Biology subjects

Anand, S.

Publications and source records attributed to Anand, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

Glioblastoma is the most prevalent and aggressive malignant tumor of the central nervous system. With a median overall survival of only one year, glioblastoma patients have a particularly poor prognosis, highlighting a clear need for novel therapeutic strategies to target this disease. Transcriptional cyclin-dependent kinases (tCDK), which phosphorylate key residues of RNA polymerase II (RNAPII) c-terminal domain (CTD), play a major role in sustaining aberrant transcriptional programs that are key to development and maintenance of cancer cells. Here, we show that either pharmacological inhibition or genetic ablation of the tCDKs, CDK12 and CDK13, markedly reduces both the proliferation and migratory capacity of glioma cells and patient-derived organoids. Using a xenograft mouse model, we demonstrate that CDK12/13 inhibition not only reduces glioma growth in vivo. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII CTD phosphorylation, which in turn disrupts transcription and cell cycle progression in glioma cells. In summary, the results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for glioblastoma. Significance statementGlioblastoma is a common, aggressive, and invasive type of brain tumor that is usually fatal. The standard treatment for glioblastoma patients is surgical resection, radiotherapy, and chemotherapy with DNA-alkylating agents, and unfortunately current treatments only extend overall survival by a few months. It is therefore critical to identify and target additional biological processes in this disease. Here, we reveal that targeting a specific transcriptional addiction for glioma cells by inhibition of CDK12/CDK13 disrupts glioma-specific transcription and cell cycle progression and has potential to provide a new therapeutic strategy for glioblastoma.

cancer biology↗

bioRxiv2023

Cholesterol-associated Locus EHBP1 Protects against NASH Fibrosis

SUMMARYHepatic free cholesterol contributes to fibrosis in nonalcoholic steatohepatitis (NASH), but how hepatic cholesterol metabolism becomes dysregulated in NASH is not completely understood. We show here that human fibrotic NASH livers have decreased EHBP1, a novel GWAS locus associated with LDL-cholesterol, and that the EHBP1 rs10496099 T>C variant in NASH patients is associated with decreased hepatic EHBP1 expression and augmented NASH fibrosis. Congruent with the human data, EHBP1 loss- and gain-of-function increases and decreases NASH fibrosis in mice, respectively. Mechanistic studies reveal that EHBP1 promotes sortilin (SORT1)-mediated PCSK9 secretion, leading to LDLR degradation, decreased LDL uptake, and reduced TAZ, a fibrogenic effector. Moreover, we show that the TNF/PPAR pathway suppresses EHBP1 in NASH. These data not only provide new mechanistic insight into the role of EHBP1 in cholesterol metabolism and NASH fibrosis by uncovering the interaction between EHBP1 and other cholesterol-related loci, including SORT1, PCSK9, and LDLR, but also elucidate a novel interplay between inflammation and EHBP1-mediated cholesterol metabolism. HighlightsHepatic EHBP1 is reduced in human and mouse fibrotic NASH EHBP1 rs10496099 T>C is associated with decreased hepatic EHBP1 and augmented NASH EHBP1 reduces LDLR, cholesterol uptake, and TAZ in hepatocytes EHBP1 promotes sortilin-mediated PCSK9 secretion in hepatocytes TNF suppresses EHBP1 expression in hepatocytes

pathology↗

bioRxiv2023

Machine Learning-Guided Antibody Engineering That Leverages Domain Knowledge To Overcome The Small Data Problem

The application of Machine Learning (ML) tools to engineer novel antibodies having predictable functional properties is gaining prominence. Herein, we present a platform that employs an ML-guided optimization of the complementarity-determining region (CDR) together with a CDR framework (FR) shuffling method to engineer affinity-enhanced and clinically developable monoclonal antibodies (mAbs) from a limited experimental screen space (order of 10^2 designs) using only two experimental iterations. Although high-complexity deep learning models like graph neural networks (GNNs) and large language models (LLMs) have shown success on protein folding with large dataset sizes, the small and biased nature of the publicly available antibody-antigen interaction datasets is not sufficient to capture the diversity of mutations virtually screened using these models in an affinity enhancement campaign. To address this key gap, we introduced inductive biases learned from extensive domain knowledge on protein-protein interactions through feature engineering and selected model hyper parameters to reduce overfitting of the limited interaction datasets. Notably we show that this platform performs better than GNNs and LLMs on an in-house validation dataset that is enriched in diverse CDR mutations that go beyond alanine-scanning. To illustrate the broad applicability of this platform, we successfully solved a challenging problem of redesigning two different anti-SARS-COV-2 mAbs to enhance affinity (up to 2 orders of magnitude) and neutralizing potency against the dynamically evolving SARS-COV-2 Omicron variants.

bioengineering↗

bioRxiv2022

Tumor cell intrinsic RIG-I activation is sufficient to drive immune mediated tumor rejection

Targeting cytosolic nucleic acid sensors is a potent approach to drive type I interferon responses and anti-tumor immunity. Recent evidence suggests that activation of retinoic acid inducible gene-I (RIG-I) using synthetic hairpin RNA agonists decreases tumor progression in multiple preclinical models. However, the role of tumor cell intrinsic RIG-I in shaping tumor cell fates and the host immune microenvironment remains unclear. Here, we show that RIG-I expression is correlated with better overall survival and a distinct immune gene signature in specific human cancers including colorectal cancer. Activation of RIG-I in breast and colorectal cancer cells is sufficient to drive tumor cell death in vitro and significantly delay tumor growth in vivo in multiple preclinical models. Importantly, the efficacy of tumor cell RIG-I activation is lost in immune deficient mice suggesting the requirement of immune responses for this effect. We observe that tumor cell intrinsic RIG-I activation elicits a robust cellular and molecular immune response. We show that tumor cell RIG-I activation also leads to induction of specific immune checkpoints including PD-L1. Using a publicly available database, we found that RIG-I expression serves as an excellent prognostic marker for responders to checkpoint immunotherapy, particularly PD-L1/PD-1 across cancers. Finally, combination of tumor cell intrinsic RIG-I activation with anti-PD-L1 led to a synergistic decrease in tumor growth in a colorectal tumor model. Our findings suggest that tumor cell intrinsic RIG-I can be targeted to enhance anti-tumor immune responses and highlights a potential strategy for anti-cancer vaccines that can invigorate the immune system.

cancer biology↗

bioRxiv2022

DNA damage-induced lncRNA MEG9 impacts angiogenesis

Endothelial cells are highly responsive to environmental changes that allow them to adapt to intrinsic and extrinsic stimuli and switch their transcriptome accordingly to go back to vascular homeostasis. Our previous data demonstrated that small non-coding-RNAs respond quickly to genotoxic stressors and determined endothelial cell fate and DNA damage response. To further understand the contribution of non-coding-RNAs, we profiled differentially expressed long non-coding RNAs in response to genotoxic stress and compared them to pro-angiogenic growth factor signaling. We identified the Maternally expressed gene 9 (MEG9) as a cytoprotective lncRNA in the endothelium. Gain and Loss-of-function studies indicate that MEG9 prevents endothelial cells from cell death, suggesting that MEG9 responses to genotoxic stress can be an adaptive and protective mechanism. Consistent with this phenotype, the knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Deletion of the MEG9 ortholog, Mirg, in mice results in increased vascular leak in Matrigel plugs and a sex and age-dependent decrease in platelets. Mechanistically, we observed that both MEG9 knockdown in vitro and Mirg-deleted mice in vivo activated common pathways, including apoptosis, clotting, and inflammation. Indeed, the proinflammatory adhesion molecule ICAM1 was significantly increased in human and mouse endothelial cells in a MEG9-dependent manner, supporting the increased vascular permeability observed on MEG9 deficient cells. Taken together, our findings illustrate how genotoxic stress responses through dynamic modulation of lncRNAs, such as MEG9, trigger adaptive mechanisms to maintain endothelial function, while loss of these molecules contributes to maladaptive responses and endothelial cell dysfunction.

cell biology↗

bioRxiv2022

HOXA9 forms a repressive complex with nuclear matrix-associated protein SAFB to maintain acute myeloid leukemia

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), where it confers poor prognosis. Characterising the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein-SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro and prolonged survival in vivo. Integrated genomic, transcriptomic and proteomic analyses further demonstrated that the HOXA9-SAFB-chromatin complex associates with NuRD and HP1{gamma} to repress the expression of factors associated with differentiation and apoptosis, including NOTCH, CEBP{delta}, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1{gamma} catalytic activity also triggered differentiation, apoptosis and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML, that may be amenable to therapeutic intervention via therapies targeting the HOXA9/SAFB interface and/or NuRD and HP1{gamma} activity. Key Points- Identification of the endogenous human HOXA9 protein interactome in AML - HOXA9 forms a repressive complex with S/MAR binding protein (SAFB) that is critical for the maintenance of AML by facilitating proliferation and preventing differentiation and cell death. - The HOXA9/SAFB (H9SB) complex represses gene expression via recruitment of NuRD and HP1{gamma}.

cancer biology↗

bioRxiv2022

Distinct selection signatures during domestication and improvement in crops: a tale of two genes in mungbean

Domestication and improvement are two crucial processes underlying the evolution of crops. Domestication transformed wild plants into a utilizable form for humans; improvement refined cultivars adapting to distinct environments and local preferences. Using whole-genome re-sequencing of Vigna radiata, we investigated the demographic history and compared the genetic footprints of domestication and improvement. The Asian wild population migrated to Australia at about 50 kya, and domestication happened in Asia about 9 kya selecting for non-shattering pods. The key candidate gene for this trait, VrMYB26a, has lower expression in cultivars, consistent with the reduced polymorphism in the promoter region reflecting hard selective sweep. The determinate stems were later selected as an improvement phenotype and associated with the gene VrDet1. Two ancient haplotypes reducing gene expression exhibit intermediate frequencies in cultivars, consistent with selection favoring independent haplotypes in soft selective sweep. Our results suggest domestication and improvement may leave different genomic signatures of selection, reflecting the fundamental differences in the two processes and highlighting the limitations of genome-scan methods relying on hard selective sweep.

plant biology↗

bioRxiv2022

Monkey Pox Virus (MPXV): Phylogenomics, Host-Pathogen Interactome, and Mutational Cascade

While the world is still managing to recover from Covid-19 pandemic, Monkeypox awaits to bring in another global outbreak as a challenge to the entire mankind. However, Covid-19 pandemic have taught us lessons to move fast in viral genomic research to implement prevention and treatment strategies. One of the important aspects in Monkeypox virus should be immediately taken up is to gather insights of its evolutionary lineage based on the genomic studies. We have thus analysed the genome sequences of reported isolates of Monkeypox in the present study through phylogenomics. Host-pathogen interactions, mutation prevalence and evolutionary dynamics of this virus were investigated for all the documented isolates. Phylogenetic exploration revealed the clustering of strain Israel 2018 (MN 648051.1) from Clade I with the four isolates reported from the recent outbreak. An in-depth scrutiny of the host-pathogen interactome identified protein E3, serine protease inhibitor-2 (SPI-2), protein K7, and cytokine response-modifying protein B (CrmB) as the major regulatory hubs. Among these, the CrmB protein (dN/dS {approx} 1.61) was detected to be operating through positive selection. It possibly attests a selective advantage with the monkeypox virus in protecting the infected cells from antiviral responses elicited by the host. Studies also revealed that CrmB protein exhibited several mutations, the majority of which were destabilizing ({Delta}{Delta}G >0). While this study identified a large number of mutations within the newly outbreak clade, it also reflected that we need to move fast with the genomic analysis of the newly detected strains from around the world to develop better prevention and treatment methods

microbiology↗

bioRxiv2022

Accurate and Fast Clade Assignment via Deep Learning and Frequency Chaos Game Representation

BackgroundSince the beginning of the COVID-19 pandemic there has been an explosion of sequencing of the SARS-CoV-2 virus, making it the most widely sequenced virus in the history. Several databases and tools have been created to keep track of genome sequences and variants of the virus, most notably the GISAID platform hosts millions of complete genome sequences, and it is continuously expanding every day. A challenging task is the development of fast and accurate tools that are able to distinguish between the different SARS-CoV-2 variants and assign them to a clade. ResultsIn this paper, we leverage the Frequency Chaos Game Representation (FCGR) and Convolutional Neural Networks (CNNs) to develop an original method that learns how to classify genome sequences that we implement into CouGaR-g, a tool for the clade assignment problem on SARS-CoV-2 sequences. On a testing subset of the GISAID, CouGaR-g achieves an 96.29% overall accuracy, while a similar tool, Covidex, obtained a 77, 12% overall accuracy. As far as we know, our method is the first using Deep Learning and FCGR for intra-species classification. Furthermore, by using some feature importance methods CouGaR-g allows to identify k-mers that matches SARS-CoV-2 marker variants. ConclusionsBy combining FCGR and CNNs, we develop a method that achieves a better accuracy than Covidex (which is based on Random Forest) for clade assignment of SARS-CoV-2 genome sequences, also thanks to our training on a much larger dataset, with comparable running times. Our method implemented in CouGaR-g is able to detect k-mers that capture relevant biological information that distinguishes the clades, known as marker variants. AvailabilityThe trained models can be tested online providing a FASTA file (with one or multiple sequences) at https://huggingface.co/spaces/BIASLab/sars-cov-2-classification-fcgr. CouGaR-g is also available at https://github.com/AlgoLab/CouGaR-g under the GPL.

bioinformatics↗

bioRxiv2022

Sphingosine-1-phosphate regulates Plasmodium histone deacetylase activity and exhibits epigenetic control over cell death and differentiation

Histone deacetylases (HDACs) play a key role in cellular processes by the regulation of gene transcription. This study contributes a novel insight how Plasmodium falciparum HDAC (PfHDAC-1) is regulated by S1P produced by host erythrocyte SphK-1. The binding of S1P with endogenous nuclear extract PfHDAC-1 and recombinant PfHDAC-1 blocks their activity. A significant modulation in transcriptional regulation of P. falciparum HDAC regulated genes resulted upon inhibition of S1P production through blocking of hSphK-1 by clinical SphK-1 inhibitor PF-543. PF-543 led to profound decrease in S1P in the parasites nuclear fraction. The significant modulation of PfHDAC-1 regulated specific candidate genes related to gametocytogenesis, virulence and proliferation was observed in parasite treated with SphK-1 inhibitor, suggesting S1P targets PfHDAC-1 and participates in epigenetic regulation of these key cellular processes. The epigenetic modulation of parasite cell growth and differentiation by host provides a novel approach for the developmenthost-targeted therapeutics.

molecular biology↗

bioRxiv2022

High-resolution lung adenocarcinoma expression subtypes identify tumors with dependencies on MET, CDK4, CDK6, and PD-L1

Lung adenocarcinoma is one of the most common cancer types with various treatment modalities. However, better biomarkers to predict therapeutic response are still needed to improve precision medicine. We utilized a consensus hierarchical clustering approach on 509 LUAD cases from TCGA to identify five robust LUAD expression subtypes. We then integrated genomic (patient and cell line) and proteomic data to help define biomarkers of response to targeted therapies and immunotherapies. This approach defined subtypes with unique proteogenomic and dependency profiles. S4-associated cell lines exhibited specific vulnerability to CDK6 and CDK6-cyclin D3 complex gene, CCND3. S3 was characterized by dependency on CDK4, immune-related expression patterns, and altered MET signaling; experimental validation showed that S3-associated cell lines responded to MET inhibitors, leading to increased PD-L1 expression. We further identified genomic features in S3 and S4 as biomarkers for enabling clinical diagnosis of these subtypes. Overall, our consensus hierarchical clustering approach identified robust tumor expression subtypes, and our subsequent integrative analysis of genomics, proteomics, and CRISPR screening data revealed subtype-specific biology and vulnerabilities. Our lung adenocarcinoma expression subtypes and their biomarkers could help identify patients likely to respond to CDK4/6, MET, or PD-L1 inhibitors, potentially improving patient outcome. SignificanceThrough integrative analysis of genomic, proteomic, and drug dependency data, we identified robust lung adenocarcinoma expression subtypes and found subtype-specific biomarkers of response, including CDK4/6, MET, and PD-L1 inhibitors.

cancer biology↗

bioRxiv2021

Genetic Subtypes of Smoldering Multiple Myeloma are associated with Distinct Pathogenic Phenotypes and Clinical Outcomes

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrated 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identified six distinct genetic subtypes. These subtypes were differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three subtypes were associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification model.

cancer biology↗

bioRxiv2021

ECLIPSER: identifying causal cell types and genes for complex traits through single cell enrichment of e/sQTL-mapped genes in GWAS loci

SummaryECLIPSER was developed to identify pathogenic cell types and cell type-specific genes that may affect complex disease susceptibility and trait variation by integrating single cell data with known GWAS loci. ECLIPSER maps genes to GWAS loci for a given complex trait based on expression and splicing quantitative trait loci (e/sQTLs) and other functional data, and tests whether the mapped genes are enriched for cell type-specific expression in particular cell types using single-cell/nucleus RNA-seq data from one or more tissues of interest. A Bayesian Fishers exact test is used to compute fold-enrichment significance. We demonstrate the application of ECLIPSER on various skin diseases and traits using snRNA-seq of healthy human skin samples. Availability and ImplementationThe source code and documentation for ECLIPSER and a Jupyter notebook for generating output tables and figures are available at https://github.com/segrelabgenomics/ECLIPSER. The source code for GWASvar2gene that maps genes to GWAS loci based on e/sQTLs is available at https://github.com/segrelabgenomics/GWASvar2gene. The analysis presented here used data from GTEx (https://gtexportal.org/home/datasets) and Open Targets Genetics (https://genetics-docs.opentargets.org/data-access/graphql-api), but can also be applied to other GWAS variant lists and QTL studies. Data used to reproduce the results of the paper are available in Supplementary data.

bioinformatics↗

bioRxiv2021

A cell-based screen identifies HDAC inhibitors and PLK inhibitor as activators of RIG-I signaling

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

cancer biology↗

bioRxiv2021

Th1/17 Cells Infiltrate Murine Cytomegalovirus-Infected Renal Allografts via Virus-Induced CCL20 and Promote Th1 Cells through IL-17A.

Cytomegalovirus (CMV) infection is associated with renal allograft failure by unknown mechanisms. In a murine renal transplant model, murine CMV (MCMV) induces intragraft infiltration of Th17 cells co-expressing Th1 cytokines, IFN-{gamma} and TNF-, but only a minority of intragraft Th17 cells are specific for MCMV antigens. Instead, MCMV promotes viral antigen-independent Th17 cell recruitment via CCL20-CCR6 and CXCL10-CXCR3 interactions. Th17 cells correlate directly with Th1 cell frequencies and inversely with Tregs in MCMV infected grafts. Pharmacologic inhibition of IL-17A reduces intragraft Th17 cells and neutrophils, increases Tregs, and reduces total but not MCMV-specific Th1 cells. Among a clinical renal transplant cohort with acute rejection, patients with CMV DNAemia had significantly higher serum IL-17A quantities compared to those without CMV DNAemia. Together, these findings indicate that CMV infection upregulates Th17 cell activity during acute rejection and suggests that inhibition of IL-17A may ameliorate CMV-associated allograft injury without impairing antiviral Th1 cells.

immunology↗

bioRxiv2021

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

Understanding the function of genes and their regulation in tissue homeostasis and disease requires knowing the cellular context in which genes are expressed in tissues across the body. Single cell genomics allows the generation of detailed cellular atlases in human tissues, but most efforts are focused on single tissue types. Here, we establish a framework for profiling multiple tissues across the human body at single-cell resolution using single nucleus RNA-Seq (snRNA-seq), and apply it to 8 diverse, archived, frozen tissue types (three donors per tissue). We apply four snRNA-seq methods to each of 25 samples from 16 donors, generating a cross-tissue atlas of 209,126 nuclei profiles, and benchmark them vs. scRNA-seq of comparable fresh tissues. We use a conditional variational autoencoder (cVAE) to integrate an atlas across tissues, donors, and laboratory methods. We highlight shared and tissue-specific features of tissue-resident immune cells, identifying tissue-restricted and non-restricted resident myeloid populations. These include a cross-tissue conserved dichotomy between LYVE1- and HLA class II-expressing macrophages, and the broad presence of LAM-like macrophages across healthy tissues that is also observed in disease. For rare, monogenic muscle diseases, we identify cell types that likely underlie the neuromuscular, metabolic, and immune components of these diseases, and biological processes involved in their pathology. For common complex diseases and traits analyzed by GWAS, we identify the cell types and gene modules that potentially underlie disease mechanisms. The experimental and analytical frameworks we describe will enable the generation of large-scale studies of how cellular and molecular processes vary across individuals and populations.

cell biology↗

bioRxiv2021

From COVID-19 to the Common Cold: Novel Host-Targeted, Pan-Respiratory Antiviral Small Molecule Therapeutics

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral lifecycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

biochemistry↗

bioRxiv2020

Mimicking the Human Tympanic Membrane: the Significance of Geometry

The human tympanic membrane (TM) captures sound waves reaching the outer ear from the environment and transforms them into mechanical motion. The successful transmission of these acoustic vibrations in varying frequency ranges is attributed to the structural architecture of the TM. However, a limited knowledge is available on the contribution of its discrete anatomical features, which is important to fabricate functional biomimetic TM replacements. This work synergizes theoretical and experimental approaches toward understanding the significance of geometry in tissue engineered TM scaffolds. Three test designs along with a plain control were chosen to decouple some of the dominant structural attributes, such as, the radial and circumferential alignment of the collagen fibrils. In silico models suggested a geometrical dependency of their mechanical and acoustical responses, where the presence of radially aligned fibers was observed to have a more prominent effect compared to their circumferential counterparts. Following which, a hybrid fabrication strategy combining electrospinning and additive manufacturing was optimized to manufacture hierarchical scaffolds within the dimensions of the native TM. The experimental characterizations conducted using macro-indentation and laser Doppler vibrometry were in line with the computational models. Finally, biological studies performed with human dermal fibroblasts and human mesenchymal stromal cells, revealed a favorable influence of scaffold hierarchy on cellular alignment and subsequent collagen deposition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/383299v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@16b3653org.highwire.dtl.DTLVardef@1bbc47forg.highwire.dtl.DTLVardef@619ad8org.highwire.dtl.DTLVardef@1fd1071_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Schematic diagram illustrating the overall flowchart of the work. 3D: three-dimensional; ES: electrospinning; FDM: fused deposition modeling; TM: tympanic membrane. C_FIG

bioengineering↗