Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-ß-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.

Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Optical Membrane Control with Red Light Enabled by Red-Shifted Photolipids.

Photoswitchable phospholipids, or "photolipids", that harbor an azobenzene group in their lipid tails are versatile tools to manipulate and control lipid bilayer properties with light. So far, the limited ultraviolet-A/blue spectral range in which the photoisomerization of regular azobenzene operates has been a major obstacle for biophysical or photopharmaceutical applications. Here, we report on the synthesis of nano- and micrometer-sized liposomes from tetra-ortho-chloro azobenzene-substituted phosphatidylcholine (termed red-azo-PC) that undergoes photoisomerization on irradiation with tissue-penetrating red light (≥630 nm). Photoswitching strongly affects the fluidity and mechanical properties of lipid membranes, although small-angle X-ray scattering and dynamic light scattering measurements reveal only a minor influence on the overall bilayer thickness and area expansion. By controlling the photostationary state and the photoswitching efficiency of red-azo-PC for specific wavelengths, we demonstrate that shape transitions such as budding or pearling and the division of cell-sized vesicles can be achieved. These results emphasize the applicability of red-azo-PC as a nanophotonic tool in synthetic biology and for biomedical applications.

The Use of Near-Infrared Spectroscopy and/or Transcranial Doppler as Non-Invasive Markers of Cerebral Perfusion in Adult Sepsis Patients With Delirium: A Systematic Review.

BACKGROUND: Several studies have previously reported the presence of altered cerebral perfusion during sepsis. However, the role of non-invasive neuromonitoring, and the impact of altered cerebral perfusion, in sepsis patients with delirium remains unclear. METHODS: We performed a systematic review of studies that used near-infrared spectroscopy (NIRS) and/or transcranial Doppler (TCD) to assess adults (≥18 years) with sepsis and delirium. From study inception to July 28, 2020, we searched the following databases: Ovid MedLine, Embase, Cochrane Library, and Web of Science. RESULTS: Of 1546 articles identified, 10 met our inclusion criteria. Although NIRS-derived regional cerebral oxygenation was consistently lower, this difference was only statistically significant in one study. TCD-derived cerebral blood flow velocity was inconsistent across studies. Importantly, both impaired cerebral autoregulation during sepsis and increased cerebrovascular resistance were associated with delirium during sepsis. However, the heterogeneity in NIRS and TCD devices, duration of recording (from 10 seconds to 72 hours), and delirium assessment methods (e.g., electronic medical records, confusion assessment method for the intensive care unit), precluded meta-analysis. CONCLUSION: The available literature demonstrates that cerebral perfusion disturbances may be associated with delirium in sepsis. However, future investigations will require consistent definitions of delirium, delirium assessment training, harmonized NIRS and TCD assessments (e.g., consistent measurement site and length of recording), as well as the quantification of secondary and tertiary variables (i.e., Cox, Mxa, MAPOPT), in order to fully assess the relationship between cerebral perfusion and delirium in patients with sepsis.

From ideal to real: a qualitative study of the implementation of in situ interprofessional simulation-based education.

BACKGROUND: Despite the widespread adoption of interprofessional simulation-based education (IPSE) in healthcare as a means to optimize interprofessional teamwork, data suggest that IPSE may not achieve these intended goals due to a gap between the ideals and the realities of implementation. METHODS: We conducted a qualitative case study that used the framework method to understand what and how core principles from guidelines for interprofessional education (IPE) and simulation-based education (SBE) were implemented in existing in situ IPSE programs. We observed simulation sessions and interviewed facilitators and directors at seven programs. RESULTS: We found considerable variability in how IPSE programs apply and implement core principles derived from IPE and SBE guidelines with some principles applied by most programs (e.g., "active learning", "psychological safety", "feedback during debriefing") and others rarely applied (e.g., "interprofessional competency-based assessment", "repeated and distributed practice"). Through interviews we identified that buy-in, resources, lack of outcome measures, and power discrepancies influenced the extent to which principles were applied. CONCLUSIONS: To achieve IPSE's intended goals of optimizing interprofessional teamwork, programs should transition from designing for the ideal of IPSE to realities of IPSE implementation.

Optical Control of Cannabinoid Receptor 2-Mediated Ca2+ Release Enabled by Synthesis of Photoswitchable Probes.

Cannabinoid receptor 2 (CB2) is a promising target for the treatment of neuroinflammation and other diseases. However, a lack of understanding of its complex signaling in cells and tissues complicates the therapeutic exploitation of CB2 as a drug target. We show for the first time that benchmark CB2 agonist HU308 increases cytosolic Ca2+ levels in AtT-20(CB2) cells via CB2 and phospholipase C. The synthesis of photoswitchable derivatives of HU308 from the common building block 3-OTf-HU308 enables optical control over this pathway with spatiotemporal precision, as demonstrated in a real-time Ca2+ fluorescence assay. Our findings reveal a novel messenger pathway by which HU308 and its derivatives affect cellular excitability, and they demonstrate the utility of chemical photoswitches to control and monitor CB2 signaling in real-time.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†.

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the ß3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Optical control of sphingosine-1-phosphate formation and function.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P1-5) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P1-3, shown through electrophysiology and Ca2+ mobilization assays. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P3-dependent pain hypersensitivity in mice. The hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to the rapidly metabolized S1P. Using lipid mass spectrometry analysis, we constructed a metabolic map of PhotoS1P and PhotoSph. The formation of these photoswitchable lipids was found to be light dependent, providing a novel approach to optically probe sphingolipid biology.

The relationship between rowing-related low back pain and rowing biomechanics: a systematic review.

BACKGROUND: Low back pain (LBP) is common in rowers. Understanding rowing biomechanics may help facilitate prevention and improve rehabilitation. OBJECTIVES: To define the kinematics and muscle activity of rowers and to compare with rowers with current or LBP history. DESIGN: Systematic review. DATA SOURCES: EMBASE, MEDLINE, Cumulative Index to Nursing and Allied Health Literature, Web of Science and Scopus from inception to December 2019. Grey literature was searched. STUDY ELIGIBILITY CRITERIA: Experimental and non-experimental designs. METHODS: Primary outcomes were kinematics and muscle activity. Modified Quality Index (QI) checklist was used. RESULTS: 22 studies were included (429 participants). Modified QI score had a mean of 16.7/28 points (range: 15-21). Thirteen studies investigated kinematics and nine investigated muscle activity. Rowers without LBP ('healthy') have distinct kinematics (neutral or anterior pelvic rotation at the catch, greater hip range of motion, flatter low back spinal position at the finish) and muscle activity (trunk extensor dominant with less flexor activity). Rowers with LBP had relatively greater posterior pelvic rotation at the catch, greater hip extension at the finish and less efficient trunk muscle activity. In both groups fatigue results in increased lumbar spine flexion at the catch, which is greater on the ergometer. There is insufficient evidence to recommend one ergometer type (fixed vs dynamic) over the other to avoid LBP. Trunk asymmetries are not associated with LBP in rowers. CONCLUSION: Improving clinicians' and coaches' understanding of safe and effective rowing biomechanics, particularly of the spine, pelvis and hips may be an important strategy in reducing incidence and burden of LBP.

Optical Control of Lysophosphatidic Acid Signaling.

Lysophosphatidic acid (LPA) is a phospholipid that acts as an extracellular signaling molecule and activates the family of lysophosphatidic acid receptors (LPA1-6). These G protein-coupled receptors (GPCRs) are broadly expressed and are particularly important in development as well as in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. Here, we report on a photoswitchable analogue of LPA, termed AzoLPA, which contains an azobenzene photoswitch embedded in the acyl chain. AzoLPA enables optical control of LPA receptor activation, shown through its ability to rapidly control LPA-evoked increases in intracellular Ca2+ levels. AzoLPA shows greater activation of LPA receptors in its light-induced cis-form than its dark-adapted (or 460 nm light-induced) trans-form. AzoLPA enabled the optical control of neurite retraction through its activation of the LPA2 receptor.

Metabolic networks of the human gut microbiota.

The human gut microbiota controls factors that relate to human metabolism with a reach far greater than originally expected. Microbial communities and human (or animal) hosts entertain reciprocal exchanges between various inputs that are largely controlled by the host via its genetic make-up, nutrition and lifestyle. The composition of these microbial communities is fundamental to supply metabolic capabilities beyond those encoded in the host genome, and contributes to hormone and cellular signalling that support the dynamic adaptation to changes in food availability, environment and organismal development. Poor functional exchange between the microbial communities and their human host is associated with dysbiosis, metabolic dysfunction and disease. This review examines the biology of the dynamic relationship between the reciprocal metabolic state of the microbiota-host entity in balance with its environment (i.e. in healthy states), the enzymatic and metabolic changes associated with its imbalance in three well-studied diseases states such as obesity, diabetes and atherosclerosis, and the effects of bariatric surgery and exercise.

Potential energy function for a photo-switchable lipid molecule.

Photo-switchable lipids are synthetic lipid molecules used in photo-pharmacology to alter membrane lateral pressure and thus control opening and closing of mechanosensitive ion channels. The molecular picture of how photo-switchable lipids interact with membranes or ion channels is poorly understood. To facilitate all-atom simulations that could provide a molecular picture of membranes with photo-switchable lipids, we derived force field parameters for atomistic computations of the azobenzene-based fatty acid FAAzo-4. We implemented a Phyton-based algorithm to make the optimization of atomic partial charges more efficient. Overall, the parameters we derived give good description of the equilibrium structure, torsional properties, and non-bonded interactions for the photo-switchable lipid in its trans and cis intermediate states, and crystal lattice parameters for trans-FAAzo-4. These parameters can be extended to all-atom descriptions of various photo-switchable lipids that have an azobenzene moiety.

Integrins and OPN localize to adhesion complexes during placentation in sheep.

Integrins and OPN are potential mediators of blastocyst attachment to the endometrium to initiate implantation. The goals were to examine the temporal/spatial pattern of expression of integrins at the endometrial-placental interface of sheep encompassing Days 9 through 80 of gestation and determine if OPN co-localizes with integrins. Results show the following: (1) αv, α4, ß1, ß3 and ß5 integrins at the apical surface of endometrial luminal epithelium (LE) from Days 11 through 16 of pregnancy that indicate a role for these integrins during implantation; (2) large, intermittent aggregates of αv, α4, α5, ß1 and ß5 integrins at the endometrial-placental interface from Days 20 through 55, suggesting adaptation to a localized tissue remodeling stage of placentation; and (3) integrin adhesion complexes (IACs) containing αv, α4, α5, ß1 and ß5 integrins precisely distribute at the apical surfaces of apposed endometrial LE and chorion along expanses of the interplacentomal endometrial-placental interface between Days 60 and 80 of gestation, suggesting engagement of these integrins with the ECM to stabilize adhesion between endometrial LE and chorion in response to the increasing mechanical stress on this interface by the increasing size of the fetus and volumes of fetal fluids. An advancement is the clear co-localization of OPN and integrins at the endometrial-placental interface throughout gestation in sheep. The comprehensive nature of these results provide evidence that integrins potentially interact with OPN to play key roles in the mechanisms required for implantation and placentation throughout pregnancy in sheep and have implications concerning implantation and placentation in other species.

Optical control of L-type Ca2+ channels using a diltiazem photoswitch.

L-type Ca2+ channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic ß-cell function and cardiac activity under optical control.

Photolipid Bilayer Permeability is Controlled by Transient Pore Formation.

Controlling the release or uptake of (bio-) molecules and drugs from liposomes is critically important for a range of applications in bioengineering, synthetic biology, and drug delivery. In this paper, we report how the reversible photoswitching of synthetic lipid bilayer membranes made from azobenzene-containing phosphatidylcholine (azo-PC) molecules (photolipids) leads to increased membrane permeability. We show that cell-sized, giant unilamellar vesicles (GUVs) prepared from photolipids display leakage of fluorescent dyes after irradiation with UV-A and visible light. Langmuir-Blodgett and patch-clamp measurements show that the permeability is the result of transient pore formation. By comparing the trans-to-cis and cis-to-trans isomerization process, we find that this pore formation is the result of area fluctuations and a change of the area cross-section between both photolipid isomers.

A Lipid Photoswitch Controls Fluidity in Supported Bilayer Membranes.

Supported lipid bilayer (SLB) membranes are key elements to mimic membrane interfaces on a planar surface. Here, we demonstrate that azobenzene photolipids (azo-PC) form fluid, homogeneous SLBs. Diffusion properties of azo-PC within SLBs were probed by fluorescence microscopy and fluorescence recovery after photobleaching. At ambient conditions, we find that the trans-to-cis isomerization causes an increase of the diffusion constant by a factor of two. Simultaneous excitation with two wavelengths and variable intensities furthermore allows to adjust the diffusion constant D continuously. X-ray reflectometry and small-angle scattering measurements reveal that membrane photoisomerization results in a bilayer thickness reduction of ∼0.4 nm (or 10%). While thermally induced back-switching is not observed, we find that the trans bilayer fluidity is increasing with higher temperatures. This change in diffusion constant is accompanied by a red-shift in the absorption spectra. Based on these results, we suggest that the reduced diffusivity of trans-azo-PC is controlled by intermolecular interactions that also give rise to H-aggregate formation in bilayer membranes.

Marijuana Use, Respiratory Symptoms, and Pulmonary Function: A Systematic Review and Meta-analysis.

Background: The health effects of smoking marijuana are not well-understood. Purpose: To examine the association between marijuana use and respiratory symptoms, pulmonary function, and obstructive lung disease among adolescents and adults. Data Sources: PubMed, Embase, PsycINFO, MEDLINE, and the Cochrane Library from 1 January 1973 to 30 April 2018. Study Selection: Observational and interventional studies published in English that reported pulmonary outcomes of adolescents and adults who used marijuana. Data Extraction: Four reviewers independently extracted study characteristics and assessed risk of bias. Three reviewers assessed strength of evidence. Studies of similar design with low or moderate risk of bias and sufficient data were pooled. Data Synthesis: Twenty-two studies were included. A pooled analysis of 2 prospective studies showed that marijuana use was associated with an increased risk for cough (risk ratio [RR], 2.04 [95% CI, 1.02 to 4.06]) and sputum production (RR, 3.84 [CI, 1.62 to 9.07]). Pooled analysis of cross-sectional studies (1 low and 3 moderate risk of bias) showed that marijuana use was associated with cough (RR, 4.37 [CI, 1.71 to 11.19]), sputum production (RR, 3.40 [CI, 1.99 to 5.79]), wheezing (RR, 2.83 [CI, 1.89 to 4.23]), and dyspnea (RR, 1.56 [CI, 1.33 to 1.83]). Data on pulmonary function and obstructive lung disease were insufficient. Limitation: Few studies were at low risk of bias, marijuana exposure was limited in the population studied, cohorts were young overall, assessment of marijuana exposure was not uniform, and study designs varied. Conclusion: Low-strength evidence suggests that smoking marijuana is associated with cough, sputum production, and wheezing. Evidence on the association between marijuana use and obstructive lung disease and pulmonary function is insufficient. Primary Funding Source: None. (PROSPERO: CRD42017059224).

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.