Non-instrumental actions can communicate roles and relationships, not just rituals.

Actions that do not have instrumental goals can communicate social goals that are not rituals. Many non-instrumental actions such as bowing or kissing communicate a commitment to or roles in dyadic relationships. What is unclear is when people understand such actions in terms of ritual and when they understand them in terms of relationships.

Therapeutic applications of extracellular vesicles: clinical promise and open questions.

This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to clinical potential and translation. Potentially useful EVs comprise diverse products of various cell types and species. EV components may also be combined with liposomes and nanoparticles to facilitate manufacturing as well as product safety and evaluation. Potential therapeutic cargoes include RNA, proteins, and drugs. Strategic issues considered herein include choice of therapeutic agent, means of loading cargoes into EVs, promotion of EV stability, tissue targeting, and functional delivery of cargo to recipient cells. Some applications may harness natural EV properties, such as immune modulation, regeneration promotion, and pathogen suppression. These properties can be enhanced or customized to enable a wide range of therapeutic applications, including vaccination, improvement of pregnancy outcome, and treatment of autoimmune disease, cancer, and tissue injury.

Stabilization of exosome-targeting peptides via engineered glycosylation.

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

Patient-Reported Outcomes and Computed Tomography Review After Minimally Invasive Fusion of the Sacroiliac Joint With Aggressive Joint Decortication and Joint Compression.

The sacroiliac joint (SIJ) is a common, underrecognized source of low back pain. We evaluated outcomes in patients undergoing sacroiliac joint fusion (SIJF) using a novel, minimally invasive SIJF system emphasizing compressive forces across an aggressively debrided SIJ. We retrospectively reviewed data from a continuous set of patients presenting to a large, tertiary care hospital from September 2017 to August 2019. All patients received the novel SIJF device. Outcomes were assessed at 8 weeks, 6 months, and 12 months using the Oswestry Disability Index (ODI) score, Numerical Rating Scale (NRS) score, Single Assessment Numerical Evaluation (SANE) score, and Patient-Reported Outcomes Measurement Information System (PROMIS) measures, plus radiographic evaluation of fusion status. Data from 75 patients were analyzed. At 8 weeks, 6 months, and 12 months, the ODI score improved by 10.5 points (P=.002), 17.4 points (P<.0001), and 23.6 points (P<.0001), respectively, while the NRS score improved by 4.6 points (P<.0001), 4.4 points (P<.0001), and 4.6 points (P<.0001), respectively. SANE scores indicated high levels of patient satisfaction (81.0%, 92.18%, and 89.2%, respectively). PROMIS physical function scores improved by 2.65 points, 3.30 points, and 3.63 points, respectively, while PROMIS mental health scores showed changes of -1.93 points, 1.57 points, and -0.47 points, respectively. A review of computed tomography scans demonstrated grade 3 fusion (complete) in 81% of cases at a mean of 371 days postoperatively. There was one revision case for a malpositioned implant. The use of a novel SIJF device emphasizing compressive forces provided early, durable improvements in patient-reported outcomes and extremely high patient satisfaction. [Orthopedics. 2024;47(2):101-107.].

Precision off-the-shelf natural killer cell therapies for oncology with logic-gated gene circuits.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations. Furthermore, TAAs are also expressed on healthy cells, leading to toxicity risk. To address these targeting challenges, we engineer natural killer (NK) cells with a multi-input gene circuit consisting of chimeric antigen receptors (CARs) controlled by OR and NOT logic gates. The OR gate kills a range of AML cells from leukemic stem cells to blasts using a bivalent CAR targeting FLT3 and/or CD33. The NOT gate protects healthy hematopoietic stem cells (HSCs) using an inhibitory CAR targeting endomucin, a protective antigen unique to healthy HSCs. NK cells with the combined OR-NOT gene circuit kill multiple AML subtypes and protect primary HSCs, and the circuit also works in vivo.

Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells.

The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)-which naturally encapsulate and transfer proteins and nucleic acids between cells-for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9-single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4+ T cells. The strategy is amenable to the targeted delivery of biologics to other cell types.

Breakthroughs, boosters, and beyond: a practical primer on current challenges with COVID-19

Identifying the extent to which breakthrough infections are contributing to the spread of COVID-19 can help guide vaccination policies and other infection prevention and control protocols to promote public health and safety. This special report summarizes key studies on COVID-19 vaccine efficacy and effectiveness and presents caveats to these studies.

Enrichment of Extracellular Vesicle Subpopulations Via Affinity Chromatography.

Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.

A Systematic Evaluation of Factors Affecting Extracellular Vesicle Uptake by Breast Cancer Cells.

Extracellular vesicles (EVs) are nanometer-scale particles that are secreted by cells and mediate intercellular communication by transferring biomolecules between cells. Harnessing this mechanism for therapeutic biomolecule delivery represents a promising frontier for regenerative medicine and other clinical applications. One challenge to realizing this goal is that to date, our understanding of which factors affect EV uptake by recipient cells remains incomplete. In this study, we systematically investigated such delivery questions in the context of breast cancer cells, which are one of the most well-studied cell types with respect to EV delivery and therefore comprise a facile model system for this investigation. By displaying various targeting peptides on the EV surface, we observed that although displaying GE11 on EVs modestly increased uptake by MCF-7 cells, neuropeptide Y (NPY) display had no effect on uptake by the same cells. In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell's environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types.

A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery.

Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general biophysical properties impact loading and delivery of RNA by EVs, we developed a platform for actively loading engineered cargo RNAs into EVs. In our system, the MS2 bacteriophage coat protein was fused to EV-associated proteins, and the cognate MS2 stem loop was engineered into cargo RNAs. Using this Targeted and Modular EV Loading (TAMEL) approach, we identified a configuration that substantially enhanced cargo RNA loading (up to 6-fold) into EVs. When applied to vesicles expressing the vesicular stomatitis virus glycoprotein (VSVG) - gesicles - we observed a 40-fold enrichment in cargo RNA loading. While active loading of mRNA-length (>1.5 kb) cargo molecules was possible, active loading was much more efficient for smaller (~0.5 kb) RNA molecules. We next leveraged the TAMEL platform to elucidate the limiting steps in EV-mediated delivery of mRNA and protein to prostate cancer cells, as a model system. Overall, most cargo was rapidly degraded in recipient cells, despite high EV-loading efficiencies and substantial EV uptake by recipient cells. While gesicles were efficiently internalized via a VSVG-mediated mechanism, most cargo molecules were rapidly degraded. Thus, in this model system, inefficient endosomal fusion or escape likely represents a limiting barrier to EV-mediated transfer. Altogether, the TAMEL platform enabled a comparative analysis elucidating a key opportunity for enhancing EV-mediated delivery to prostate cancer cells, and this technology should be of general utility for investigations and applications of EV-mediated transfer in other systems.

Regulation of the IL-10-driven macrophage phenotype under incoherent stimuli.

Macrophages are ubiquitous innate immune cells that play a central role in health and disease by adopting distinct phenotypes, which are broadly divided into classical inflammatory responses and alternative responses that promote immune suppression and wound healing. Although macrophages are attractive therapeutic targets, incomplete understanding of this functional choice limits clinical manipulation. While individual stimuli, pathways, and genes involved in macrophage functional responses have been identified, how macrophages evaluate complex in vivo milieus comprising multiple divergent stimuli remains poorly understood. Here, we used combinations of "incoherent" stimuli-those that individually promote distinct macrophage phenotypes-to elucidate how the immunosuppressive, IL-10-driven macrophage phenotype is induced, maintained, and modulated under such combinatorial stimuli. The IL-10-induced immunosuppressive phenotype was largely insensitive to co-administered IL-12, which has been reported to modulate macrophage phenotype, but maintaining the immunosuppressive phenotype required sustained exposure to IL-10. Our data implicate the intracellular protein, BCL3, as a key mediator of the IL-10-driven phenotype. Notably, co-administration of IFN-γ disrupted an IL-10-mediated positive feedback loop that may reinforce the immunosuppressive phenotype. This novel combinatorial perturbation approach thus generated new insights into macrophage decision making and local immune network function.

Modulating the frequency and bias of stochastic switching to control phenotypic variation.

Mechanisms that control cell-to-cell variation in gene expression ('phenotypic variation') can determine a population's growth rate, robustness, adaptability and capacity for complex behaviours. Here we describe a general strategy (termed FABMOS) for tuning the phenotypic variation and mean expression of cell populations by modulating the frequency and bias of stochastic transitions between 'OFF' and 'ON' expression states of a genetic switch. We validated the strategy experimentally using a synthetic fim switch in Escherichia coli. Modulating the frequency of switching can generate a bimodal (low frequency) or a unimodal (high frequency) population distribution with the same mean expression. Modulating the bias as well as the frequency of switching can generate a spectrum of bimodal and unimodal distributions with the same mean expression. This remarkable control over phenotypic variation, which cannot be easily achieved with standard gene regulatory mechanisms, has many potential applications for synthetic biology, engineered microbial ecosystems and experimental evolution.

Canadian pharmacy practice residents' projects: publication rates and study characteristics.

BACKGROUND: Research projects are a key component of pharmacy residents' education. Projects represent both a large investment of effort for each resident (up to 10 weeks over the residency year) and a large body of research (given that there are currently over 150 residency positions in Canada annually). Publication of results is a vital part of the dissemination of information gleaned from these projects. OBJECTIVES: To determine the publication rate for research projects performed under the auspices of accredited English-language hospital pharmacy residency programs in Canada and to describe the study characteristics of residency projects performed in Ontario from 1999/2000 to 2008/2009. METHODS: Lists of residents and project titles for the period of interest were obtained from residency coordinators. PubMed, CINAHL, the Canadian Journal of Hospital Pharmacy, and Google were searched for evidence of publication of each project identified, as an abstract or presentation at a meeting, a letter to the editor, or a full-text manuscript. The library holdings of the University of Toronto were reviewed to determine study characteristics of the Ontario residency projects. RESULTS: For the objective of this study relating to publication rate, 518 projects were included. The overall publication rate was 32.2% (60 [35.9%] as abstracts and 107 [64.1%] as full-text manuscripts). Publication in pharmacy-specific journals (66 [61.7%] of 107 full-text manuscripts) was more frequent than publication in non-pharmacy-specific journals. The publication rate of projects as full-text manuscripts remained stable over time. Of the 202 Ontario residency projects archived in the University of Toronto's library, most were cohort studies (83 [41.1%]), and the most common topic was efficacy and/or safety of a medication (46 [22.8%]). CONCLUSIONS: Most hospital pharmacy residents' projects were unpublished, and the publication rate of projects as full-text manuscripts has not increased over time. Most projects were observational studies. Increasing publication rates and creating a central database or repository of residency projects would increase the dissemination and accessibility of residents' research.

CONTEXTE: Les projets de recherche sont un élément clé de la formation des résidents en pharmacie. Ils représentent à la fois un investissement important en temps pour chaque résident (jusqu'à 10 semaines au cours de l'année de résidence) et une imposante masse de recherche (compte tenu qu'il y a actuellement plus de 150 postes de résidence au Canada chaque année). La publication des résultats est une partie essentielle de la diffusion de l'information issue de ces projets. OBJECTIFS: Déterminer le taux de publication des projets de recherche menés sous l'égide des programmes anglophones de résidence en pharmacie d'hôpital au Canada et décrire les caractéristiques expérimentales des projets menés dans le cadre de la résidence en Ontario entre 1999­2000 et 2008­2009. MÉTHODES: Les listes des résidents et des titres de projets de recherche pour la période en question ont été obtenues des coordonnateurs des programmes de résidence. Des recherches ont été effectuées dans PubMed, CINAHL, le Journal canadien de la pharmacie hospitalière et Google pour corroborer la publication de chaque projet recensé, sous forme de résumé ou de présentation lors d'un congrès, d'une lettre à la rédaction ou d'un manuscrit complet. Les archives de la bibliothèque de l'Université de Toronto ont été examinées pour déterminer les caractéristiques expérimentales des projets de résidence menés en Ontario. RÉSULTATS: En ce qui a trait à l'objectif de cette étude relatif au taux de publication, 518 projets ont été recensés. Le taux de publication global était de 32,2 % (60 [35,9 %] sous forme de résumés et 107 [64,1 %] sous forme de manuscrits complets). La publication dans des revues spécialisées en pharmacie (66 [61,7 %] des 107 manuscrits complets) était plus fréquente que dans des revues non spécialisées en pharmacie. Le taux de publication des projets sous forme de manuscrits complets est demeuré stable dans le temps. Des 202 projets de résidence archivés dans la bibliothèque de l'Université de Toronto, la plupart étaient des études de cohorte (83 [41,1 %]) et le sujet le plus courant était l'efficacité ou l'innocuité d'un médicament (46 [22,8 %]). CONCLUSIONS: La plupart des projets de recherche des résidents en pharmacie hospitalière n'ont pas été publiés et le taux de publication des projets sous forme de manuscrits complets n'a pas augmenté dans le temps. La plupart des projets étaient des études observationnelles. L'augmentation des taux de publication et la création d'une base de données centrale ou d'un dépôt pour les projets de résidence permettrait d'accroître la diffusion de la recherche effectuée par les résidents et son accessibilité. [Traduction par l'éditeur].