Interactions of SARS-CoV-2 and MERS-CoV fusion peptides measured using single-molecule force methods.

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

Formation of versus Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity.

Liquid-liquid phase separation (LLPS), the spontaneous formation of contiguous liquid phases with distinct compositions, has been long known in chemical systems and more recently recognized as a ubiquitous feature of cell biology. We describe a system involving biologically relevant components, synthetic peptides, and total yeast RNA, that has enabled us to explore factors that underlie phase separation. Coulombic complementarity between a cationic peptide and anionic RNA is necessary but not sufficient for formation of a condensed phase in our system. In addition to a net positive charge, the peptide must present the proper type of cationic moiety. Guanidinium groups, as found in the Arg side chain, support phase separation, but ammonium groups, as found in the Lys side chain, or dimethylguanidinium groups, as found in post-translationally modified Arg side chains, do not support phase separation in our system. However, the cationic groups that do not support phase separation via interaction with RNA can nevertheless enable recruitment to a condensed phase, which reveals that the network of forces governing condensed phase formation can differ from the network of forces governing recruitment to such a phase. We introduce a new method for measuring the concentrations of components in condensed phases based on fluorine-containing additives and 19F NMR.

Exploring long-term volunteerism in a community family service centre in Singapore: A Focused Ethnographic Study.

Volunteerism contributes significantly to the social development of a country. Although the rate of volunteerism has steadily increased over the years, the numbers of regular volunteers remains small. While the existing literature has elucidated individuals' motivations for volunteerism, research is lacking on their motivations and challenges in sustaining long-term volunteerism. A focused ethnographic approach was adopted in this study to explore 20 participants' motivations and challenges towards long-term volunteerism. Purposive sampling was used to recruit participants in one single-family service centre in Singapore from October to December 2018. Data were collected through covert observations and semi-structured interviews. Field notes, observational data and findings from the interviews were triangulated and analysed through thematic analysis. In this study, a central exhaustive description of the volunteers 'providing help while receiving good deeds' is established. This was supported with three main themes revolving around the volunteers: fulfilling life goals, deriving meaning from experiences and striking a balance in the life. These themes characterised the motivations and challenges faced by them amidst their voluntary works. They reported that the satisfaction and fulfilment through volunteering had brought personal growth, well-being and happiness to them. The sense of purpose through volunteering further enhanced their experiences. Finally, some volunteers reported that volunteering enabled them to strike a balance in their own life by engaging it as a form of solace. The findings suggest that sustained volunteerism is a multidimensional construct involving the interplay of different factors in the individual's life. By understanding motivations underlying long-term volunteerism, voluntary organisations can create volunteering opportunities that dovetail with volunteers' personal goals, thus boosting their satisfaction and incentivising their sustained engagement. Additionally, the organisations can hold regular bonding activities to promote rapport among their volunteers, beneficiaries and staff, thus encouraging the volunteers to persevere on their volunteering journey.

Targeting connexin43 expression accelerates the rate of wound repair.

The repair of tissue damage is a key survival process in all organisms and involves the coordinated activation of several cell types. Cell-cell communication is clearly fundamental to this process, and a great deal is known about extracellular communication within the wound site via cytokines. Here we show that direct cell-cell communication through connexin 43 (Cx43) gap junction channels also plays a major role in the wound healing process. In two different wound healing models, incisional and excisional skin lesions, we show that a single topical application of Cx43 antisense gel brings about a transient downregulation of Cx43 protein levels, and this results in a dramatic increase in the rate of wound closure. Cx43 knockdown reduces inflammation, seen both macroscopically, as a reduction in swelling, redness, and wound gape, and microscopically, as a significant decrease in neutrophil numbers in the tissue around the wound. One long-term consequence of the improved rate of healing is a significant reduction in the extent of granulation tissue deposition and the subsequent formation of a smaller, less distorted, scar. This approach is likely to have widespread therapeutic applications in other injured tissues and opens up new avenues of research into improving the wound healing process.

Dynamic changes in connexin expression correlate with key events in the wound healing process.

Wound healing is a complex process requiring communication for the precise co-ordination of different cell types. The role of extracellular communication through growth factors in the wound healing process has been extensively documented, but the role of direct intercellular communication via gap junctions has scarcely been investigated. We have examined the dynamics of gap junction protein (Connexins 26, 30, 31.1 and 43) expression in the murine epidermis and dermis during wound healing, and we show that connexin expression is extremely plastic between 6 hours and 12 days post-wounding. The immediate response (6 h) to wounding is to downregulate all connexins in the epidermis, but thereafter the expression profile of each connexin changes dramatically. Here, we correlate the changing patterns of connexin expression with key events in the wound healing process.