Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization.

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.

Promoting the strengths and resilience of an Indigenous community through photovoice.

In the face of negative media attention, community members and Elders from the First Nation community of Maskwacis identified the importance of promoting community strengths and reframing perceptions of their community. Two research questions were addressed: (1) How do youth in Maskwacis view their community strengths? and (2) To what extent can photographs be used as a tool for reframing perceptions of an Indigenous community? A community-based participatory research approach was used for the current photovoice project, through which two Elders and eleven youth were engaged. Indigenous photographers trained youth participants, and Elders mentored youth to capture photographs that represented their community's strengths and resilience. Youth selected forty photos to display at a total of six photo exhibits, where feedback was gathered from 392 attendees using brief questionnaires. The content of their photos was analyzed using thematic analysis. This resulted in four themes that described the strengths of Maskwacis, reflecting the community's strong relationships, commitment to culture, the beautiful natural world that is a part of Maskwacis, and the community's ability to look toward the future. In addition, findings provide foundational support for the use of photographs to reframe perceptions of an Indigenous community. The majority (93%) of survey respondents provided examples of ways that viewing the photos had positively changed their perceptions of Maskwacis. This study prompts consideration of the strengths and resilience of other Indigenous communities facing similar social and health issues. Therefore, findings are highly relevant to paediatric health care providers seeking to provide culturally responsive care.

Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

As phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2α kinases. In the crystal structure of the PKR-eIF2α complex, the C-terminal lobe of the kinase contacts eIF2α on a face remote from Ser51, leaving Ser51 ∼ 20 Å from the kinase active site. PKR mutations that cripple the eIF2α-binding site impair phosphorylation; here, we identify mutations in eIF2α that restore Ser51 phosphorylation by PKR with a crippled substrate-binding site. These eIF2α mutations either disrupt a hydrophobic network that restricts the position of Ser51 or alter a linkage between the PKR-docking region and the Ser51 loop. We propose that the protected state of Ser51 in free eIF2α prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases, whereas docking of eIF2α on PKR induces a conformational change that regulates the degree of Ser51 exposure and thus restricts phosphorylation to the proper kinases.

The molecular organization of cypovirus polyhedra.

Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 A crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5-12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen-antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Optimizing Inpatient Nutrition Care of Adult Patients with Inflammatory Bowel Disease in the 21st Century.

Malnutrition is highly prevalent in inflammatory bowel disease (IBD) patients and disproportionately affects those admitted to hospital. Malnutrition is a risk factor for many complications in IBD, including prolonged hospitalization, infection, greater need for surgery, development of venous thromboembolism, post-operative complications, and mortality. Early screening for malnutrition and prompt nutrition intervention if indicated has been shown to prevent or mitigate many of these outlined risk factors. There are many causes of malnutrition in IBD including reduced oral food intake, medications, active inflammation, and prior surgical resections. Hospitalization can further compound pre-existing malnutrition through inappropriate diet restrictions, nil per os (NPO) for endoscopy and imaging, or partial bowel obstruction, resulting in "post-hospital syndrome" after discharge and readmission. The aim of this article is to inform clinicians of the prevalence and consequences of malnutrition in IBD, as well as available screening and assessment tools for diagnosis, and to offer an organized approach to the nutritional care of hospitalized adult IBD patients.

Malnutrition Impacts Health-Related Quality of Life in Cirrhosis: A Cross-Sectional Study.

BACKGROUND: To explore the influence of nourishment state measured by various nutrition assessment tools (NATs) on health-related quality of life (HRQoL) in a pretransplant population with cirrhosis. METHODS: We collected demographic, nutrition assessment, and disease specific data on 81 patients. HRQoL was measured with the Short-Form 36 and divided into 8 subscales. Significant relationships between NATs and HRQoL were examined using independent sample t-tests, χ2 analyses, correlations, and multiple and logistic regression adjusted for age and gender. RESULTS: Study mean age was 54.2 years (SD 10.4 years), and 57% were male. Subjective Global Assessment (SGA) was significantly related to all HRQoL subscales, except bodily pain and mental health. In the adjusted regression models, general health, vitality, and social functioning were all significantly lower in patients with poorer nutrition status measured using SGA (adjusted R2 = 11%, ß = -0.34, p < 0.01; adjusted R2 = 8%, ß = -0.27, P < 0.05; and adjusted R2 = 12%, ß = -0.38, P < 0.01, Q4 respectively). Physical functioning improved as hand grip strength increased (adjusted R2 = 20%, ß = 0.36, P < 0.01). MELDNa demonstrated a significant negative relationship with role-emotional (adjusted R2 = 3%, ß = 0.25, P < 0.05), and mid-arm circumference did not demonstrate any significant relationships with HRQoL. CONCLUSIONS: Malnutrition assessed by SGA is associated with lower HRQoL in patients with cirrhosis. Future research should identify if nutrition interventions can effectively improve HRQoL in cirrhosis patients.

Purification, crystallization and preliminary X-ray analysis of truncated and mutant forms of VP4 protease from infectious pancreatic necrosis virus.

In viruses belonging to the Birnaviridae family, virus protein 4 (VP4) is the viral protease responsible for the proteolytic maturation of the polyprotein encoding the major capsid proteins (VP2 and VP3). Infectious pancreatic necrosis virus (IPNV), the prototype of the aquabirnavirus genus, is the causative agent of a contagious disease in fish which has a large economic impact on aquaculture. IPNV VP4 is a 226-residue (24.0 kDa) serine protease that utilizes a Ser/Lys catalytic dyad mechanism (Ser633 and Lys674). Several truncated and mutant forms of VP4 were expressed in a recombinant expression system, purified and screened for crystallization. Two different crystal forms diffract beyond 2.4 A resolution. A triclinic crystal derived from one mutant construct has unit-cell parameters a = 41.7, b = 69.6, c = 191.6 A, alpha = 93.0, beta = 95.1, gamma = 97.7 degrees. A hexagonal crystal with space group P6(1)22/P6(5)22 derived from another mutant construct has unit-cell parameters a = 77.4, b = 77.4, c = 136.9 A.

Insect virus polyhedra, infectious protein crystals that contain virus particles.

High-resolution atomic structures have been reported recently for two types of viral polyhedra, intracellular protein crystals produced by ubiquitous insect viruses. Polyhedra contain embedded virus particles and function as the main infectious form for baculoviruses and cypoviruses, two distinct classes of viruses that infect mainly Lepitoptera species (butterflies and moths). Polyhedra are extremely stable and protect the virus particles once released in the environment. The extensive crystal contacts observed in the structures explain the remarkable stability of viral polyhedra and provide hints about how these crystals dissolve in the alkaline midgut, releasing embedded virus particles to infect feeding larvae. The stage is now set to answer intriguing questions about the in vivo crystallization of polyhedra, how virus particles are incorporated into polyhedra, and what determines the size and shape of the crystals. Large quantities of polyhedra can be obtained from infected larvae and polyhedra can also be produced using insect cell expression systems. Modified polyhedra encapsulating other entities in place of virus particles have potential applications as a means to stabilize proteins such as enzymes or growth factors, and the extremely stable polyhedrin lattice may provide a framework for future engineered micro-crystal devices.

Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture.

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.