Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

Respiratory virus surveillance in hospitalized children less than two-years of age in Kenema, Sierra Leone during the COVID-19 pandemic (October 2020- October 2021).

Globally, viral pathogens are the leading cause of acute respiratory infection in children under-five years. We aim to describe the epidemiology of viral respiratory pathogens in hospitalized children under-two years of age in Eastern Province of Sierra Leone, during the second year of the SARS-CoV-2 pandemic. We conducted a prospective study of children hospitalized with respiratory symptoms between October 2020 and October 2021. We collected demographic and clinical characteristics and calculated each participant´s respiratory symptom severity. Nose and throat swabs were collected at enrollment. Total nucleic acid was purified and tested for multiple respiratory viruses. Statistical analysis was performed using R version 4.2.0 software. 502 children less than two-years of age were enrolled. 376 (74.9%) had at least one respiratory virus detected. The most common viruses isolated were HRV/EV (28.2%), RSV (19.5%) and PIV (13.1%). Influenza and SARS-CoV-2 were identified in only 9.2% and 3.9% of children, respectively. Viral co-detection was common. Human metapneumovirus and RSV had more than two-fold higher odds of requiring O2 therapy while hospitalized. Viral pathogen prevalence was high (74.9%) in our study population. Despite this, 100% of children received antibiotics, underscoring a need to expand laboratory diagnostic capacity and to revisit clinical guidelines implementation in these children. Continuous surveillance and serologic studies among more diverse age groups, with greater geographic breadth, are needed in Sierra Leone to better characterize the long-term impact of COVID-19 on respiratory virus prevalence and to better characterize the seasonality of respiratory viruses in Sierra Leone

Respiratory virus detection by month (October 2020 to October 2021).

Respiratory virus detection by month (October 2020 to October 2021).</p

Total number of viral pathogens identified and their associated co-detected viruses.

Total number of viral pathogens identified and their associated co-detected viruses.</p

Demographic and clinical characteristics of children under two-years, by whether a virus was detected on PCR, hospitalized with respiratory symptoms, Kenema, October 2020-October 2021.

Demographic and clinical characteristics of children under two-years, by whether a virus was detected on PCR, hospitalized with respiratory symptoms, Kenema, October 2020-October 2021.</p

This file is the inclusivity in global research checklist.

This file is the inclusivity in global research checklist.</p

Demographic and clinical characteristics of children under two-years hospitalized with respiratory symptoms, Kenema, October 2020-October 2021.

Demographic and clinical characteristics of children under two-years hospitalized with respiratory symptoms, Kenema, October 2020-October 2021.</p