Miracle-dz/UniFAR-FTUs huggingface dataset:
Toward Cardiac Regeneration: Combination of Pluripotent Stem Cell-Based Therapies and Bioengineering Strategies

Cardiovascular diseases represent the major cause of morbidity and mortality worldwide. Multiple studies have been conducted so far in order to develop treatments able to prevent the progression of these pathologies. Despite progress made in the last decade, current therapies are still hampered by poor translation into actual clinical applications. The major drawback of such strategies is represented by the limited regenerative capacity of the cardiac tissue. Indeed, after an ischaemic insult, the formation of fibrotic scar takes place, interfering with mechanical and electrical functions of the heart. Hence, the ability of the heart to recover after ischaemic injury depends on several molecular and cellular pathways, and the imbalance between them results into adverse remodeling, culminating in heart failure. In this complex scenario, a new chapter of regenerative medicine has been opened over the past 20 years with the discovery of induced pluripotent stem cells (iPSCs). These cells share the same characteristic of embryonic stem cells (ESCs), but are generated from patient-specific somatic cells, overcoming the ethical limitations related to ESC use and providing an autologous source of human cells. Similarly to ESCs, iPSCs are able to efficiently differentiate into cardiomyocytes (CMs), and thus hold a real regenerative potential for future clinical applications. However, cell-based therapies are subjected to poor grafting and may cause adverse effects in the failing heart. Thus, over the last years, bioengineering technologies focused their attention on the improvement of both survival and functionality of iPSC-derived CMs. The combination of these two fields of study has burst the development of cell-based three-dimensional (3D) structures and organoids which mimic, more realistically, the in vivo cell behavior. Toward the same path, the possibility to directly induce conversion of fibroblasts into CMs has recently emerged as a promising area for in situ cardiac regeneration. In this review we provide an up-to-date overview of the latest advancements in the application of pluripotent stem cells and tissue-engineering for therapeutically relevant cardiac regenerative approaches, aiming to highlight outcomes, limitations and future perspectives for their clinical translation.
Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes

CRISPR-Cas systems employ ribonucleoprotein complexes to identify nucleic acid targets with complementarity to bound CRISPR RNAs. Analyses of the high diversification of these effector complexes suggest that they can exhibit a wide spectrum of target requirements and binding affinities. Therefore, streamlined analysis techniques to study the interactions between nucleic acids and proteins are necessary to facilitate the characterization and comparison of CRISPR-Cas effector activities. Bio-layer Interferometry (BLI) is a technique that measures the interference pattern of white light that is reflected from a layer of biomolecules immobilized on the surface of a sensor tip (bio-layers) in real time and in solution. As streptavidin-coated sensors and biotinylated oligonucleotides are commercially available, this method enables straightforward measurements of the interaction of CRISPR-Cas complexes with different targets in a qualitative and quantitative fashion. Here, we present a general method to carry out binding assays with the Type I-Fv complex from Shewanella putrefaciens and the Type I-F complex from Shewanella baltica as model effectors. We report target specificities, dissociation constants and interactions with the Anti-CRISPR protein AcrF7 to highlight possible applications of this technique.
Body size and tree species composition determine variation in prey consumption in a forest‐inhabiting generalist predator

Due to physiological, behavioural or ecological mechanisms, generalist predators may show individual variation in prey consumption. This individual specialization can result from both environmental and trait variation, with especially body size strongly connected to diet. Environmental variation can thus affect consumer-predator interactions by the joint action of changes in prey community composition and predator size. We studied whether and how changes in forest environmental conditions, such as variation in tree species identity and forest fragmentation, affect predator-prey interactions. More specifically we anticipate tree diversity or spatial context to impact the predator diet directly via prey availability and prey size, but also indirectly through shifts in predator size. We used the orb-weaving spider Araneaus diadematus inhabiting forest fragments differing in edge distance, tree diversity and tree species as a model species. The species’ diet was quantified by an unprecedented metabarcoding campaign. A total of 983 spiders were subjected to molecular diet analysis, from in total of 53 forest plots. A. diadematus showed to be a generalist predator, with 298 prey items detected in total. At the individual level, we found large spiders to consume less different species, but consistently larger species. Tree species composition affected both prey species richness and size in the spider’s diet, although tree diversity per se had no influence on the consumed prey. Edges had an indirect effect on the spider diet as spiders closer to the forest edge were larger and therefore consumed larger prey. We attribute the structural complexity of the understory related to tree species composition as a driver of prey composition and its size distribution as reflected in the predator’s diet. Although large spiders may specialize on large prey, we found no firm evidence for either ecological opportunism or strong specialization. Finally, we conclude that intraspecific size variation and tree species composition define the consumed prey of this generalist predator, and that the many feeding interactions of this spider underlie the species’ success in a large diversity of habitats.
Cadherin clusters stabilized by a combination of specific and nonspecific cis-interactions

We demonstrate a combined experimental and computational approach for the quantitative characterization of lateral interactions between membrane-associated proteins. In particular, weak, lateral (cis) interactions between E-cadherin extracellular domains tethered to supported lipid bilayers, were studied using a combination of dynamic single-molecule Förster Resonance Energy Transfer (FRET) and kinetic Monte Carlo (kMC) simulations. Cadherins are intercellular adhesion proteins that assemble into clusters at cell-cell contacts through cis- and trans- (adhesive) interactions. A detailed and quantitative understanding of cis-clustering has been hindered by a lack of experimental approaches capable of detecting and quantifying lateral interactions between proteins on membranes. Here single-molecule intermolecular FRET measurements of wild-type E-cadherin and cis-interaction mutants combined with simulations demonstrate that both nonspecific and specific cis-interactions contribute to lateral clustering on lipid bilayers. Moreover, the intermolecular binding and dissociation rate constants are quantitatively and independently determined, demonstrating an approach that is generalizable for other interacting proteins.
Chronic high dietary sucrose induces sexually dimorphic metabolic adaptations in liver and adipose tissue

Almost all effective treatments for non-alcoholic fatty liver disease (NAFLD) involve reduction of adiposity, which suggests the metabolic axis between liver and adipose tissue is essential to NAFLD development. Since excessive dietary sugar intake may be an initiating factor for NAFLD, we have characterized the metabolic effects of liquid sucrose intake at concentrations relevant to typical human consumption in mice. We report that sucrose intake induces sexually dimorphic effects in liver, adipose tissue, and the microbiome; differences concordant with steatosis severity. We show that when steatosis is decoupled from impairments in insulin responsiveness, sex is a moderating factor that influences sucrose-driven lipid storage and the contribution of de novo fatty acid synthesis to the overall hepatic triglyceride pool. Our findings provide physiologic insight into how sex influences the regulation of adipose-liver crosstalk and highlight the importance of extrahepatic metabolism in the pathogenesis of diet-induced steatosis and NAFLD.
A Biophysical Model for Plant Cell Plate Development

Plant cell division involves de novo formation of a ‘cell plate’ that partitions the cytoplasm of the dividing cell. Cell plate formation is directed by orchestrated delivery of cytokinetic vesicles via the phragmoplast, vesicle fusion, and membrane maturation to the nascent cell wall by the timely deposition of polysaccharides such as callose, cellulose, and crosslinking glycans. In contrast to the role of endomembrane protein regulators the role of polysaccharides, particularly callose, in cell plate development is poorly understood. It has been suggested that the transient accumulation of callose provides an anisotropic spreading force which helps the transition of earlier, membrane-network cell plate stages into a more mature fenestrated sheet stage. Here we present a biophysical model based on the Helfrich free energy for membranes that models this spreading force. We show that proper cell plate development in the model is possible, depending upon the selection of the bending modulus, with a two-dimensional spreading force parameter of between 2 – 6pN/nm, an osmotic pressure difference of 2 – 10kPa, and a range of spontaneous curvature between 0 – 0.04nm−1. With these conditions, we can achieve stable membrane conformations in agreement with observed sizes and morphologies corresponding to intermediate stages of cell plate development. Altogether, our mathematical model predicts that a spreading force generated by callose and/or other polysaccharides, coupled with a concurrent decrease in spontaneous curvature, is vital for the transition of a membrane network to a nearly mature cell plate. Significance Statement Plant cell division features the development of a unique membrane network called the cell plate that matures to a cell wall which separates the two daughter cells. During cell plate development, callose, a β-1-3 glucan polymer, is transiently synthesized at the cell plate only to be replaced by cellulose in mature stages. The role for this transient callose accumulation at the cell plate is unknown. It has been suggested that callose provides mechanical stability, as well as a spreading force that widens and expands tubular and fenestrated cell plate structures to aid the maturation of the cell plate. Chemical inhibition of callose deposition results in the failure of cell plate development supporting this hypothesis. This publication establishes the need for a spreading force in cell plate development using a biophysical model that predicts cell plate development in the presence and the absence of this force. Such models can potentially be used to decipher for the transition/maturation of membrane networks upon the deposition of polysaccharide polymers.
Physical constrains and functional plasticity of cellulases: Linear scaling relationships for a heterogeneous enzyme reaction

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.
Sequencing and annotation of the endangered wild buffalo (Bubalus arnee) mitogenome for taxonomic and hybridization assessment

The wild water buffalo (Bubalus arnee) is one of the most endangered and least studied large bovid in the Indian subcontinent. India retains 90% of the global population as two fragmented populations in Assam and Chhattisgarh, both threatened by habitat loss and degradation, hunting, disease from livestock, and hybridization with the domestic buffalos. For the first time, we sequenced the 16,357 bp long mitogenome of pure wild water buffalo from both populations. The annotated genes included 13 protein-coding genes, 22 tRNA, two ribosomal genes, and a non-coding control region. Comparative mitogenome analyses showed both populations are genetically similar but significantly different from domestic buffalo. We also identified structural differences in seven tRNA secondary structures between both species. Both wild and domestic water buffalo formed sister clades which were paraphyletic to genus Bos. This study provides baseline information on wild buffalo mitogenome for further research on phylogeny, phylogeography and hybrid assessment and help conserving this endangered species.
High-Resolution Metabolic Mapping of the Cerebellum Using a Zoomed Magnetic Resonance Spectroscopic Imaging

Purpose The human cerebellum plays an important role in functional activity cerebrum which is ranging from motor to cognitive activities since due to its relaying role between spinal cord and cerebrum. The cerebellum poses many challenges to magnetic resonance spectroscopic imaging (MRSI) due to the caudal location, the susceptibility to physiological artifacts and partial volume artifact due to its complex anatomical structure. Thus, in present study, we propose a high-resolution MRSI acquisition scheme for the cerebellum. Methods A zoomed or reduced-field of view (rFOV) metabolite-cycled full-intensity magnetic resonance spectroscopic imaging (MRSI) at 3T with a nominal resolution of 62.5 μL was developed. Single-slice rFOV MRSI data were acquired from the cerebellum of 5 healthy subjects with a nominal resolution of 2.5□×□2.5□mm2 in 9□minutes 36. Spectra were quantified with LCModel. A spatially unbiased atlas template of the cerebellum was used for analyzing metabolite distributions in the cerebellum. Results The high quality of the achieved spectra enabled to generate a high-resolution metabolic map of total N-acetylaspartate, total creatine, total choline, glutamate+glutamine and myo-inositol with Cramér-Rao lower bounds below 50%. A spatially unbiased atlas template of the cerebellum-based region of interest (ROIs) analysis resulted in spatially dependent metabolite distributions in 9 ROIs. The group-averaging across subjects in the Montreal Neurological Institute-152 template space allowed to generate a very high-resolution metabolite maps in the cerebellum. Conclusion These findings indicate that very high-resolution metabolite probing of cerebellum is feasible using rFOV or zoomed MRSI at 3T.
A previously uncharacterized gene in SARS-CoV-2 illuminates the functional dynamics and evolutionary origins of the COVID-19 pandemic

Understanding and preventing the emergence of novel viruses requires an accurate and comprehensive understanding of their genomes. One under-investigated class of functional genomic elements is overlapping genes (OLGs), which allow a single stretch of nucleotides to encode two distinct proteins in different reading frames. Viral OLGs are common and have been associated with the origins of pandemics, but are still widely overlooked. We investigate ​de novo OLG candidates in SARS-CoV-2 and identify a new gene here named ORF3c​. ​ORF3c has been documented elsewhere but is unnamed, unannotated, or conflated with ​ORF3b of other SARS-related betacoronaviruses (sarbecoviruses). In fact, ​ORF3c is not homologous to ​ORF3b​, as the two genes occupy different genomic positions and reading frames. We find that ​ORF3c exhibits clear ​evidence of translation from ribosome profiling and important immunological properties. We then conduct an evolutionary analysis of ORF3c at three levels: between-species, between-host, and within-host. Specifically, 21 representative sarbecovirus genomes show ​ORF3c is also present in some pangolin-CoVs but not more closely related bat-CoVs; 3,978 SARS-CoV-2 genomes reveal ​ORF3c gained a new stop codon (G25563U) that rose drastically in frequency during the current COVID-19 pandemic; and 401 deeply sequenced samples of SARS-CoV-2 demonstrate the recurrence of this mutation in multiple hosts. Surprisingly, the newly gained ​ORF3c stop codon hitchhiked early with haplotype 241U/3037U/14408U/23403G (Spike-D614G), which appears to drive the European pandemic spread. Our results liken ​ORF3c to other important viral accessory genes recombined, lost, split, or truncated before or during outbreaks, including ​ORF3b and ​ORF8 in sarbecoviruses. OLGs deserve considerably more attention, as their rapid evolution may be more important than is currently appreciated in the emergence of zoonotic viruses.
Osteoarthritis: articular chondrocyte inflammatory signaling leads to enhanced gene expression and function of mechanotransduction channel Piezo1 as a pathogenic feed-forward mechanism

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies (1, 2). We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury (3, 4). We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) upregulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+, at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically-induced deformation-microtrauma. As intracellular substrates of this novel OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. In ensemble, these signaling and genetic re-programming events represent a novel and detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA. Significance Statement Osteoarthritis affecting weight-bearing joints is a global health problem, causing loss of mobility and enormous healthcare costs. Disease-modifying approaches are lacking. Here, we report a new cellular mechanism of inflammatory signaling in chondrocytes, the cellular substrate of cartilage. We show how osteoarthritis-relevant levels of interleukin-1α reprogram articular chondrocytes so that they become more susceptible to mechanical trauma, which chondrocytes sense via Piezo1/2 mechanosensitive ion channels. We uncover that IL-1α enhances gene expression of Piezo1 in primary articular chondrocytes underlying Piezo1 gain-of-function. We elucidate the new signaling pathway, from membrane to nucleus, including transcription factors that enhance Piezo1-expression. We also define detrimental effects of gain-of-function of Piezo1, for mechanotransduction and at-rest, that suggest this new reprogramming mechanism to contribute to osteoarthritis pathogenesis.
Democratising “Microscopi”: a 3D printed automated XYZT fluorescence imaging system for teaching, outreach and fieldwork

Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing “Microscopi”, an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device. The light path can easily be switched between different imaging modalities. The open source Python-based control software allows the hardware to be driven as an integrated imaging system. Furthermore, the microscope is fully customisable, which also enhances its value as a learning tool. Here, we describe the basic design and demonstrate imaging performance for a range of easily sourced specimens. Highlights Portable, low cost, self-build from 3D printed and commodity components Multimodal imaging: bright field, dark field, pseudo-phase and fluorescence Automated XYZt imaging from a tablet or smartphone via a simple GUI Wide ranging applications in teaching, outreach and fieldwork Open source hardware and software design, allowing user modification
Influence of Supraphysiologic Biomaterial Stiffness on Ventricular Mechanics and Myocardial Infarct Reinforcement

Injectable intramyocardial biomaterials have promise to limit adverse ventricular remodeling through mechanical and biologic mechanisms. While some success has been observed by injecting materials to regenerate new tissue, optimal biomaterial stiffness to thicken and stiffen infarcted myocardium to limit adverse remodeling has not been determined. In this work, we present an in-vivo study of the impact of biomaterial stiffness over a wide range of stiffness moduli on ventricular mechanics. We utilized injectable methacrylated polyethylene glycol (PEG) hydrogels fabricated at 3 different mechanical moduli: 5 kPa (low), 25 kPa (medium/myocardium), and 250 kPa (high/supraphysiologic). We demonstrate that the supraphysiological high stiffness favorably alters post-infarct ventricular mechanics and prevents negative tissue remodeling. Lower stiffness materials do not alter mechanics and thus to be effective, must instead target biological reparative mechanisms. These results may influence rationale design criteria for biomaterials developed for infarct reinforcement therapy.
Chromosome-scale genome assembly of Fusarium oxysporum strain Fo47, a fungal endophyte and biocontrol agent

Here, we report a chromosome-level genome assembly of Fusarium oxysporum strain Fo47 (12 pseudomolecules; contig N50: 4.52Mb), generated using a combination of PacBio long-read, Illumina pair-ended and Hi-C sequencing data. Although F. oxysporum causes vascular wilt to over 100 plant species, the strain Fo47 is classified as an endophyte and widely used as a biocontrol agent for plant disease control. The Fo47 genome carries a single accessory chromosome of 4.23 Mb, compared to the reference genome of F. oxysporum f.sp. lycopersici strain Fol4287. The high-quality assembly and annotation of the Fo47 genome will be a valuable resource for studying the mechanisms underlying the endophytic interactions between F. oxysporum and plants, as well as deciphering the genome evolution of the F. oxysporum species complex.