Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants.

The emergence of new escape mutants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-angiotensin-converting enzyme 2 interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif of the spike protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, using mCherry fluorescence-based reporter assay, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.

Presence of actin binding motif in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking.

Type VI secretion systems (T6SS) plays a crucial role in Vibrio cholerae mediated pathogenicity. Tip of T6SS is homologous to gp27/gp5 complex or tail spike of T4 bacteriophage. VgrG-1 of V. cholerae T6SS is unusual among other VgrG because its effector domain is trans-located into the cytosol of eukaryotic cells with an additional actin cross-linking domain (ACD) at its C terminal end. ACD of VgrG-1 (VgrG-1-ACD) causes T6SS dependent host cell cytotoxicity through actin cytoskeleton disruption to prevent bacterial engulfment by macrophages. ACD mediated actin cross-linking promotes survival of the bacteria in the small intestine of humans, along with other virulence factors; establishes successful infection with the onset of diarrhoea in humans. Our studies demonstrated VgrG-1-ACD can bind to actin besides actin cross-linking activity. Computational analysis of ACD revealed the presence of actin binding motif (ABM). Mutations in ABM lead to loss of actin binding in vitro. VgrG-1-ACD having the mutated ABM cannot cross-link actin efficiently in vitro and manifests less actin cytoskeleton disruption when transfected in HeLa cells.