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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2024

Dispersion indices for universal quantification of fluorescently-labelled subcellular structure spatial distributions

Image analysis of subcellular structures and biological processes relies on specific, context-dependent pipelines, which are labor-intensive, constrained by the intricacies of the specific biological system, and inaccessible to broader applications. Here we introduce the application of dispersion indices, a statistical tool traditionally employed by economists, to analyze the spatial distribution and heterogeneity of subcellular structures. This computationally efficient high-throughput approach, termed GRID (Generalized Readout of Image Dispersion), is highly generalizable, compatible with open-source image analysis software, and adaptable to diverse biological scenarios. GRID readily quantifies diverse structures and processes to include autophagic puncta, mitochondrial clustering, and microtubule dynamics. Further, GRID is versatile, applicable to both 2D cell cultures and 3D multicellular organoids, and suitable for high-throughput screening and performance metric measurements, such as half-maximal effective concentration (EC50) values. The approach enables mechanistic analysis of critical subcellular structure processes of relevance for diseases ranging from metabolic and neuronal diseases to cancer as well as a first-pass screening method for identifying biologically active agents for drug discovery. Statement of SignificanceCurrent methods for image analysis in microscopy are tailored to specific biological contexts, which creates challenges in implementation and efficiency for researchers studying a diverse range of subcellular processes. Our application of dispersion indices, traditionally used in economics, offers a universal framework for high throughput quantification of biological structures, enabling easier and more consistent analysis across various biological contexts. By transforming pixel intensity and count into meaningful statistical measures, our method simplifies the quantification of subcellular structures such as autophagic puncta, microtubule dynamics, and mitochondrial clustering. This approach accelerates the quantitative analysis of sub-cellular processes in disease as well as imaged-based drug discovery. Classification: Bioengineering, Cell Biology, Applied Biological Sciences

cell biology↗

bioRxiv2024

CXCL12 chemokine dimer signaling modulates acute myelogenous leukemia cell migration through altered receptor internalization

Acute myeloid leukemia (AML) is a malignancy of immature myeloid blast cells with stem-like and chemoresistant cells being retained in the bone marrow through CXCL12-CXCR4 signaling. Current CXCR4 inhibitors that mobilize AML cells into the bloodstream have failed to improve patient survival, likely reflecting persistent chemokine receptor localization on target cells. Here we characterize the signaling properties of CXCL12-locked dimer (CXCL12-LD), a bioengineered variant of the naturally occurring oligomer of CXCL12. CXCL12-LD, in contrast to wild-type or locked monomer variants, was unable to induce chemotaxis in AML cells. CXCL12-LD binding to CXCR4 decreased G protein, {beta}-arrestin, and intracellular calcium mobilization signaling pathways, and indicated the locked dimer is a partial agonist of CXCR4. Despite these partial agonist properties, CXCL12-LD increased CXCR4 internalization compared to wildtype and monomeric CXCL12. Analysis of a previously published AML transcriptomic data showed CXCR4 positive AML cells co-express genes involved in survival, proliferation, and maintenance of a blast-like state. The CXCL12-LD partial agonist effectively mobilized stem cells into the bloodstream in mice suggesting a potential role for their use in targeting CXCR4. Together, our results suggest that enhanced internalization by CXCL12-LD partial agonist can avoid pharmacodynamic tolerance and may identify new avenues to better target G protein coupled receptors.

cancer biology↗

bioRxiv2024

Genomes of nitrogen-fixing eukaryotes reveal a non-canonical model of organellogenesis

Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina: nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.

evolutionary biology↗

bioRxiv2024

A Hybrid Type I and II Polyketide Synthases Yields Distinct Aromatic Polyketides

Bacterial aromatic polyketides are compounds with multiple aromatic rings synthesized by bacterial type II polyketide synthases (PKSs), some of which have been developed into clinical drugs. Compounds containing aromatic polyketides synthesized by a hybrid type I and type II PKSs are extremely rare. Here, we report the discovery of a gene cluster encoding both modular type I PKS, type II PKS and KAS III through extensive bioinformatics analysis, leading to the characterization of the hybrid polyketide, spirocycline A. The structure of spirocycline A is unprecedented among all aromatic polyketides, featuring a unique starter unit, four spirocycles, and forming a dimer. Biosynthetic studies indicate that the starter unit of this molecule is synthesized by type I PKS in collaboration with two trans-acting ketoreductase (KR) and enoylreductase (ER). It is then transferred by KAS III to the type II PKS system, which then synthesizes the tricyclic aromatic polyketide backbone. The subsequent formation of the spirocycle and dimerization is carried out by four redox enzymes encoded in the gene cluster. Overall, the discovery of spirocycline A provides a new approach for identifying novel aromatic polyketides and offers potential enzymatic tools for the bioengineering of these hybrid polyketides. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/610196v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@5a7efaorg.highwire.dtl.DTLVardef@14665d5org.highwire.dtl.DTLVardef@c7b8f2org.highwire.dtl.DTLVardef@3f1e6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Description of a novel extremophile green algae, Chlamydomonas pacifica, and its potential as a biotechnology host

We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42{degrees}C), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, motility and phototaxis. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a GC content of 61%. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/611117v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ead226org.highwire.dtl.DTLVardef@18af631org.highwire.dtl.DTLVardef@291c2borg.highwire.dtl.DTLVardef@f860ac_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2024

Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or S-layer, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle core, a three-helix bundle arm, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

microbiology↗

bioRxiv2024

PIEZO-dependent mechano-sensing of the niche is essential for intestinal stem cell fate decision and maintenance

Stem cells continuously perceive and respond to various environmental signals to maintain homeostasis. In addition to biochemical factors, the stem cell niche is subjected to mechanical and physical cues. However, it remains unclear how stem cells can sense mechanical signals from their niche in vivo. Since intestinal stem cells constantly and directly face the external environment, we investigated the roles of mechano-sensing PIEZO ion channels in the gut stem cell niche. By employing mouse genetics and performing single-cell RNAseq analysis, we revealed the absolute requirement for PIEZO channels in intestinal stem cell (ISC) state dynamics and maintenance. In vivo measurement of basement membrane region stiffness demonstrated that ISCs reside in a more rigid microenvironment at the bottom of the crypt. Using 3D and 2D organoid systems combined with bioengineered substrates and a cell stretching device, we found that PIEZO channels are activated by high extracellular matrix stiffness and tissue tension to modulate ISC behavior. This study delineates the mechanistic cascade of PIEZO channel activation in ISCs from the upstream extracellular stimuli through the downstream signaling activation that coordinates stem cell fate decision and maintenance.

cell biology↗

bioRxiv2024

An Iterative Approach to Polish the Nanopore Sequencing Basecalling for Therapeutic RNA Quality Control

Nucleotide modifications deviate nanopore sequencing readouts, therefore generating artifacts during the basecalling of sequence backbones. Here, we present an iterative approach to polish modification-disturbed basecalling results. We show such an approach is able to promote the basecalling accuracy of both artificially-synthesized and real-world molecules. With demonstrated efficacy and reliability, we exploit the approach to precisely basecall therapeutic RNAs consisting of artificial or natural modifications, as the basis for quantifying the purity and integrity of vaccine mRNAs which are transcribed in vitro, and for determining modification hotspots of novel therapeutic RNA interference (RNAi) molecules which are bioengineered (BioRNA) in vivo.

bioinformatics↗

bioRxiv2024

The Precise Basecalling of Short-Read Nanopore Sequencing

The nanopore sequencing of short sequences, whose lengths are typically less than 0.3kb therefore comparable with Illumina sequencing techniques, has recently gained wide attention. Here, we design a scheme for training nanopore basecallers that are specialized for short biomolecules. With bioengineered RNA (BioRNA) molecules as examples, we demonstrate the superior accuracy of basecallers trained by our scheme.

bioinformatics↗

bioRxiv2024

Mesoscale Modelling of Fibrin Clots: The Interplay between Rheology and Microstructure at the Gel Point

This study presents a numerical model for incipient fibrin-clot formation that captures characteristic rheological and microstructural features of the clot at the gel point. Using a mesoscale-clustering framework, we evaluate the effect of gel concentration or gel volume fraction and branching on the fractal dimension, the gel time, and the viscoelastic properties of the clots. We show that variations in the gel concentration of our model can reproduce the effect of thrombin in the formation of fibrin clots. In particular, the model reproduces the fractal dimensions dependency on gel concentration and the trends in elasticity and gelation time with varying thrombin concentrations. This approach allows us to accurately recreate the gelation point of fibrin-thrombin gels, highlighting the intricate process of fibrin polymerization and gel network formation. This is critical for applications in the clinical and bioengineering fields where precise control over the gelation process is required.

biophysics↗

bioRxiv2024

Rubisco packaging and stoichiometric composition of a native β-carboxysome

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within {beta}-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the {beta}-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in {beta}-carboxysomes, which is instrumental for Rubisco encapsulation and {beta}-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire {beta}-carboxysome. This study and recent findings on the {beta}-carboxysome structure provide insights into the assembly principles and structural variation of {beta}-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

biochemistry↗

bioRxiv2024

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes towards a Purkinje Fiber Fate

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro, demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

developmental biology↗

bioRxiv2024

Robust and highly efficient transformation method for a minimal Mycoplasma cell

Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining of the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, CFU, and transformation efficiency, we found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early-exponential growth phase. Notably, we attained the transformation efficiency up to 4.4 x 10-2 transformants per cell per g plasmid DNA. We developed a method to obtain several hundred to several thousand transformants with less than 0.2 mL of cultured cells and 10 ng of plasmid DNA. Moreover, we established a transformation method using frozen stock of transformation-ready cells. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCEMycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand cellular process of life and for bioengineering because of their simple metabolism, small genomes and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtainable only 10 nanograms of plasmid DNA, which is around one-thousandth amount required for traditional mycoplasma transformations. Moreover, we established a convenient method using frozen stocks of transformation-ready cells. These improved methods play a crucial role in further studies using minimal cells.

microbiology↗

bioRxiv2024

Enzymatic Protein Fusions with 100% Product Yield

The protein ligase Connectase can be used to fuse proteins to small molecules, solid carriers, or other proteins. Compared to other protein ligases, it offers greater substrate specificity, higher catalytic efficiency, and catalyzes no side reactions. However, its reaction is reversible, resulting in only 50% fusion product from two equally abundant educts. Here, we present a simple method to reliably obtain 100% fusion product in 1:1 conjugation reactions. This method is efficient for protein-protein or protein-peptide fusions at the N-or C-termini. It enables the generation of defined and completely labeled antibody conjugates with one fusion partner on each chain. The reaction requires short incubation times with small amounts of enzyme and is effective even at low substrate concentrations and at low temperatures. With these characteristics, it presents a valuable new tool for bioengineering.

biochemistry↗

bioRxiv2024

A multi-step completion process model of cell plasticity

Plasticity is the potential for cells or cell populations to change their phenotypes and behaviours in response to internal or external cues. A common way to study a plasticity programme is to track the underlying molecular changes over time, by collecting omics time-series data. However, there remains a lack of mathematical models to elucidate and predict molecular behaviours in a plasticity programme using omics time-series data. Here we report a new mathematical framework that models cell plasticity as a multi-step completion process, where the system moves from the initial state along a path guided by multiple intermediate attractors until the final state (i.e. a new homeostasis) is reached. In benchmarking tests, we show that our developed Control Point (CP) model fits omics time-series data and identifies attractor states that are well-aligned with prior knowledge. Importantly, we show that the CP model can make non-trivial predictions such as the molecular outcomes of blocking a plasticity programme from reaching completion, in a quantitative and time-resolved manner. Significance statementCell plasticity is fundamental to a wide range of complex biological processes, for example in embryonic development, immunology, regenerative medicine, aging, cancer, and others. We developed a new mathematical model that leverages omics time-series data to understand and make accurate predictions about cell plasticity. This can lead to new discoveries and open up new avenues of research in medicine and bioengineering.

systems biology↗

bioRxiv2024

Tunable Low-Rate Genomic Recombination with Cre-lox in E. coli: A Versatile Tool for Synthetic Biology and Environmental Sensing

The ability to induce heritable genomic changes in response to environmental cues is valuable for environmental biosensing, for experimentally probing microbial ecology and evolution, and for synthetic biology applications. Site-specific recombinases provide a route to genetic memory via targeted DNA modifications, but their high specificity and efficiency are offset by leaky expression and limited tunability in prokaryotes. We developed a tightly regulated, titratable Cre recombinase system for Escherichia coli that achieves low recombination rates and minimal basal activity. Implemented on both plasmids and the chromosome, the latter showed superior retention of genetic memory across generations. These features make the system broadly useful for environmental biosensing and other applications. To demonstrate applicability to environmental biosensing, we developed a whole-cell recombination-based biosensor for arsenite, a toxic and ubiquitous pollutant that is primarily mobilized in anoxic environments such as flooded soils, sediments, and aquifers. However, existing arsenite whole-cell biosensors face limitations in sensitivity and workflow in anaerobic settings. Our biosensor reliably recorded anoxic arsenite exposure as a stable genetic memory for delayed fluorescence readout in aerobic conditions, with detection sensitivity comparable to conventional wet chemical methods. By decoupling exposure from measurement, this approach offers a foundation for arsenite biosensing under field-relevant conditions, including redox variability and other physicochemical gradients, without the constraints of anoxic measurement. More broadly, the ability to induce low-rate, heritable genetic changes expands the genetic toolkit for environmentally responsive systems, with applications in environmental monitoring, bioproduction, bioengineering, as well as experimental studies of microbial ecology, evolution, and host-microbe interactions. IMPORTANCEArsenic is a toxic and globally prevalent pollutant, mobilized primarily under anoxic conditions where detection is challenging. Whole-cell biosensors offer a promising route for monitoring bioavailable arsenic in situ, but their development has largely focused on aerobic conditions, with anoxic assays limited by sensitivity and workflow constraints. Genetic tools that enable heritable, low-frequency genomic changes in bacteria can expand biosensor capabilities by recording transient exposures and supporting applications in environmental monitoring, synthetic biology, and quantitative microbial population dynamics research. Here, we developed a tightly regulated, chemically inducible Cre-lox system in Escherichia coli that enables recombination at low, tunable rates. We demonstrate its utility by constructing an arsenite biosensor that reliably detects low concentrations and records exposures under both aerobic and anoxic conditions. This approach is broadly applicable for biosensors designed for field deployment and for experiments investigating microbial ecology and evolution, where controllable genetic diversification may be desirable.

synthetic biology↗

bioRxiv2024

Light-guided actin polymerization drives directed motility in protocells

Motility is a hallmark of lifes dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, building a minimal platform to test this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 {micro}m/min, within the range of adherent mammalian cells. Notably, our findings reveal the requirements of both branched and linear actin networks for efficient membrane protrusions. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

synthetic biology↗

bioRxiv2024

Building neurovascular tissue from autologous blood for modeling brain-activity

There are no faithful individualized stem cell-based bioengineered neuro-vascularized models that can recapitulate the physiological hemodynamic phenomenon of neuro-vascular coupling (NVC)-the principal behind BOLD (blood oxygen level-dependent) signal in functional neuroimaging, thereby dissuading the research in exploring the brain activity-based investigative studies in neurological/neurosensory diseases. This encouraged us to establish a preclinical optoacoustic (Hb/dHb hemoglobin/deoxyhemoglobin) imaging-competent in vitro neuro-vascularized model by employing a novel cellular reprograming PITTRep (Plasma Induced Transcriptomics/ epi-Transcriptomics Reprograming) approach. The current reprograming approach is based on coaxing autologous blood components to ecto-mesodermal lineage intermediates that can subsequently self-pattern into neurovascular tissue by harnessing the hemorheological properties of RBCs. The nature of blood flow is non-Newtonian and is a function of RBC concentration /haematocrit when they flow through the regions of low shear rates as seen in cerebral microcirculation. The current reprograming approach is a modification of our previous cellular reprograming approach that employed a Newtonian plasma fluid. The autologous blood-derived neurovascular tissue is free from exogenous genetic modification, external growth factors, and induced pluripotent stem cell (iPSC) derivation. This model uniquely integrates functional vasculature and neurogenesis. The current reprogramming approach resulted (in part) serendipitously while testing a potential (yet completely unexplored) hypothesis of haemodynamic reprograming by leveraging the fluid mechanic feature of blood erythrocytes as seen in thrombus formation during cerebral ischemic stroke, that is characterized by physiologically intriguing yet clinically meaningful neurological recovery (neuroplasticity) during an early time window. The current study attempted to induce "a post stroke-like model" of adult neurogenesis with functional synaptogenesis by instructing autologous blood components into thrombus formation through incorporation of erythrocytes in varying concentrations. We tried to instruct adult neurogenesis and neuroplasticity (a relatively non-resilient phenomenon under in vitro conditions) by co-induction of a neuro-vascular niche (NVN). These NVNs are marked by dendrites, synapses, astrogliosis, microglia activation, and growth factor signaling, thus phenocopying molecular and cellular aspects of post-stroke recovery window. The induction of neuro-vascularized niches and functional neuro-vascular coupling (NVC) was characterized by confocal microscopy, scanning electron microscopy, proteomic profiling, and Hb/dHb spectra based optoacoustic imaging. The blood thrombus formation was checked by rotational thromboelastometry (ROTEM), and switching of adult-to-embryonic hemoglobin was confirmed by routine hemoglobin typing. We also attempted to establish patient-specific neuro-vascularized niches from autologous blood of sensorineural hearing loss (SNHL) patients. The individualized neovascularised tissues are intended to be employed for investigating deregulated synaptic plasticity/ long term potentiation underlying poor auditory comprehension outcomes in school going kids suffering from SNHL that greatly compromises their academic performance and socio-behavioural-cognitive development. The attendant multiomics of patient-specific NVNs may have potential implications in developing stem-cell based therapies for neurosensory and cerebrovascular diseases.

neuroscience↗