Neurological disorders of COVID-19 can be explained in terms of “loss or gain of function” of a solution for the nervous system

Background: Covid-19 infection causes several neurological disorders. Even though it is still not known how the brain works, there are models of its operational mechanism. Semblance hypothesis has derived a mechanism of nervous system functions whose keystone structural change is inter-postsynaptic LINK (IPL) formed by a spectrum of interactions between spines that belong to different neurons. These interactions range from close contact between membranes of spines to partial and complete hemifusion between them.Recent developments: Covid-19 spike protein is a fusion protein that in addition to generating fusion pores in the host cell membrane for viral entry, also forms inter-cellular fusion between host cells. When virus exits a cell by vesicle exocytosis, it can cause fusion of the hemifused area of IPL, which is a bilayer structure,. An adaptation mechanism that prevents progression of hemifusion to fusion is expected to deteriorate during aging and may prevent reversal of IPL fusion. Since analyses have shown that transcriptomes of even adjacent neurons of same type are different in the cortex, inter-cellular fusion can lead to protein precipitation within spines that can lead to spine loss and eventual neuronal death. Furthermore, formation of non-specific IPLs can lead to “gain of function” changes responsible for some of the neurological features of COVID-19. Where next? Since neurological symptoms of COVID-19 can be explained in terms of “loss or gain of function” of the derived structure-function mechanism of IPL, it motivates undertaking verification of IPLs. Expected size of an IPL is nearly ten square nanometers. It is necessary to conduct dedicated studies using advanced microscopic methods to verify formation of IPLs and invent methods to study units of inner sensations at the inter-LINKed spines and their computations.

Grafting Methionine on 1F1 Ab Increases the Broad-Activity on HA Structural-Conserved Residues of H1, H2, and H3 Influenza a Viruses

A high level of mutation enables the influenza A virus to resist antibiotics previously effective against the influenza A virus. A portion of the structure of hemagglutinin HA is assumed to be well-conserved to maintain its role in cellular fusion, and the structure tends to be more conserved than sequence. We designed peptide inhibitors to target the conserved residues on the HA surface, which were identified based on structural alignment. Most of the conserved and strongly similar residues are located in the receptor-binding and esterase regions on the HA1 domain In a later step, fragments of anti-HA antibodies were gathered and screened for the binding ability to the found conserved residues. As a result, Methionine amino acid got the best docking score within the −2.8 Å radius of Van der Waals when it is interacting with Tyrosine, Arginine, and Glutamic acid. Then, the binding affinity and spectrum of the fragments were enhanced by grafting hotspot amino acid into the fragments to form peptide inhibitors. Our peptide inhibitor was able to form in silico contact with a structurally conserved region across H1, H2, and H3 HA, with the binding site at the boundary between HA1 and HA2 domains, spreading across different monomers, suggesting a new target for designing broad-spectrum antibody and vaccine. This research presents an affordable method to design broad-spectrum peptide inhibitors using fragments of an antibody as a scaffold.

Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3’UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3’UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif “AUUUA”. Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3’UTR, indicating that the AU-rich elements of the 3’UTR are not contributing to RNA stability. Instead, the three “AUUUA” motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Detecting and controlling dye effects in single-virus fusion experiments

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.Statement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

A mechanism of nervous system functions capable to have evolved: by generating an inducible variant of inter-cellular fusion

A realistic hope has been that from ontogeny, it may become possible to understand the steps of evolutionary changes that may assist in understanding the mechanism of nervous system functions. However, explaining how first-person internal sensations are formed in the nervous system makes this approach very difficult. In this context, if it becomes possible to derive an operational principle by using constraints from observations at different levels, then it will enable examining whether it is possible to arrive at its specific circuit features from single neuronal cells using simple steps of introducing variations and selection. In this context, semblance hypothesis is examined. Inter-neuronal inter-spine interaction leading to the formation of inter-postsynaptic functional LINK (IPL) is necessary for generating units of internal sensations and their computation. The results show its suitability as an evolved mechanism. Significant neuronal and spine loss during ontogeny indicate that following these events, IPLs resulted from the selection of a suitable variation. This can only be achieved through transient inter-neuronal inter-spine fusion, which leads to inducible molecular changes for keeping the spines separate by arresting fusion at the stage of hemifusion. The importance of sustaining this one-time induced IPL mechanism for retaining cognitive functions throughout life is discussed.

Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion

Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic compartment, and release the cargo for degradation. The mechanism of autophagosome–vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome–vacuole fusion. We found that this process requires the Atg14–Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome–vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process.

The microprotein Minion controls cell fusion and muscle formation

Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, the functional repertoire of this microproteome remains to be determined1. In skeletal muscle, proper development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process2,3. Here we report the identification of a small ORF encoding an essential skeletal muscle specific microprotein we term Minion (microprotein inducer of fusion). Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally with marked reduction in fused muscle fibers. This fusogenic activity is conserved to the human Minion ortholog, previously annotated as a long noncoding RNA. Loss-of-function studies demonstrate that Minion is the factor providing muscle specific fusogenic function for the transmembrane protein Myomaker4. Remarkably, we demonstrate that co-expression of Minion and Myomaker is sufficient to induce rapid cytoskeletal rearrangement and homogeneous cellular fusion, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component program for the induction of mammalian cell fusion, enabling both research and translational applications. Importantly, these data also significantly expand the known functions of smORF-encoded microproteins, an under-explored source of proteomic diversity.

Abstract 362: Deletion of Gata4 and Gata6 in the c-Kit Lineage Augments Cellular Fusion in vivo

Cell therapy for treatment of ischemic heart disease has been under rigorous evaluation in recent years. Heart-derived c-Kit cells showed promising results in clinical trials with scar reduction and improved ejection fraction following coronary infusion. We previously developed a c-Kit lineage tracing model showing that these cells very rarely convert into de novo cardiomyocytes. To potentially reduce this rate to zero, and unequivocally evaluate cellular fusion processes in vivo, we deleted the cardiogenic transcription factors Gata4 and Gata6 in c-Kit cells using a Cre-loxP approach (Kit-Gata4/6 KO). We used the tamoxifen inducible Kit-MerCreMer allele crossed into Gata4/6 homozygous LoxP targeted background and the Rosa26-eGFP reporter, which were given tamoxifen at weaning to delete Gata4 and Gata6 in all c-Kit expressing cells and show them and their progeny as eGFP positive. Unexpectedly, we observed a greater than 10-fold increase in Kit lineage-traced cardiomyocytes in some Kit-Gata4/6 KO mice compared to Kit only controls with up to 4 months of treatment. Exploration of other tissues revealed a dramatic increase in presumed Kit-lineage traced skeletal muscle fibers in Kit-Gata4/6 KO mice as well. However, investigation of this effect suggested that this increase in presumed Kit-lineage traced cardiomyocytes and skeletal muscle fibers was due to an alteration in the immune cell compartment of these mice that generated greater rates of fusion. Indeed, analysis of Kit-derived hematopoietic lineages present in the heart showed a 6-fold increase in leukocyte infiltration (CD45 + ) and a 60-fold increase in dendritic cells (CD11c + ) from Kit-Gata4/6 KO mice. Kit-eGFP + bone marrow was also transplanted into mice expressing a membrane tomato reporter. Kit-Gata4/6 KO bone marrow showed a 3-fold increase in Kit-eGFP cells, although all these myocytes had the membrane tomato tag indicting that these events were all due to fusion. Hence, alterations in c-Kit cell activity likely impacts the activity of the immune system and the fusigenic activity of derived cells.