Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

The Vascularization of the Skin of the Atlantic Hagfish, Myxine glutinosa L. as Revealed by Scanning Electron Microscopy of Vascular Corrosion Casts

The vascularization of three different (A, B, C) skin regions (from the level of the heart to the cloaca including dorsal, lateral and ventral skin areas) of the Atlantic hagfish, Myxine glutinosa L. was studied by scanning electron microscopy of vascular corrosion casts. Vessel variables were measured either from semithin sections (diameters) or from vascular corrosion casts (diameters, lengths) and total blood capacities as well as vessel surfaces per unit skin area (mm2) were calculated. There are no significant differences in the number of subepidermal capillary meshes (ranging from 164 to 185 meshes per micrograph) in areas A, B or C nor in vessel lengths. The average vessel length per mm2 is 32 mm. Assuming an average diameter of 22.3 μm these vessels have an average surface of 2.24 mm2 and a volume of 12.5 nanoliters (nl). In contrary weighing two pieces ( 5 mm times 5 mm in size) of the whole skin vascular bed - knowing the density of the casting medium -results in only one fifth of that volume. Overestimation of vessel lengths and diameters by measuring casted structures from micrographs on the one hand and inaccuracies in weighing or dissection of casted skin pieces on the other hand are discussed as sources of observed differences

Mood and fluency: The case of pronunciation ease, liking and trust

We explored the impact of mood on the judgemental consequences of word pronounceability in six Experiments (1 preregistered, total N = 1183). Positive and negative mood was induced via video clips (all but Experiment 4) and subliminal affective primes (Experiment 4). Additionally, participants were presented with easy‐ and difficult‐to‐pronounce letter strings. These were framed as target words to be judged for liking (Experiments 1-2), as names of eBay sellers to be judged for trustworthiness (Experiments 3-5), or as either seller names or passwords to provoke opposing interpretations of pronunciation fluency (Experiment 6). While pronounceability showed a robust effect across experiments, mood did not modulate the judgemental use of (Experiments 1-4), the correction for (Experiment 5) and the interpretation (Experiment 6) of word pronounceability. In conclusion, the judgemental effects of pronounceability persist despite the presence of more objective and task‐pertinent cues, resist judgemental correction and remain unaffected by affective states

Quality of CAD-CAM inlays placed on aged resin-based composite restorations used as deep margin elevation: a laboratory study

OBJECTIVES To assess the impact of the age of resin-based composite (RBC) restorations used for deep margin elevation (DME) on the marginal quality and fracture resistance of inlays. MATERIALS AND METHODS Permanent human molars with direct RBC restorations, involving the mesial, occlusal, and distal surface (MOD), were allocated to four groups (each n = 12). Half of the teeth underwent thermomechanical loading including 240,000 occlusal load cycles and 534 thermal cycles (TML, 5 °C/55 °C; 49 N, 1.7 Hz). With RBC left in one proximal box as DME, all teeth received MOD inlays, made from lithium disilicate (LDS) or a polymer-infiltrated ceramic network material (PICN). The restored teeth underwent TML including 1.2 million occlusal cyclic loadings and 2673 thermal cycles. The marginal quality was assessed at baseline and after both runs of TML. Load-to-fracture tests were performed. The statistical analysis comprised multiple linear regressions (α = 0.05). RESULTS Simulated aging of RBC restorations had no significant effect on the marginal quality at the interface between the RBC and the tooth and the RBC and the inlay (p ≥ 0.247). Across time points, higher percentages of non-continuous margin were observed between the inlay and the tooth than between the tooth and the RBC (p ≤ 0.039). The age of the DME did not significantly affect the fracture resistance (p ≥ 0.052). CONCLUSIONS Artificial aging of RBC restorations used for DME had no detrimental effect on the marginal quality and fracture resistance of LDS and PICN inlays. CLINICAL RELEVANCE This laboratory study suggests that-in select cases-intact, direct RBC restorations not placed immediately before the delivery of an indirect restoration may be used for DME

Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

© 2014 Sitdikova, Fuchs, Kainz, Weiger and Hermann. Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S

Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

© 2014 Sitdikova, Fuchs, Kainz, Weiger and Hermann. Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO) or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Methods: Single channel recordings of GH3, GH4 and GH4 STREX cells were used to analyze channel open probability, amplitude and open dwell times. H2S was measured with ananion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only11-13%, i.e. 34-41 μM is effective as H2S in solution. GH3, GH4 and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S

Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

Endodontic management of traumatized permanent teeth : a comprehensive review

The pulp plays a key role in the treatment of traumatic dental injuries (TDIs) and is strongly associated with the outcome, particularly in severe cases. A correct pulp diagnosis is essential as it forms the basis for developing the appropriate management strategy. However, many TDIs are complex, and their treatment requires a profound knowledge of the physiological and pathological responses of the affected tissues. This comprehensive review will look at the dentine-pulp complex and its interaction with the surrounding tissues following TDIs. The literature up to 2020 was reviewed based on several searches on PubMed and the Cochrane Library using relevant terms. In addition to the recently revised guidelines of the International Association of Dental Traumatology, this article aims to provide background information with a focus on endodontic aspects and to gather evidence on which a clinician can make decisions on the choice of the appropriate endodontic approach for traumatized permanent teeth.Peer reviewe

Trends in complementary/alternative medicine use by breast cancer survivors: Comparing survey data from 1998 and 2005

BACKGROUND: Use of complementary and alternative medicine (CAM) by women with breast cancer is often said to be increasing, yet few data exist to confirm this commonly held belief. The purpose of this paper is to compare overall patterns of CAM use, as well as use of specific products and therapies at two different points in time (1998 vs 2005) by women diagnosed with breast cancer. METHODS: Surveys were mailed to women randomly selected from the Ontario Cancer Registry (Canada) in the spring of 1998 (n = 557) and again in the spring of 2005(n = 877). RESULTS: The response rates were 76.3% in 1998 and 63% in 2005. In 1998, 66.7% of women reported using either a CAM product/therapy or seeing a CAM therapist at some time in their lives as compared with 81.9% in 2005 (p = 0.0002). Increases were seen in both use of CAM products/therapies (62% in 1998 vs. 70.6% in 2005) and visits to CAM practitioners (39.4% of respondents in 1998 vs 57.4% of respondents in 2005). Women in 2005 reported that 41% used CAM for treating their breast cancer. The most commonly used products and practitioners for treating breast cancer as reported in 2005 were green tea, vitamin E, flaxseed, vitamin C, massage therapists and dietitians/nutritionists. CONCLUSION: CAM use (both self-medication with products and visits to CAM practitioners) increased significantly from 1998 to 2005. Now that more than 80% of all women with breast cancer report using CAM (41% in a specific attempt to management their breast cancer), CAM use can no longer be regarded as an "alternative" or unusual approach to managing breast cancer