Uncovering the gaps: A systematic mixed studies review of quality of life measures in extremity soft tissue sarcoma.

The health-related quality of life of patients with extremity soft tissue sarcoma (STS) is not precisely captured by current patient-reported outcome measures. Although functional impairment is central to their concerns, multiple sources of distress, emotional restoration, coping strategies, and somatic symptoms are crucial in approaching patients with extremity STS.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Integrating intracellular nanovesicles into integrin trafficking pathways and beyond.

Membrane traffic controls the movement of proteins and lipids from one cellular compartment to another using a system of transport vesicles. Intracellular nanovesicles (INVs) are a newly described class of transport vesicles. These vesicles are small, carry diverse cargo, and are involved in multiple trafficking steps including anterograde traffic and endosomal recycling. An example of a biological process that they control is cell migration and invasion, due to their role in integrin recycling. In this review, we describe what is known so far about these vesicles. We discuss how INVs may integrate into established membrane trafficking pathways using integrin recycling as an example. We speculate where in the cell INVs have the potential to operate and we identify key questions for future investigation.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Intracellular nanovesicles mediate α5ß1 integrin trafficking during cell migration.

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5ß1 integrins in INVs.

Viral use and subversion of membrane organization and trafficking.

Membrane trafficking is an essential cellular process conserved across all eukaryotes, which regulates the uptake or release of macromolecules from cells, the composition of cellular membranes and organelle biogenesis. It influences numerous aspects of cellular organisation, dynamics and homeostasis, including nutrition, signalling and cell architecture. Not surprisingly, malfunction of membrane trafficking is linked to many serious genetic, metabolic and neurological disorders. It is also often hijacked during viral infection, enabling viruses to accomplish many of the main stages of their replication cycle, including entry into and egress from cells. The appropriation of membrane trafficking by viruses has been studied since the birth of cell biology and has helped elucidate how this integral cellular process functions. In this Review, we discuss some of the different strategies viruses use to manipulate and take over the membrane compartments of their hosts to promote their replication, assembly and egress.

Psychological functioning, coping styles and their relationship to appraisal of physical limitations following invasive surgical procedures for soft-tissue sarcoma: A qualitative study.

BACKGROUND AND OBJECTIVES: This study explored psychological functioning and coping styles in adult patients with soft-tissue sarcoma who underwent surgical procedures in a single expert sarcoma medical center in Canada. METHODS: This is a qualitative study with three formats of data collection. The interview guide was based on theoretical health-related quality of life model. We began the investigation with 2 online and 2 in-person focus groups. Four individual semistructured interviews were added to further explore emerging themes. Data were analyzed using inductive thematic networks approach. RESULTS: Twenty-eight adults (13 female, 24-75 years of age) participated. In the domain of psychological functioning we identified three main themes; changes in mood, worry, and body image concerns. In the domain of coping styles, we identified four adaptive coping styles; positive reframing and optimism, finding a purpose, being proactive, and using humor. Among the maladaptive coping styles, we found passive acceptance, and avoidance and denial. CONCLUSIONS: Psychological well-being can be contingent on physical functioning and coping styles in adults with soft-tissue sarcoma. Both psychological and physical function impact quality of life. Patients with more physical limitations, psychological distress and maladaptive coping styles should be monitored for their well-being.

Tumor protein D54 defines a new class of intracellular transport vesicles.

Transport of proteins and lipids from one membrane compartment to another is via intracellular vesicles. We investigated the function of tumor protein D54 (TPD54/TPD52L2) and found that TPD54 was involved in multiple membrane trafficking pathways: anterograde traffic, recycling, and Golgi integrity. To understand how TPD54 controls these diverse functions, we used an inducible method to reroute TPD54 to mitochondria. Surprisingly, this manipulation resulted in the capture of many small vesicles (30 nm diameter) at the mitochondrial surface. Super-resolution imaging confirmed the presence of similarly sized TPD54-positive structures under normal conditions. It appears that TPD54 defines a new class of transport vesicle, which we term intracellular nanovesicles (INVs). INVs meet three criteria for functionality. They contain specific cargo, they have certain R-SNAREs for fusion, and they are endowed with a variety of Rab GTPases (16 out of 43 tested). The molecular heterogeneity of INVs and the diverse functions of TPD54 suggest that INVs have various membrane origins and a number of destinations. We propose that INVs are a generic class of transport vesicle that transfer cargo between these varied locations.

Unintended perturbation of protein function using GFP nanobodies in human cells.

Tagging a protein of interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed 'dongles', that could add, for example, an FKBP tag to a GFP-tagged protein of interest, enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools.This article has an associated First Person interview with the first author of the paper.

New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision.

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the ß2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.

Nurses' perceptions of caring for parents of children with chronic medical complexity in the pediatric intensive care unit.

OBJECTIVES: The aim of this study was to explore nurses' perceptions of caring for parents of children with medical complexity [CMC] in the pediatric intensive care unit [PICU]. RESEARCH METHODOLOGY: An interpretive descriptive design was used to explore nurses' perceptions of caring for parents of CMC in the PICU. Semi-structured interviews were conducted with ten nurses. Interview data were collected and analyzed using qualitative inductive content analysis. FINDINGS: Nurses revealed that their experiences of caring for parents of CMC evolved over time as they learned to tailor a caregiving partnership based on trust. Although various circumstances could challenge this partnership, nurses strove to maintain and nurture it through self-reflection and optimal communication. Three themes were identified in the data that captured PICU nurses' perceptions: (i) "Thrown to the wolves": Adjusting to a new caregiving role; (ii) "Getting to know each other": Merging caregiving roles; (iii) "Keeping connected": Working to preserve the partnership. CONCLUSIONS: Findings shed new light on the importance of a trusting nurse-parent partnership in caring for parents of CMC in the PICU. Results will be used to develop strategies to enhance this partnership, with the goal of supporting parents and staff in their caregiving roles.

The psychosocial experience of individuals living with osteogenesis imperfecta: a mixed-methods systematic review.

PURPOSE: Osteogenesis imperfecta (OI) is a genetic disorder (prevalence: 1:10,000), leading to bone fragility, frequent fractures, and varying degrees of physical limitations. Despite a substantial amount of research on the genetics, pathophysiology, and treatments related to OI, there remains a paucity of knowledge concerning the lived psychosocial experience of the OI population. This mixed-methods systematic review aimed to review, appraise, and synthesize the literature on the psychosocial experience of children and adults with OI with the goal of identifying implications for research, practice, and policy-making. METHODS: Using a systematic methodology, quantitative, qualitative, and mixed-methods studies were accessed through database searching, screened, assessed for eligibility, and appraised. Data from the selected studies fulfilling the eligibility and quality criteria were extracted and synthesized using thematic analysis with an inductive approach. RESULTS: A total of four qualitative and 20 quantitative studies, with various study designs and methodologies ranging in quality, were included in the review (n = 800; comprising 610 children and 175 adults with OI types I, III, IV, and V, ten parents and five healthcare professionals). Six themes were identified: intellectual feats, isolation and feeling different, fear of fractures, coping with challenges, adapting by learning new skills, and social relationships. CONCLUSION: These findings highlighted key aspects of the experiences of children and adults with OI and will be essential for improving the quality and direction of research, tailoring clinical interventions addressing the psychosocial needs and quality of life of individuals with OI, and raising awareness among caregivers, healthcare professionals, administrators, and policy-makers associated with the OI population.