Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Oligodendrocyte calcium signaling promotes actin-dependent myelin sheath extension.

Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length are thought to precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation remains incompletely understood. Here, we use genetic tools to attenuate oligodendrocyte calcium signaling during myelination in the developing mouse CNS. Surprisingly, genetic calcium attenuation does not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation causes myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduces actin filaments in oligodendrocytes, and an intact actin cytoskeleton is necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a cellular mechanism required for accurate CNS myelin formation and may provide mechanistic insight into how oligodendrocytes respond to neuronal activity to sculpt and refine myelin sheaths.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and a key component of the cytoskeleton. A range of small molecules have emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Amongst these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390 - 490 nm pulsed light and rapidly relaxes to the inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated by live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery.

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes.

Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.

Developing curricular-content and systems-related impact indicators for intellectual disability awareness training for acute hospital settings: A modified International Delphi Survey.

AIM: To identify, and reach consensus on, curricular-content and delivery methods, as well as ways to maximize the impact of intellectual disability awareness training programmes in acute hospital settings. BACKGROUND: With the continuing evidence of avoidable deaths and unwarranted variations in the quality of care to people with an intellectual disability in acute hospitals, it could be purported that current training provided to hospital staff appears to be making a minimal difference in the care provided to this population. DESIGN: A two-round modified Delphi survey was conducted between June 2020-January 2021. METHODS: International experts from primary healthcare and hospital settings, and intellectual disability health fields participated in the survey. Initial curricular-content items were developed from the literature, and based on the combined clinical and academic experience base of the authors. Items were evaluated in terms of agreement/consensus, importance and stability of responses. There were 57 expert responses in Round 1 and 45 in Round 2. RESULTS: The consensus was reached with regard to 55 of 65 curricular-content indicators relating to Aims, Design, Content and Delivery. Ten curricular-content indicators failed to be agreed on relating to the mode of training delivery. With regard to systems-related impact indicators, 28 out of 31 reached consensus. The expert panel identified and agreed on seven system barriers that could obstruct the successful implementation of the awareness training programmes in acute hospital settings. CONCLUSIONS: This is the first international Delphi survey to agree on curricular-content and identify systems-related facilitators for intellectual disability awareness training. Potential system barriers have been highlighted which could be addressed by systemic improvement. Implications for developing, and robustly testing the efficacy of, intellectual disability awareness training programmes are discussed, as are the implications for other cognitively impaired populations. IMPACT: In order to maximize the impact, investment in acute hospital staff education will need to be accompanied by wider changes to systems and structures concerning the governance of service provision for people with an intellectual disability.

Modeling myelin: A toolkit for exploring myelin's mysteries in vitro.

In the myelin field, there is a lack of reliable in vitro tools to study myelination, especially using human cells. In this issue of Developmental Cell, James et al. present a guide to generating human iPSC-derived "myelinoids"-3D models of myelination that reliably achieve mature myelin formation.

Barriers to Creating Scalable Business Models for Digital Health Innovation in Public Systems: Qualitative Case Study.

BACKGROUND: Health systems are increasingly looking toward the private sector to provide digital solutions to address health care demands. Innovation in digital health is largely driven by small- and medium-sized enterprises (SMEs), yet these companies experience significant barriers to entry, especially in public health systems. Complex and fragmented care models, alongside a myriad of relevant stakeholders (eg, purchasers, providers, and producers of health care products), make developing value propositions for digital solutions highly challenging. OBJECTIVE: This study aims to identify areas for health system improvement to promote the integration of innovative digital health technologies developed by SMEs. METHODS: This paper qualitatively analyzes a series of case studies to identify health system barriers faced by SMEs developing digital health technologies in Canada and proposed solutions to encourage a more innovative ecosystem. The Women's College Hospital Institute for Health System Solutions and Virtual Care established a consultation program for SMEs to help them increase their innovation capacity and take their ideas to market. The consultation involved the SME filling out an onboarding form and review of this information by an expert advisory committee using guided considerations, leading to a recommendation report provided to the SME. This paper reports on the characteristics of 25 SMEs who completed the program and qualitatively analyzed their recommendation reports to identify common barriers to digital health innovation. RESULTS: A total of 2 central themes were identified, each with 3 subthemes. First, a common barrier to system integration was the lack of formal evaluation, with SMEs having limited resources and opportunities to conduct such an evaluation. Second, the health system's current structure does not create incentives for clinicians to use digital technologies, which threatens the sustainability of SMEs' business models. SMEs faced significant challenges in engaging users and payers from the public system due to perverse economic incentives. Physicians are compensated by in-person visits, which actively works against the goals of many digital health solutions of keeping patients out of clinics and hospitals. CONCLUSIONS: There is a significant disconnect between the economic incentives that drive clinical behaviors and the use of digital technologies that would benefit patients' well-being. To encourage the use of digital health technologies, publicly funded health systems need to dedicate funding for the evaluation of digital solutions and streamlined pathways for clinical integration.

Identifying optimal frameworks to implement or evaluate digital health interventions: a scoping review protocol.

INTRODUCTION: Digital health interventions (DHIs) are defined as health services delivered electronically through formal or informal care. DHIs can range from electronic medical records used by providers to mobile health apps used by consumers. DHIs involve complex interactions between user, technology and the healthcare team, posing challenges for implementation and evaluation. Theoretical or interpretive frameworks are crucial in providing researchers guidance and clarity on implementation or evaluation approaches; however, there is a lack of standardisation on which frameworks to use in which contexts. Our goal is to conduct a scoping review to identify frameworks to guide the implementation or evaluation of DHIs. METHODS AND ANALYSIS: A scoping review will be conducted using methods outlined by the Joanna Briggs Institute reviewers' manual and will conform to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews. Studies will be included if they report on frameworks (ie, theoretical, interpretive, developmental) that are used to guide either implementation or evaluation of DHIs. Electronic databases, including MEDLINE, EMBASE, CINAHL and PsychINFO will be searched in addition to grey literature and reference lists of included studies. Citations and full text articles will be screened independently in Covidence after a reliability check among reviewers. We will use qualitative description to summarise findings and focus on how research objectives and type of DHIs are aligned with the frameworks used. ETHICS AND DISSEMINATION: We engaged an advisory panel of digital health knowledge users to provide input at strategic stages of the scoping review to enhance the relevance of findings and inform dissemination activities. Specifically, they will provide feedback on the eligibility criteria, data abstraction elements, interpretation of findings and assist in developing key messages for dissemination. This study does not require ethical review. Findings from review will support decision making when selecting appropriate frameworks to guide the implementation or evaluation of DHIs.

Soft extracellular matrix enhances inflammatory activation of mesenchymal stromal cells to induce monocyte production and trafficking.

Mesenchymal stromal cells (MSCs) modulate immune cells to ameliorate multiple inflammatory pathologies. Biophysical signals that regulate this process are poorly defined. By engineering hydrogels with tunable biophysical parameters relevant to bone marrow where MSCs naturally reside, we show that soft extracellular matrix maximizes the ability of MSCs to produce paracrine factors that have been implicated in monocyte production and chemotaxis upon inflammatory stimulation by tumor necrosis factor-α (TNFα). Soft matrix increases clustering of TNF receptors, thereby enhancing NF-κB activation and downstream gene expression. Actin polymerization and lipid rafts, but not myosin-II contractility, regulate mechanosensitive activation of MSCs by TNFα. We functionally demonstrate that human MSCs primed with TNFα in soft matrix enhance production of human monocytes in marrow of xenografted mice and increase trafficking of monocytes via CCL2. The results suggest the importance of biophysical signaling in tuning inflammatory activation of stromal cells to control the innate immune system.

Mobile App for Improved Self-Management of Type 2 Diabetes: Multicenter Pragmatic Randomized Controlled Trial.

BACKGROUND: As the increasing prevalence of type 2 diabetes mellitus has put pressure on health systems to appropriately manage these patients, there have been a growing number of mobile apps designed to improve the self-management of diabetes. One such app, BlueStar, has been shown to significantly reduce hemoglobin A1c (HbA1c) levels in small studies and is the first app in the United States to receive Food and Drug Administration approval as a mobile prescription therapy. However, the impact of the app across real-world population among different clinical sites and health systems remains unclear. OBJECTIVE: The primary objective of this study was to conduct a pragmatic randomized controlled trial of the BlueStar mobile app to determine if app usage leads to improved HbA1c levels among diverse participants in real-life clinical contexts. We hypothesized that this mobile app would improve self-management and HbA1c levels compared with controls. METHODS: The study consisted of a multicenter pragmatic randomized controlled trial. Overall, 110 participants randomized to the immediate treatment group (ITG) received the intervention for 6 months, and 113 participants randomized to the wait-list control (WLC) group received usual care for the first 3 months and then received the intervention for 3 months. The primary outcome was glucose control measured by HbA1c levels at 3 months. Secondary outcomes assessed intervention impact on patient self-management, experience of care, and self-reported health utilization using validated scales, including the Problem Areas in Diabetes, the Summary of Diabetes Self-Care Activities, and the EuroQol-5D. Intervention usage data were collected directly from the app. RESULTS: The results of an analysis of covariance controlling for baseline HbA1c levels did not show evidence of intervention impact on HbA1c levels at 3 months (mean difference [ITG-WLC] -0.42, 95% CI -1.05 to 0.21; P=.19). Similarly, there was no intervention effect on secondary outcomes measuring diabetes self-efficacy, quality of life, and health care utilization behaviors. An exploratory analysis of 57 ITG participants investigating the impact of app usage on HbA1c levels showed that each additional day of app use corresponded with a 0.016-point decrease in participants' 3-month HbA1c levels (95% CI -0.03 to -0.003). App usage varied significantly by site, as participants from 1 site logged in to the app a median of 36 days over 14 weeks (interquartile range [IQR] 10.5-124); those at another site used the app significantly less (median 9; IQR 6-51). CONCLUSIONS: The results showed no difference between intervention and control arms for the primary clinical outcome of glycemic control measured by HbA1c levels. Although there was low usage of the app among participants, results indicate contextual factors, particularly site, had a significant impact on overall usage. Future research into the patient and site-specific factors that increase app utilization are needed. TRIAL REGISTRATION: Clinicaltrials.gov NCT02813343; https://clinicaltrials.gov/ct2/show/NCT02813343 (Archived by WebCite at https://clinicaltrials.gov/ct2/show/NCT02813343).

Community health workers on a college campus: Effects on influenza vaccination.

OBJECTIVE: To assess the impact of a campus community health worker program (HealthPALs) on student influenza vaccination. PARTICIPANTS: Undergraduate students at a northeastern US university (enrollment 6650), influenza seasons 2011-2012 through 2015-2016. METHODS: Study design: Difference-in-differences analysis of student vaccination at campus dormitory influenza clinics during intervention vs. baseline. INTERVENTION: In the first intervention year, HealthPALs conducted in-person peer outreach at several campus dormitory flu clinics. Subsequent years, HealthPALs conducted an enhanced intervention, with the addition of a personalized, dormitory-specific social media campaign appealing to students' community identity. RESULTS: The initial intervention increased vaccinations by 66% (IRR = 1.66, 95%CI 1.39-1.97) at intervention clinics relative to control. The enhanced intervention increased vaccinations by 85% (IRR = 1.85, 95%CI 1.75-1.96). CONCLUSION: Community health workers can be a highly effective, low-cost strategy for increasing influenza vaccination among college students. This model could also be used to address other campus health challenges where student engagement is key.

Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery.

Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel's mechanical properties. Here, we report a microfluidic-based method for encapsulating single cells in an approximately six-micrometre layer of alginate that increases the proportion of cell-containing microgels by a factor of ten, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that, for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.

Excitatory amino acid antagonists in the rostral ventromedial medulla inhibit mesencephalic morphine analgesia in rats.

Supraspinal opioid analgesia is mediated in part by connections between the periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Morphine analgesia elicited from the PAG is respectively decreased by selective serotonergic and opioid receptor antagonists administered into the RVM, and increased by RVM neurotensin antagonists. Since glutamate and excitatory amino acid (EAA) receptors are also active in the RVM, the present study evaluated whether either competitive (AP7) or non-competitive (MK-801) N-methyl-D-aspartate (NMDA) antagonists or a kainate/AMPA (CNQX) antagonist microinjected into the RVM altered morphine (2.5 micrograms) analgesia elicited from the PAG as measured by the tail-flick and jump tests. Mesencephalic morphine analgesia was markedly reduced on both tests after RVM pretreatment with either AP7 (0.01-1 microgram, 0.08-7.8 nmol) or MK-801 (0.03-3 micrograms, 0.04-4.4 nmol). In contrast, small but significant reductions in mesencephalic morphine analgesia occurred on the jump test following CNQX (0.5 microgram, 2.2 nmol) in the RVM. NMDA antagonists did not markedly alter either basal nociceptive thresholds following RVM administration, or mesencephalic morphine analgesia following administration into medullary placements lateral or dorsal to the RVM. These data implicate EAA and particularly NMDA receptors in the RVM in modulating the transmission of opioid pain-inhibitory signals from the PAG.