Search

Background\ud SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown.\ud \ud Methods\ud In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR.\ud \ud Results\ud A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding.\ud \ud Conclusions\ud Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2.

Purpose Laminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing. Methods Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell–associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging. Results LaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities. Conclusions These data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

The laminin N terminus (LaNt) proteins are a family of laminin and netrin related proteins derived by alternative splicing from laminin encoding genes. There are few reports to date on LaNt protein function. However, one family member, LaNt α31, has been shown to regulate epidermal keratinocyte migration and adhesion, although the mechanism for these effects is unknown. Based on its protein architecture, we predicted that LaNt α31 would influence laminin organisation. To test this prediction, we induced expression of GFP tagged LaNt α31 via adenoviral delivery into human epidermal and corneal keratinocytes. Consistent with the LaNt/laminin interaction hypothesis, LaNt α31 GFP codistributed with laminin β3 in the extracellular matrix and formed a complex together as indicated by co-immunoprecipitation, while live cell assays revealed the proteins to be deposited together during new matrix synthesis. Moreover, the induced expression of LaNt α31 led to changes to laminin α3 organisation into tight clusters compared with wide arcs in control cells. These changes were associated with early maturation of hemidesmosomes and mislocalization of focal adhesion complexes. At the cellular level, epithelial cells expressing LaNt α31 GFP displayed decreased scratch closure and single cell motility rates and increased cell spread area. These differences were rescued through the provision of a pre-formed cell-derived matrix. Together these data identify a new mechanism that influences the early stages of laminin matrix assembly and adds the LaNt proteins as new players in defining cell-to-matrix adhesion and migration characteristics. Grant support This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BB/L020513/1, KH), Fight For Sight (New lecturers’ award, KH), British Skin Foundation (PhD studentship, KH) and National Institute of Arthritis Musculoskeletal and Skin (K99/R00 1K99AR060242, KH). Highlights LaNt α31 is a new mediator of laminin-dependent processes. LaNt α31 codistributes and is deposited with laminin 332 in the extracellular matrix of epithelial cells. Increased LaNt α31 expression leads to changes in laminin 332 organisation into tight clusters compared with broad tracks. Epithelial cells expressing increased LaNt α31 exhibit early hemidesmosome maturation and mislocalization of focal adhesion complexes. LaNt α31 overexpression decreases scratch wound closure rates and single cell migration rates of epithelial keratinocytes which is rescues through the provision of a preformed matrix. This manuscript identifies a new way in which laminin deposition and organisation is controlled, via LaNt α31. Specifically, these data demonstrate that this relatively unstudied laminin-gene splice isoform, induces the formation of laminin 332 clusters during times of new matrix synthesis, leading to changes in cell-matrix adhesion and behaviour.

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.