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Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Purpose: Critical bending moment (CBM), defined as the bending moment at which the external nonaxial load applied overcomes screw joint preload and causes loss of contact between the mating surfaces of the implant screw joint components, was measured for four different implants and their single-tooth replacement abutments. Materials and Methods: CBM at the implant-abutment screw joint for four implant-abutment test groups was measured in vitro at 80%, 100%, and 120% of the manufacturers' recommended torque levels. Regular-platform implants with their corresponding single-tooth abutments were used. Microstrain was measured while known loads were applied to the abutment at known distances from the implant-abutment interface. Strain instrumentation was used to record the strain data dynamically to determine the point of gap opening. All torque applications and strain measurements were repeated five times for the five samples in each group. Results: For the Brånemark/CeraOne assemblies, the mean CBMs were 72.14 Ncm, 102.21 Ncm, and 119.13 Ncm, respectively, at 80%, 100%, and 120% of the manufacturer's recommended torque. For the Replace/Easy assemblies, mean CBMs were 86.20 Ncm, 109.92 Ncm, and 120.93 Ncm; for the Biomet 3i/STA assemblies, they were 67.97 Ncm, 83.14 Ncm, and 91.81 Ncm; and for the Lifecore/COC assemblies, they were 58.32 Ncm, 76.79 Ncm, and 78.93 Ncm. Two-way analysis of variance revealed significant effects for the test groups and torque levels. Subsequent tests confirmed that significant differences existed between test groups and torque levels. Conclusion: The results appear to confirm the primary role of the compressive preload imparted by the abutment screw in maintaining screw joint integrity. CBM was found to differ among implant systems and torque levels. Torque levels recommended by the manufacturer should be followed to ensure screw joint integrity. [ABSTRACT FROM AUTHOR]

Introduction: Genome-Wide Association Studies suggest glutathione S transferase C terminal domain (GSTCD) may play a role in development of Chronic Obstructive Pulmonary Disease. We aimed to define the potential role of GSTCD in airway inflammation and contraction using precision cut lung slice (PCLS) from wild-type (GSTCD+/+) and GSTCD knockout mice (GSTCD-/-). Methods: PCLS from age and gender matched GSTCD+/+ and GSTCD-/- mice were prepared using a microtome. Contraction was studied after applying either a single dose of Methacholine (Mch) (1 μM) or different doses of Mch (0.001 to 100 μM). Each slice was then treated with lipopolysaccharide (LPS) or vehicle (PBS) for 24 hours. PCLS contraction in the same airway was repeated before and after stimulation. Levels of TNFα production was also measured. Results: There were no differences in contraction of PCLS from GSTCD+/+ and GSTCD-/- mice in response to Mch (EC50 of GSTCD+/+ vs GSTCD-/- animals: 100.0±20.7 vs 107.7±24.5 nM, p = 0.855, n = 6 animals/group). However, after LPS treatment, there was a 31.6% reduction in contraction in the GSTCD-/- group (p = 0.023, n = 6 animals). There was no significant difference between PBS and LPS treatment groups in GSTCD+/+ animals. We observed a significant increase in TNFα production induced by LPS in GSTCD-/- lung slices compared to the GSTCD+/+ LPS treated slices. Conclusion: GSTCD knockout mice showed an increased responsiveness to LPS (as determined by TNFα production) that was accompanied by a reduced contraction of small airways in PCLS. These data highlight an unrecognised potential function of GSTCD in mediating inflammatory signals that affect airway responses. [ABSTRACT FROM AUTHOR]

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. [ABSTRACT FROM AUTHOR]

Significant progress has been made in the last decade in the development of optogenetic effectors and sensors that can be deployed to understand complex biological signaling in mammals at a molecular level, without disrupting the distributed, lineage specific signaling circuits that comprise nuanced physiological responses. A major barrier to the widespread exploitation of these imaging tools, however, is the lack of readily available genetic reagents that can be easily combined to probe complex biological processes. Ideally, one could envision purpose-produced mouse lines expressing optically compatible sensors and effectors, sensor pairs in distinct lineages, or sensor pairs in discrete subcellular compartments, such that they could be crossed to enable in vivo imaging studies of unprecedented scientific power. Such lines could also be combined with mice to determine the alteration in signaling accompanying targeted gene deletion or addition. In order to address this lack, the National Heart Lung and Blood Institute has recently funded an optogenetic resource designed to create optically compatible, combinatorial mouse lines that will advance NHLBI research. Here we review recent advances in optogenetic sensor and effectors and describe the rationale and goals for the establishment of the Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus). [ABSTRACT FROM AUTHOR]

We examined the myogenic response to infarction in neonatal and adult mice to determine the role of c-kit+ cardiovascular precursor cells (CPC) that are known to be present in early heart development. Infarction of postnatal day 1-3 c-kitBAC-EGFP mouse hearts induced the localized expansion of (c-kit)EGFP+ cells within the infarct, expression of the c-kit and Nkx2.5 mRNA, myogenesis, and partial regeneration of the infarction, with (c-kit)EGFP+ cells adopting myogenic and vascular fates. Conversely, infarction of adult mice resulted in a modest induction of (c-kit)EGFP+ cells within the infarct, which did not express Nkx2.5 or undergo myogenic differentiation, but adopted a vascular fate within the infarction, indicating a lack of authentic CPC. Explantation of infarcted neonatal and adult heart tissue to scid mice, and adoptive transfer of labeled bone marrow, confirmed the cardiac source of myogenic (neonate) and angiogenic (neonate and adult) cells. FACS-purified (c-kit)EGFP+/(αMHC)mCherry- (noncardiac) cells from microdissected infarcts within 6 h of infarction underwent cardiac differentiation, forming spontaneously beating myocytes in vitro; cre/LoxP fate mapping identified a noncardiac population of (c-kit)EGFP+ myocytes within infarctions, indicating that the induction of undifferentiated precursors contributes to localized myogenesis. Thus, adult postinfarct myogenic failure is likely not due to a context-dependent restriction of precursor differentiation, and c-kit induction following injury of the adult heart does not define precursor status. [ABSTRACT FROM AUTHOR]

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca 2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca 2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca 2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca 2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca 2+ but not depletion of intracellular Ca 2+ stores prevented histone-induced Ca 2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca 2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca 2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca 2+ signaling. We found that histones induce an influx of Ca 2+ in ECs that does not cause vasodilation but instead causes Ca 2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Significant progress has been made in the last decade in the development of optogenetic effectors and sensors that can be deployed to understand complex biological signaling in mammals at a molecular level, without disrupting the distributed, lineage specific signaling circuits that comprise nuanced physiological responses. A major barrier to the widespread exploitation of these imaging tools, however, is the lack of readily available genetic reagents that can be easily combined to probe complex biological processes. Ideally, one could envision purpose-produced mouse lines expressing optically compatible sensors and effectors, sensor pairs in distinct lineages, or sensor pairs in discrete subcellular compartments, such that they could be crossed to enable in vivo imaging studies of unprecedented scientific power. Such lines could also be combined with mice to determine the alteration in signaling accompanying targeted gene deletion or addition. In order to address this lack, the National Heart Lung and Blood Institute has recently funded an optogenetic resource designed to create optically compatible, combinatorial mouse lines that will advance NHLBI research. Here we review recent advances in optogenetic sensor and effectors and describe the rationale and goals for the establishment of the Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).