ADAMTSL4 on vascular smooth muscle cell function relevant to spontaneous coronary artery dissection Internal Medicine Cardiology Santhi K. Ganesh Spontaneous coronary artery dissection (SCAD) is the cause of 1.7-4% of acute coronary syndromes (ACS), with a strongly sex-biased prevalence, with more frequency occurrence in female individuals. In a whole exome sequencing study of SCAD, we recently reported exonic variants in ADAMTSL4 that were predicted to be deleterious by in silico analysis. We and others have previously reported a robustly associated common variant, rs12740679 at chromosome 1q21.2, identified by genome-wide associations study of SCAD that demonstrated a colocalized expression quantitative trait locus effect on ADAMTSL4, with reduced gene expression associated with the SCAD risk allele. Expression of myc-tagged ADAMTSL4 construct of the coding variant ADAMTSL4 c.2263dupG(p.Gly758Trpfs*59) in 293T cells demonstrated 50% reduction in the secretion of ADAMTSL4 from the transfected cells, as compared to the ADAMTSL4 WT-transfected control. Based upon this finding, which is concordant with the GWAS risk allele association with reduced ADAMTSL4 expression, we modeled these effects by transient siRNA transfection of female human coronary artery smooth muscle cells (huCASMCs) and performed in vitro studies to understand the function of ADAMTSL4 in the huCASMC extracellular matrix. After achieving knock down of ADAMTSL4 mRNA expression following transfection we observed a significant increase in cell migration and wound healing. Cell adhesion and contraction was quantified using a traction force microscopy (TFM) assay, and a significant increase in traction force was observed following ADAMTSL4 loss of function. Biomaterial properties were assessed via tensile testing engineered tissue rings (ETR) created with ADAMTSL4 siRNA transfected female huCASMCs, showed a significant decrease in ultimate tensile strength, elastic modulus, and failure strength following candidate gene knock down. A microfibril assay using ADAMTSL4 siRNA transfected huCASMCs cultured in conditioned media with excess secreted ADAMTSL4 WT or ADAMTSL4 c.2263dupG(p.Gly758Trpfs*59) via immunofluorescence staining showed a decrease in FBN1 binding and fibrillogenesis when cells were cultured in the ADAMTSL4 variant-conditioned media.
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Identifying the Fe-S cluster intermediate X-S with rapid mitochondrial immunopurification and unbiased metabolic profiling Tslil Ast Iron-sulfur (Fe-S) clusters are small inorganic cofactors involved in many vital biological processes across the tree of life, from archaea and bacteria to fungi and humans. These clusters cannot be taken up from their environment and must instead be synthesized de-novo within cells. In eukaryotic cells, two dedicated biosynthesis machineries are found in the mitochondrial matrix and the cytosol, forming these essential cofactors. A direct dependency between these two machineries exists. The mitochondrial Fe-S scaffold machinery synthesizes an unknown sulfur-containing molecule called X-S in the matrix. X-S is then exported to the cytosol by the mitochondrial transporter ABCB7, where the cytosolic machinery utilizes it for cluster formation. Disruption of this relay pathway can elicit several diseases and disorders, leading to poor biological functionality and viability. Despite the critical role of X-S, its molecular identity remains unknown, posing a significant barrier to understanding and potentially mitigating the cytosolic effects of its loss. To bridge this gap, we applied a rapid and high-purity mitochondrial isolation method on genetically modified cells, followed by unbiased metabolomics to resolve this sought-after and essential intermediate. This work thus aims to extend basic cofactor biology knowledge and shed light on potential novel therapeutic interventions for Fe-S cluster-associated diseases.
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Unraveling the mechanisms of -synuclein aggregation in models of Parkinson’s disease Medicine Prof. Simone Engelender The accumulation and aggregation of α-synuclein is a central element of Parkinson's disease (PD), but the molecular mechanisms responsible for these processes are not fully understood. Currently, there is no neuronal model that consistently shows α-synuclein aggregation in neurons. Previous neuronal models using toxins such as MPP+ or α-synuclein pre-formed fibrils which have not been able to fully mimic the disease. We now sought to mimic and investigate in neurons the effect of various pathological conditions thought to influence PD, including oxidative stress and impaired blood supply. Several studies have shown that brain vasculature is significantly impaired in prodromal PD, suggesting that the supply of nutrients may also be impaired in early stages of the disease. In agreement, we found that withdrawal of amino acids from the culture medium promotes robust aggregation of α-synuclein in primary neurons, which begins in the processes and progresses temporally into the soma of neurons. Expression of wild-type or disease mutant A53T aggregates upon amino acid starvation, suggesting that this effect may occur in both familial and sporadic disease. In addition, amino acid starvation promotes much less pronounced aggregation of other neurodegenerative disease proteins, including the wild-type and disease mutants tau and TDP-43. We also found that aggregation of α-synuclein in neurons is prevented by the addition of amino acids to a non-enriched medium, but once aggregation has started, the process is irreversible, even after the addition of amino acids. Finally, we do not observe robust aggregation of endogenous α-synuclein, suggesting that the decrease in amino acid support may represent an event secondary to the prior accumulation of α-synuclein in the brain. Understanding the mechanisms underlying α-synuclein aggregation in PD is critical for the development of targeted therapies. Our findings suggest that maintaining amino acid homeostasis may be important to prevent or slow the disease progression.
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Self-Correcting Brain Computer Interface for Multiple Commands Using Error Related Potentials Technion Autonomous Systems Program (TASP) Prof. Miriam Zacksenhouse Electroencephalogram (EEG) based Brain-Computer Interfaces (BCIs) enables direct communication between the brain and external devices. Despite their potential to revolutionize various fields including mobility, communication, and rehabilitation, their effectiveness is limited due to high error-rate. A promising strategy to reduce the error-rate involves detection of error-related potentials (ErrPs) that reflect error processing in the brain. By integrating machine learning models to classify and rectify errors in real-time, BCIs can achieve higher levels of accuracy and robustness. This strategy is particularly challenging yet crucial in multi-class BCIs, as even upon detecting an ErrP, the correct command remains undecided among multiple options. This research presents the development and evaluation of a novel self-correcting BCI composed of a basic Movement Classifier (bMC) and an enhanced Movement Classifier (eMC), designed to classify five movements. The eMC involves an Error Classifier (EC), based on the ErrP evoked in response to the bMC decision, and a Combined Classifier (CC) that uses features extracted from both bMC and EC. The ongoing experiment comprises three stages: (1) bMC training (N=10), (2) eMC training (N=15), and (3) self-correcting BCI evaluation. Promising results demonstrate an average online bMC accuracy of 67.6% across the 15 participants. Moreover, in offline analysis the eMC improved accuracy for all participants (0 - 14.75%) with significant improvement of 4.5% in average accuracy and 10% in the median accuracy. Current work focuses on assessing the self-correcting BCI online, while future experiments will assess its power for motor imagery. Our work suggests that ErrPs contain relevant information regarding the type of error and can be used to develop self-correcting BCIs. Thus, our work presents a significant step toward the development of more accurate, reliable, and user-friendly non-invasive BCIs. Acknowledgement: This work supported by Dan and Betty Kahn Foundation (grant #2029755), ADRI (grant #2033709) and PMRI (grant #2031643).
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A novel decoy peptide that prevents α-synuclein pathology in Parkinson's disease models Biochemistry Prof. Engelender Simone Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor and non-motor symptoms. Motor symptoms such as tremors and rigidity are caused by degeneration of dopaminergic neurons in the substantia nigra. Non-motor symptoms such as constipation and dementia are due to dysfunction of additional neurons. Motor and non-motor symptoms are caused by accumulation and aggregation of α-synuclein. α-Synuclein also aggregates in other neurodegenerative diseases, including Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA). There is currently no treatment to prevent α-synuclein pathology. In PD, the available treatment is symptomatic and relies on the replenishment of dopamine in the brain to improve the motor symptoms. We have previously shown that α-synuclein is monoubiquitinated, leading to the proteasomal degradation of α-synuclein. We also found that an additional post-translational modification, SUMOylation, compete with and decrease α-synuclein monoubiquitination, leading to reduced proteasomal degradation and α-synuclein accumulation. Therefore, we sought to develop a technology to decrease α-synuclein SUMOylation. In this framework, we developed a cell-penetrating peptide that works as a decoy to reduce α-synuclein SUMOylation. We found that this decoy peptide facilitates the degradation of α-synuclein and decreases the levels of endogenous and pathological -synuclein in neurons. We also found that the decoy peptide is specific towards α-synuclein as it does not decrease the levels of other neurodegenerative diseases such as tau and general protein SUMOylation. The decoy peptide crosses the blood-brain barrier and decreases the levels of endogenous α-synuclein in the brain. Most importantly, we found that the decoy peptide reduces α-synuclein pathology when administered intraperitoneally in the PD mouse model of α-synuclein pre-formed fibrils (α-SynPFF). We believe that the decoy peptide we developed may be a viable strategy to decrease aggregated α-synuclein in the disease and may represent a disease-modifying treatment for PD and perhaps other α-synucleinopethies.
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Modeling Early Familial Alzheimer’s Disease in Human Brain Organoids Molecular Neuroscience Orly Reiner Alzheimer’s Disease (AD) is characterized by molecular hallmarks such as hyperphosphorylated tau/neurofibrillary tangles, extracellular amyloid-β plaques, and immune cell activation. Familial AD (fAD), a genetic form of the disease, progresses predictably after these hallmarks appear, yet early-stage mechanisms remain unclear despite their implications for neurodevelopment and initial patient changes. Traditional animal models, often reliant on overexpressed mutant human transgenes, fail to fully replicate AD hallmarks and the mechanisms leading up to them. In contrast, human brain organoids derived from pluripotent stem cells (PSCs) with fAD mutations present these hallmarks during early neurodevelopment. Utilizing fluorescent imaging and highly reactive sensors, live organoids can be monitored continuously, providing real-time insights into tissue states without the need to sacrifice valuable samples. Our study aims to use a diverse panel of isogenic PSCs with differing genetic backgrounds, each harbouring distinct fAD mutations to model early fAD stages. Our goals include uncovering the early pathological events leading to hallmark development and identifying other potential abnormalities preceding these hallmarks. We aim to understand how these early events contribute to the progression to more advanced AD stages. Preliminary results demonstrate the feasibility of our system, including the presentation of AD hallmarks such as amyloid-β and neurofibrillary tangles. This research will enhance our understanding of early fAD mechanisms present before hallmarks.
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Single Cell Multiomics in CHARGE iPSC-derived Neural tube model reveals defective cranial neural crest cell development Pediatrics Donna M. Martin Background CHARGE syndrome is characterized by Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary tract anomalies, and Ear malformations including deafness and vestibular disorders, occurring in 1/10,000 – 1/15,000 births worldwide. Pathogenic variants in the chromodomain helicase DNA binding 7 (CHD7) gene are the major cause of CHARGE and occur in ~90% of affected individuals. Several malformations observed in individuals with CHARGE are attributed to abnormalities in neural crest cell (NCC) defects. Defects in neural crest formation and migration have been observed in animal models of CHARGE and human induced pluripotent stem cells (iPSCs)-derived NCCs, yet the specific underlying mechanisms are not well understood. Objectives To explore the function of CHD7 in cranial NCC development using a 3D model culture system. Methods We generated 3D microfluidic neural tube-like structures (μNTLS) with rostral-caudal/dorsal-ventral patterning from CHARGE-iPSCs with CHD7 pathogenic variant c.2254A>T, and isogenic corrected iPSCs (IC-iPSCs). To explore the function of CHD7 in cranial NCC development, we explored the single cell transcriptome and cis-regulatory landscape of cranial CH-μNTLS and IC-μNTLS using 10x single cell multiome analysis. Results and Conclusions CHARGE 3D μNTLS exhibited cranial NCC defects. CHD7 haploinsufficiency was associated with disrupted expression and cis-regulatory element chromatin accessibility of specific transcription factors implicated in NCC delamination/epithelial-mesenchymal transition (EMT). Upregulated and more accessible genes were involved in negative regulation of EMT (FOXA2, SPRY1, SPRY2, IL17RD and SPRED1) and extracellular matrix organization (FBLN1, MMP16, ADAMTS9, ADAMTS12, HMCN1, ADAMTS19) while downregulated and less accessible genes encoded cell-cell adhesion proteins (CDH2/6/12/18, PCDH7/9/17/11X/11Y) and extracellular matrix structural proteins (COL4A5, COL11A1, VCAN, LTBP1, EDIL3, MMRN1, FRAS1). In addition, amoeboid type cell migration was reduced in CHARGE cranial NCCs. Our findings shed light on the molecular basis by which CHD7 regulates NCC delamination/EMT and helps to uncover potential therapeutic target genes and pathways.
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Faculty posters:
Immunomodulating mechanisms of human neural stem cell transplantation in a transgenic Alzheimer’s disease mouse model Neurology Eva Feldman Stem cells can simultaneously correct multiple pathways associated with Alzheimer’s disease (AD) pathogenesis; however, stem cell therapeutic mechanisms remain undefined. We transplanted a human neural stem cell (hNSC) line (HK532-IGF1, Palisade Bio) into the hippocampus fimbria fornix (6 total injections, 180,000-600,000 total cells) of the 5XFAD mouse model of AD. hNSC transplantation restored spatial memory learning curves of 5XFAD mice in Morris water maze testing, mimicking performance of wild-type animals. However, there were no changes in amyloid-beta burden, as assessed by immunofluorescence and ELISA. Spatial transcriptomics (Visium, 10X Genomics) on hemibrains elucidated non-amyloid-mediated hNSC therapeutic mechanisms. In hippocampal subregions, we discovered 1061 differentially expressed genes (DEGs; p < 0.05) that were upregulated in 5XFAD animals but were in turn normalized upon hNSC treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these DEGs revealed significant impact on protein processing (“ubiquitin mediated proteolysis”, “protein export”, and “proteosome”), “long-term potentiation,” and “metabolic pathways.” A previously published subset of plaque induced genes were normalized upon hNSC transplantation, particularly within microglia. A reduction in stage 1 and stage 2 disease associated microglia (DAM) was seen, without affecting homeostatic microglia populations. Ligand-receptor interaction analysis (CellChat) showed hNSCs interacted particularly with glia (astrocytes, oligodendrocytes, and microglia, including stage 1 and stage 2 DAM) along many pathways. Pathologic signaling between hippocampus and DAM upregulated in the 5XFAD model was again normalized upon hNSC transplantation. In summary, transplantation of hNSCs rescued memory in 5XFAD mice. No change in amyloid plaque burden was noted, but spatial transcriptomics revealed many genes dysregulated in 5XFAD that were normalized with hNSC transplantation, particularly within hippocampus. hNSCs interacted with and normalized populations of stage 1 and stage 2 DAM. Our results suggest that hNSCs exert beneficial effects in part by modulating microglia-mediated neuroinflammation in AD.
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Long term high-fat feeding; connecting metabolism, cognitive impairment, and altered microglial morphology Neurology Eva Feldman Obesity and metabolic dysfunction (Metabolic Syndrome, prediabetes, diabetes) increase the risk of later life cognitive impairment, including dementias such as Alzheimer’s disease. One common etiology that might underly increased risk is immune system dysregulation and inflammation. Specifically, microglial dependent mechanisms are thought to play an important role in pathology. However, the precise immune and inflammatory mechanisms promoting cognitive decline secondary to obesity and metabolic dysfunction are unclear. We fed male BL6 mice either 60% high fat diet (HFD) or 10% standard diet (SD) for 1 year. Body weight, glucose tolerance, and cognition were assessed over time. Cognition was evaluated using Morris water maze, social recognition, and puzzle box. After 1 year of feeding, mice were given an intraperitoneal injection of lipopolysaccharide as an immune challenge or saline as a control. Activation of hippocampal microglia was assessed via immunohistochemistry and analysis of microglial morphology. Correlation analyses were preformed to understand associations between metabolic, cognitive, and microglial morphology parameters. HFD feeding caused early increases in weight and glucose intolerance, which persisted throughout the study. Cognitive deficits were also observed in HFD animals and were similarly persistent. In response to immune challenge, hippocampal microglia in SD mice had a normal morphological response indicative of activation. However, microglia of HFD mice had no change in morphology in response to lipopolysaccharide injection. In our correlation analysis, body weight in particular was predictive of cognitive changes, especially for later time points. Additionally, changes in morphology were significantly correlated with cognitive outcomes indicating that higher degrees of microglial activation associate with worse cognitive outcomes. Overall, these data indicate HFD feeding causes early and persistent cognitive impairment associated with obesity and confirm a role for microglia in HFD-induced cognitive impairment. Indeed, microglial data strongly indicate a role for inflammation and inflammatory pathways in HFD-induced cognitive impairment.
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Association between urinary metals and amyotrophic lateral sclerosis (ALS) survival Neurology Eva Feldman Objective: This study aimed to investigate the potential association between urinary metal levels and survival in patients with amyotrophic lateral sclerosis (ALS). Methods: A survival analysis was conducted using a Cox proportional hazards regression model with 136 ALS cases. Urinary metals including copper, nickel, tin, and zinc were measured in the study participants. Results: Our survival analysis demonstrated that increased levels of urinary copper (HR=1.34, 95% CI: 1.13-1.59, adjusted P=0.007), nickel (HR=1.27, 95% CI: 1.10-1.47, adjusted P=0.007), tin (HR=1.24, 95% CI: 1.06-1.44, adjusted P=0.030), and zinc (HR=1.53, 95% CI: 1.26-1.86, adjusted P<0.001) were significantly associated with a higher hazard of mortality in patients with ALS. Notably, these hazard ratios correspond to one standard deviation increase in log-transformed metal levels. Conclusion: Our findings suggest that increased levels of urinary copper, nickel, tin, and zinc may be associated with decreased survival rates in patients with ALS. Further investigation is needed to better understand the underlying mechanism of these associations.
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Immune Changes Precede Amyotrophic Lateral Sclerosis Progression Neurology Eva Feldman The immune system contributes to amyotrophic lateral sclerosis (ALS) progression, but it is unclear which immune pathways contribute to downstream ALS progression and which alterations result from upstream neurodegeneration. Identifying upstream immune mechanisms is crucial for developing immune-based ALS therapies, as these causative mechanisms will alter subsequent disease pathology. We previously showed that altered immune cell levels and surface marker expression correlate with changes in ALS disease score in human subjects. We therefore hypothesize that immune system changes correlate with subsequent changes in ALS and can help predict the future disease course, making the underlying mechanisms ideal targets for drug development. In a cohort of 50 control and 56 ALS subjects, we measured both immune markers and ALS functional rating scale-revised (ALSFRS-R) scores at monthly intervals. Immune cell levels, natural killer (NK) cell subpopulations levels, and NK cell surface marker expression was assessed using flow cytometry, and changes in immune levels were assessed in control and ALS subjects over time. Next, in ALS subjects a linear mixed effect model – adjusted for covariates including age, sex, site of onset, and El Escorial criteria –correlated changes in ALSFRS-R with immune system changes occurring up to six months before or after. Consistent with our previous reports, immune cells like CD4 T cells decreased over time, as did several markers expressed by NK cells including CD11a, CD11b, CD38, CX3CR1, and NKp30. Interestingly, we found that increases in NK cell markers CD11a, CD38, and CX3CR1 associated with smaller subsequent decreases in ALSFRS-R – indicating slower disease progression – while increases in NKp30 associated with larger subsequent decreases in ALSFRS-R. Together, these findings demonstrate that not only do changes in specific immune metrics predict subsequent ALS progression, but that NK cells may play a more complex role in ALS pathophysiology than previously thought.
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Dysfunction of the neuroglial lactate shuttle in metabolic syndrome contributing to cognitive and memory impairment Neurology Eva Feldman The metabolic syndrome (MetS) is a worldwide health concern affecting 30% of the population in the United States. MetS precipitates severe neurological complications including dementia. However, the mechanisms linking MetS to dementia are not fully understood. We hypothesized that oligodendrocytes, mainly studied in the context of myelination, exert a metabolic supportive role for CNS neurons. This role is particularly critical in case of brain metabolic dysfunction such as in MetS and ageing. Young (5-week old) and old (12-month old) male mice were fed either standard diet (SD, 10% fat) or high fat diet (HFD, 60% fat) for 24 weeks followed by assessment of metabolic parameters, single cell RNA-sequencing (scRNA-seq). Furthermore, human oligodendrocytes cells (HOG) were cultured in media with or without palmitate to simulate MetS in vitro. Our results show that both young and old mice on HFD gained more weight and suffered from impaired glucose tolerance compared to SD controls. Clustering of scRNA-seq data of oligodendrocytes from young mice brains revealed 3 different clusters of mature oligodendrocytes (MOL). One of the mature oligodendrocytes clusters, MOL2, showed differential expression of metabolic and mitochondrial genes between HFD and SD mice. We used two different concentrations of palmitate to treat HOG in vitro. Chronic treatment with high palmitate concentration (150 µM for 24 hr) triggered insulin resistance and downregulated MCT1 protein expression while acute treatment with low palmitate concentration (50 µM for 6 hr) did not precipitate these changes. Jointly, these results indicate that oligodendrocytes have a metabolic supportive role through shuttling energy substrates to neurons to respond to any metabolic disturbances such as in MetS and ageing.
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Stress and Its Impact on the Development of Diabetes in Obese Individuals: A Comprehensive Analysis Institute of Endocrinology, Diabetes & Metabolism, Rambam Medical College Dave Bridges Chronic psychosocial stress and obesity are both risk factors for type 2 diabetes. In this study we evaluated data from the Michigan Genomics Initiative, a hospital-based cohort for the associations between perceived stress, obesity and type 2 diabetes prevalence. Perceived stress was positively associated with type 2 diabetes, and this effect was enhanced in participants with obesity. Via multivariate modelling of this cross-sectional association, accounting for differences in gender, age, race/ethnicity, and socioeconomic status we found that perceived stress enhanced type 2 prevalence by 13% above that expected by obesity or stress alone. Similar analyses evaluating the effects of obesity and self-reported psychosocial stress demonstrated additive but not synergistic effects on other obesity related comorbidities. These data support the hypothesis that obesity and chronic psychosocial stress augment the risk of type 2 diabetes.
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Combinatorial transcription factor gene therapy induces cochlear hair cell regeneration Otolaryngology, Head and Neck Surgery Raphael Using transgenesis, a combination of transcription factors (TFs) was found to regulate hair cell (HC) development and to induce transdifferentiation of supporting cells into new HCs. Here we tested the combinatorial approach in vivo in mature ears, using two lesion models. One model was the flat epithelium (FE), a condition characterized by the loss of both HCs and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which was induced in guinea pigs. The other model was the DTR mouse, where an injection of diphtheria toxin (DT) leads to degeneration of all HCs leaving behind supporting cells that appear morphologically differentiated.
An adenovirus vector containing gene inserts for Gfi1, Atoh1, Pou4f3, Six1 (GAPS) and a venus reporter, or the reporter alone, was injected into the deafened ears. One or 2 months later, cochleae were labeled with antibodies against Myosin VIIa, and Sox2, and tissues assessed for presence of new hair cell like cells (HCLC) using fluorescence microscopy.
All groups treated with the GAPS vector exhibited large variability in the number of HCLC with some animals displaying no new cells. In guinea pigs, the FE was negative for Sox2, and HCLCs could be detected in the scala tympani side of the basilar membrane. In DT-deafened DTR mice, HCLCs appeared predominantly in the area of the organ of Corti.
In conclusion, the GAPS cocktail of transcription factor transgenes can convert mesothelial cells to HCLCs in guinea pigs with FE and in the deaf DTR mouse.
An adenovirus vector containing gene inserts for Gfi1, Atoh1, Pou4f3, Six1 (GAPS) and a venus reporter, or the reporter alone, was injected into the deafened ears. One or 2 months later, cochleae were labeled with antibodies against Myosin VIIa, and Sox2, and tissues assessed for presence of new hair cell like cells (HCLC) using fluorescence microscopy.
All groups treated with the GAPS vector exhibited large variability in the number of HCLC with some animals displaying no new cells. In guinea pigs, the FE was negative for Sox2, and HCLCs could be detected in the scala tympani side of the basilar membrane. In DT-deafened DTR mice, HCLCs appeared predominantly in the area of the organ of Corti.
In conclusion, the GAPS cocktail of transcription factor transgenes can convert mesothelial cells to HCLCs in guinea pigs with FE and in the deaf DTR mouse.
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SARS-CoV-2 evolution is driven by parallel forces driving the sequence of the Spike – ACE2 protein-protein interaction Biomolecular Sciences Gideon Schreiber With the emergence of the COVID-19 epidemic, we decided to use our expertise in protein-protein interactions to generate a drug that will block ACE2, the receptor of the Spike protein. Our logic was that by blocking the receptor, we will avoid the ability of the virus to evolve circumventing the effect of neutralizing antibodies. Using an enhanced yeast display system engineered by us 1, we succeeded to increase the binding affinity of the receptor binding domain (RBD) of the Spike protein by 1000-fold, to obtain pM binding affinity between RBD and ACE2. 2 Indeed, we showed that the inhibitor, termed RBD-62 was successful in reducing viral titers of CoV-2 in a rhesus model by over 1000-fold, and by this reducing all parameters of the disease 3. Moreover, we showed that RBD-62 is equally effective against all variants of CoV-2, while neutralizing Abs lost reactivity rapidly (2, 3). However, what was more surprising was that the mutations that established themselves during in vitro evolution mimicked the evolution of CoV-2. This includes mutations found to be critical in alpha, beta, gamma, omicron and recently BA2.75 variants. 2, 4, 5 Moreover, we also predicted the epistatic affinity enhancement driven by the double mutation N501Y+Q498R, which allowed for re-organization of the RBD binding interface and the accumulation of a large number of new immune evasion mutations in omicron and its sub-variants, maintaining nM binding between S-CoV-2 and ACE2. 5 Our work clearly shows that CoV-2 evolution is constrained by four major driving forces: (1) Maintaining structural integrity of S-CoV-2; (2) Binding the ACE2 cellular receptor with nM affinity; (3) Avoiding humoral immune responses; and (4) maintaining infectivity. CoV-2 adaptation to our humoral immune system involves mostly the spike protein (S-CoV-2), with the vast majority of neutralizing Abs binding the receptor binding domain (RBD). This realization provides the basis for future antigen development that will provide vaccination against not yet seen variants of CoV-2 and other viruses.
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YAP mediates apoptosis through failed integrin adhesion reinforcement faculty of medicine Haguy Wolfenson Introduction: Tumorigenesis is powered by transformed cell growth characterized as anchorage-independent growth, linked with improper rigidity sensing of the extracellular matrix (ECM). In contrast, normal, anchorage-dependent cells properly sense ECM rigidity and respond by proliferating on stiff matrices and activating apoptosis on soft matrices. However, the mechanobiological mechanism underlying this anchorage/rigidity dependence is poorly understood.
Methods and aims: In this study, we used anchorage/rigidity-dependent fibroblast cells as well as the breast cancer cell line MDA-MB-231 (MDA-WT) which we modified to become anchorage/rigidity dependent by expressing Tropomyosin 2.1 (Tpm 2.1) and studied the responses of these cells to different rigidities.
Results and conclusions: Our studies show that in anchorage/rigidity-dependent cells on soft matrices, YAP is recruited to small adhesions, phosphorylated at the Y357 residue, and translocated into the nucleus, ultimately leading to apoptosis. In contrast, Y357 phosphorylation levels are dramatically low in large adhesions on stiff matrices. Furthermore, mild attenuation of actomyosin contractility allows adhesion growth on soft matrices, leading to reduced Y357 phosphorylation levels and resulting in cell growth. These findings indicate that failed adhesion reinforcement drives rigidity-dependent apoptosis through YAP, and that this decision is not determined solely by ECM rigidity, but rather by the balance between cellular forces and ECM rigidity.
Methods and aims: In this study, we used anchorage/rigidity-dependent fibroblast cells as well as the breast cancer cell line MDA-MB-231 (MDA-WT) which we modified to become anchorage/rigidity dependent by expressing Tropomyosin 2.1 (Tpm 2.1) and studied the responses of these cells to different rigidities.
Results and conclusions: Our studies show that in anchorage/rigidity-dependent cells on soft matrices, YAP is recruited to small adhesions, phosphorylated at the Y357 residue, and translocated into the nucleus, ultimately leading to apoptosis. In contrast, Y357 phosphorylation levels are dramatically low in large adhesions on stiff matrices. Furthermore, mild attenuation of actomyosin contractility allows adhesion growth on soft matrices, leading to reduced Y357 phosphorylation levels and resulting in cell growth. These findings indicate that failed adhesion reinforcement drives rigidity-dependent apoptosis through YAP, and that this decision is not determined solely by ECM rigidity, but rather by the balance between cellular forces and ECM rigidity.
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