Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes

Jefferson Digital Commons quarterly report: January-March 2020

This quarterly report includes: New Look for the Jefferson Digital Commons Articles COVID-19 Working Papers Educational Materials From the Archives Grand Rounds and Lectures JeffMD Scholarly Inquiry Abstracts Journals and Newsletters Master of Public Health Capstones Oral Histories Posters and Conference Presentations What People are Saying About the Jefferson the Digital Common

High-content analysis of sequential events during the early phase of influenza A virus infection

Influenza A virus (IAV) represents a worldwide threat to public health by causing severe morbidity and mortality every year. Due to high mutation rate, new strains of IAV emerge frequently. These IAVs are often drug-resistant and require vaccine reformulation. A promising approach to circumvent this problem is to target host cell determinants crucial for IAV infection, but dispensable for the cell. Several RNAi-based screens have identified about one thousand cellular factors that promote IAV infection. However, systematic analyses to determine their specific functions are lacking. To address this issue, we developed quantitative, imaging-based assays to dissect seven consecutive steps in the early phases of IAV infection in tissue culture cells. The entry steps for which we developed the assays were: virus binding to the cell membrane, endocytosis, exposure to low pH in endocytic vacuoles, acid-activated fusion of viral envelope with the vacuolar membrane, nucleocapsid uncoating in the cytosol, nuclear import of viral ribonucleoproteins, and expression of the viral nucleoprotein. We adapted the assays to automated microscopy and optimized them for high-content screening. To quantify the image data, we performed both single and multi-parametric analyses, in combination with machine learning. By time-course experiments, we determined the optimal time points for each assay. Our quality control experiments showed that the assays were sufficiently robust for high-content analysis. The methods we describe in this study provide a powerful high-throughput platform to understand the host cell processes, which can eventually lead to the discovery of novel anti-pathogen strategies

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in aphosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidasemember of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. Anull mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene inLeishmaniamajor. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigotestages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds asynthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] as shown in an electrophoretic mobility shiftassay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved intranslation initiation and elongation. Significantly, we found a strong reduction ofde novoprotein biosynthesis in transgenicparasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentiallyimplicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.Fil: Norris Mullins, Brianna. University Of Notre Dame-Indiana; Estados UnidosFil: VanderKolk, Kaitlin. University Of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Joyce, Michelle V.. University Of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University Of Notre Dame-Indiana; Estados Unido

Role of a bacterial dynamin-like protein DynA in resistance to environmental stress response

Many bacterial species contain dynamin-like proteins (DLPs). DLPs show a high level of functional diversification. DynA in Bacillus subtilis is a two-headed DLP, mediating nucleotide-independent membrane tethering in vitro and contributing to the innate immunity of bacteria against membrane stress and phage infection. However, so far, the functional mechanisms of the bacterial DLP are poorly understood. Here, the biochemical characteristics of DynA-induced membrane fusion, the mechanisms of DynA-mediated phage resistance and protection effect to antibiotic treatment. To uncover the biochemical characteristics of DynA-induced membrane fusion, we employed content mixing and lipid mixing assays in reconstituted systems to study if DynA induces membrane full fusion. Further, the individual contribution of its subunits in membrane fusion and the role of GTP hydrolysis of DynA were analysed in detail. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in the absence of GTP. Moreover, DynA-induced membrane fusion in vitro was a thermo-promoted slow response and showed phospholipid preferences. The D1 subunit of DynA was crucial for membrane fusion, whereas it cannot stabilize the tethered membrane without the help of the D2 subunit. At low temperatures, GTPase activity promoted dissociation of membrane clusters induced by DynA. Surprisingly, the digestion of DynA after hemifusion mediated an instant rise of content exchange, supporting the assumption that disassembly of DynA is the elemental power for fusion-through-hemifusion. Next, the role of DynA in phage resistance was analyzed. We found that the presence of DynA could delay cell lysis after phage infection, thereby slowing down the release of phage progeny from the host cells. During the process, DynA formed static clusters on the cell membrane. It seems that DynA supports the cell membrane and delays rupture. Using transmission electron microscopy (TEM), the protein structure of DynA in solution was analyzed. DynA likely formed dimers and trimers, showing a four-corner and hexagonal shape. In a screening approach, we searched for novel genes that could have a role in phage defence. A B. subtilis 168 single-gene deletion library was screened. DagK and ypmB were found to be related to bacteriophage resistance. The presence of DagK can help bacteria resist phage attachment, replication, and host cell lysis. And the role of YpmB against bacteriophages needs to be further verified and characterized.Viele Bakterienarten enthalten Dynamin-ähnliche Proteine (DLPs). DLPs weisen ein hohes Maß an funktionaler Diversifikation auf. DynA in Bacillus subtilis ist ein zweiköpfiges DLP, das in vitro die nukleotidunabhängige Membranbindung vermittelt und zur angeborenen Immunität von Bakterien gegen Membranstress und Phageninfektion beiträgt. Bisher sind die Funktionsmechanismen des bakteriellen DLP jedoch kaum bekannt. Hier wurden die biochemischen Eigenschaften der DynA-induzierten Membranfusion, die Mechanismen der DynA-vermittelten Phagenresistenz und die Schutzwirkung gegen Antibiotika in Detail untersucht. Um die biochemischen Eigenschaften der DynA-induzierten Membranfusion aufzudecken, verwendeten wir Inhaltsmischungs- und Lipidmischungsassays in rekonstituierten Systemen, um zu untersuchen, ob DynA die vollständige Membranfusion induziert. Ferner wurden der individuelle Beitrag seiner Untereinheiten zur Membranfusion und die Rolle der GTP-Hydrolyse von DynA detailliert analysiert. Unsere Ergebnisse basierend auf dem Fluoreszenzresonanzenergietransfer (FRET) zeigten, dass DynA selbst in Abwesenheit von GTP eine Vermischung des Liposomeninhalts induzieren kann. Darüber hinaus war die DynA-induzierte Membranfusion in vitro eine thermisch geförderte langsame Reaktion und zeigte Phospholipidpräferenzen. Die D1-Untereinheit von DynA war entscheidend für die Membranfusion, wohingegen sie die angebundene Membran ohne die Hilfe der D2-Untereinheit nicht stabilisieren kann. Bei niedrigen Temperaturen förderte die GTPase-Aktivität die Dissoziation von durch DynA induzierten Membranclustern. Überraschenderweise vermittelte der Verdau von DynA nach der Hemifusion einen sofortigen Anstieg des Inhaltsaustauschs, was die Annahme stützt, dass die Zerlegung von DynA die elementare Kraft für die Fusion durch Hemifusion ist. Als nächstes wurde die Rolle von DynA bei der Phagenresistenz analysiert. Wir fanden heraus, dass das Vorhandensein von DynA die Zelllyse nach einer Phageninfektion verzögern und dadurch die Freisetzung von Phagennachkommen aus den Wirtszellen verlangsamen kann. Während des Prozesses bildete DynA statische Cluster auf der Zellmembran. Es scheint, dass DynA die Zellmembran unterstützt und den Bruch verzögert. Unter Verwendung eines Transmissionselektronenmikroskops (TEM) wurde die Proteinstruktur von DynA in Lösung analysiert. DynA bildete wahrscheinlich Dimere und Trimere, die eine viereckige und sechseckige Form zeigten. In einem Screening-Ansatz suchten wir nach neuen Genen, die eine Rolle bei der Phagenabwehr spielen könnten. Eine B. subtilis 168-Einzelgen-Deletionsbibliothek wurde gescreent. Es wurde festgestellt, dass dagK und ypmB mit der Bakteriophagenresistenz zusammenhängen. Das Vorhandensein von DagK kann Bakterien helfen, die Anhaftung, und die Replikation zu behindern. Zudem wird die Lyse von Wirtszellen vermindert. Und die Rolle von YpmB gegen Bakteriophagen muss weiter verifiziert und charakterisiert werden

Gait Verification using Knee Acceleration Signals

A novel gait recognition method for biometric applications is proposed. The approach has the following distinct features. First, gait patterns are determined via knee acceleration signals, circumventing difficulties associated with conventional vision-based gait recognition methods. Second, an automatic procedure to extract gait features from acceleration signals is developed that employs a multiple-template classification method. Consequently, the proposed approach can adjust the sensitivity and specificity of the gait recognition system with great flexibility. Experimental results from 35 subjects demonstrate the potential of the approach for successful recognition. By setting sensitivity to be 0.95 and 0.90, the resulting specificity ranges from 1 to 0.783 and 1.00 to 0.945, respectively

Morphological Profiling for Drug Discovery in the Era of Deep Learning

Morphological profiling is a valuable tool in phenotypic drug discovery. The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high-throughput. These efforts have facilitated understanding of compound mechanism-of-action (MOA), drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering- and deep learning-based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.Comment: 44 pages, 5 figure, 5 table