SCN cytosolic Ca2+ levels exhibit a peak during the day, when both action potential firing and Ca2+ channel activity are increased, and are decreased at night, correlating with a reduction in firing rate. In this study, we employ a single-color fluorescence anisotropy reporter (FLARE), Venus FLARE-Cameleon, and polarization inverted selective-plane illumination microscopy to measure rhythmic changes in cytosolic Ca2+ in SCN neurons. Using this technique, the Ca2+ channel subtypes contributing to intracellular Ca2+ at the peak and trough of the circadian cycle were assessed using a pharmacological approach with Ca2+ channel inhibitors. Peak (218 ± 16 nM) and trough (172 ± 13 nM) Ca2+ levels were quantified, indicating a 1.3-fold circadian variance in Ca2+ concentration. Inhibition of ryanodine-receptor-mediated Ca2+ release produced a larger relative decrease in cytosolic Ca2+ at both time points compared to voltage-gated Ca2+channels. These results support the hypothesis that circadian Ca2+ rhythms in SCN neurons are predominantly driven by intracellular Ca2+ channels, although not exclusively so. The study provides a foundation for future experiments to probe Ca2+ signaling in a dynamic biological context using FLAREs.Circadian rhythms in mammals are coordinated by the central clock in the brain, located in the suprachiasmatic nucleus (SCN). Multiple molecular and cellular signals display a circadian variation within SCN neurons, including intracellular Ca Ca2+ is required for SCN neurons to maintain 24-h rhythms in clock gene expression  including L-, N-, P/Q-, R-, and T-type channels (2+ release from intracellular stores in the endoplasmic reticulum (ER) is mediated by ryanodine receptors (RyR2 and RyR3)   , 37, 38. (IP3Rs) . Prior smic Ca2+ ,40,41. H2+ channels to daytime (peak) and nighttime (trough) Ca2+ levels in SCN neurons from intact brain slice cultures. Polarization inverted selective-plane illumination microscopy (piSPIM) was used to measure Ca2+ concentration within SCN using a ratiometric, neuronally expressed Ca2+ sensor, Venus FLARE-Cameleon (Venus-cp172Venus FLARE-Cameleon)  . The Venameleon) . Ca2+ coWild-type C57BL/6J mice were bred in a standard 12:12-h light-dark cycle. Male and female mice were killed for experiments via decapitation at postnatal day 4. All procedures involving mice were conducted in accordance with the University of Maryland School of Medicine Animal Care and Use Guidelines and approved by the Institutional Animal Care and Use Committee.μm) were made on a manual tissue chopper  in ice-cold dissection medium containing bicarbonate-free Dulbecco’s modified Eagle’s medium , 10 mM HEPES (pH 7.3), 100 U/mL penicillin/streptomycin , and 2 mM L-glutamine . Slices containing the SCN  were cultured as organotypic interface explants  was added to culture medium starting on culture day 2 to inhibit glial cell growth. Immediately after plating, slices were transduced with 1 μL of adeno-associated viral vector  containing Venus-cp172Venus FLARE-Cameleon biosensor DNA . Neuron-specific expression was driven by the human synapsin 1 (hSyn1) promoter (2) with 100% of the culture medium exchanged every ∼72 h.For piSPIM experiments, SCN slices were plated onto filter membranes  in 35-mm culture dishes  with 1.2 mL culture medium containing minimal essential medium , 25 mM HEPES (pH 7.3), 25% horse serum , 28 mM D-glucose , 10 U/mL penicillin/streptomycin , and 2 mM L-glutamine . Cytosine nsor DNA   and maintained in culture medium as described in A subset of slices transduced with Venus FLARE-Cameleon AAVs were plated onto multielectrode arrays on culture day 10 as described previously ,45. SCN 2, 20 mM HEPES, 5 mM D-glucose, and 2.5 mM CaCl2 (pH 7.20 ± 0.01 at 35°C). Fluorescence anisotropy imaging was performed on a polarization inverted selective-plane illumination microscopy (piSPIM) microscope with stage-scanning capability assembled and aligned as described previously (P) and perpendicular (S) polarizations. The microscope was housed in an environmentally controlled incubator  maintained at 37°C. Automated stage and piezo focus control hardware elements were purchased from Applied Scientific Instruments . Camera and piezo electronics were controlled using Micromanager software (available at https://micro-manager.org/) . Samples were excited in 10-s (KCl experiments) or 30-s (Ca2+ inhibitor experiments) intervals with a 488-nm laser. Images were collected from a ∼170 × 340 × 20 μm area within the center of the SCN, which was visually identified under brightfield illumination at 4× magnification using the optic chiasm and third ventricle as reference landmarks. After baseline control images were acquired, imaging solution (100–200 μL) was removed from the bath, mixed with the appropriate amount of drug stocks or dimethyl sulfoxide (DMSO) , and reapplied to the bath chamber. The temperature of the bath solution was 35 ± 0.1°C.Imaging experiments were conducted in 6-h time windows using the peak and trough of firing rhythms as the reference point, in which images were obtained between 5 h before to 1 h after the time of the peak or the trough in action potential firing. Filter sections with SCN slices were excised, rinsed in phosphate-buffered saline, transferred to the microscope chamber, and equilibrated for 20–30 min in 6 mL of prewarmed imaging solution containing 125 mM NaCl, 8 mM NaOH, 5 mM KCl, 1 mM MgCleviously , 47, 48.er.org/)  on a Z84P and S channels. Corresponding P and S image stacks (512 × 1024 pixels) were aligned using a Python script and separated into individual images. The median grayscale value of the background intensities for each image was calculated and subtracted. An adaptive local thresholding method was used to obtain a binary clipping mask to separate cell signals from image background. The local threshold value for each pixel was calculated using the Gaussian-weighted sum of the neighborhood pixel intensities (r) were calculated using pixel intensities above the threshold value from the corresponding background-subtracted P and S images using the equation  was measured using an isotropic fluorescein solution and calculated to account for the difference between P and S channel transmission efficiencies as previously described . Scripts for automated image alignment, background subtraction, pixel thresholding, and r-value calculations were executed in Python.The g-factor constant  was prepared and split into two volumes. CaCl2 and NaOH were added to one volume and diluted to obtain a 1× Ca2+-EGTA solution containing 125 mM NaCl, 44 mM NaOH, 5 mM KCl, 2 mM KOH, 1 mM MgCl2, 20 mM HEPES, 1.8 mM 2-deoxy-D-glucose, 5 mM EGTA, 5 mM CaCl2, 0.01 mM rotenone, 0.01 mM ionomycin, and 0.01 mM cyclopiazonic acid (CPA) (pH 7.20 ± 0.01 at 35°C). NaOH and HCl were added to the second volume to produce a final 1× EGTA (zero free Ca2+) solution containing 125 mM NaCl, 44 mM NaOH, 5 mM KCl, 2 mM KOH, 1 mM MgCl2, 20 mM HEPES, 1.8 mM 2-deoxy-D-glucose, 5 mM EGTA, 0.01 mM rotenone, 0.01 mM ionomycin, and 0.01 mM CPA (pH 7.20 ± 0.01 at 35°C). The appropriate quantities of Ca2+-EGTA and EGTA solutions were mixed to obtain solutions with known free Ca2+ concentrations calculated with WebMaxC standard (available online at https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). SCN slices were equilibrated in Ca2+ buffer solutions at least 20 min before imaging. All imaging solutions were prepared with Ca2+-free liquid-chromatography mass-spectrometry (LC-MS)-grade water . The dissociation constant (Kd) and Hill coefficient (n) were determined by fitting a plot of the r vs. Ca2+ concentration data in Prism (GraphPad Software) with the equationCan et al. . To ensut-tests (two tailed) were used to determine significant differences in anisotropy values and Ca2+ concentrations between peak and trough time points. One-way ANOVA with Bonferroni’s post hoc tests were used to determine significant differences in ΔCa2+ between conditions at each time point. Paired t-tests (two tailed) were used to test for changes in anisotropy and Ca2+ concentration between baseline and drug conditions for individual SCN slices at each condition. Significant differences in GCaMP6f fluorescence across multiple peak and trough time points were tested with a two-way, repeated-measures ANOVA and Bonferroni’s post hoc test using the F/Fmax-values from individual cells for all slices across time points.Statistical tests were performed in Prism v8.4 (Graphpad Software). Changes in anisotropy values across baseline time points were tested with a two-way analysis of variance (ANOVA) with repeated measures. Student’s μM nimodipine , 10 μM dantrolene , 30 μM CPA , 3 μM ω-conotoxin GVIA , 200 nM ω-agatoxin IVA , 30 μM NiCl2 , 10 μM ionomycin , 10 μM rotenone , and 50 mM KCl . Reagent stocks (1000×) were prepared in DMSO  or water  and stored at −20°C. KCl was prepared as a 4 M HEPES-buffered stock solution.Pharmacological reagents were used at final concentrations of 10 2+ was also rhythmic under these experimental conditions using the Ca2+ sensor GCaMP6f  and transduced with AAVs containing Venus FLARE-Cameleon cDNAs . Volumetric images of the polarized fluorescence signals were collected from a cubic area of the SCN (A iii). Anisotropy histograms were fitted with a Gaussian distribution to calculate a single mean anisotropy value encompassing signals from all cells within the imaging region.Fluorescent signals from neurons expressing the Venus FLARE-Cameleon biosensor were clearly detectable within the SCN A ii. Vo the SCN . A local the SCN A iii. A2+ concentration can be sensitive to variations in temperature and pH . For in situ calibration experiments, ionomycin, which permeabilizes the cell membrane to Ca2+; rotenone, an ATP inhibitor; and CPA, a SERCA-ATPase inhibitor, were added to the bath solution to limit homeostatic compensation and promote the clamping of intracellular Ca2+ concentration (d = 230 nM) and hillslope (n = −1.0) values obtained from this in situ Ca2+ calibration curve were similar to those previously reported for this sensor in vitro (B). The maximal (Rmax) and minimal (Rmin) anisotropy values were 0.259 and 0.184 in 0 and 1 mM Ca2+, respectively. These results indicated that the Venus FLARE-Cameleon reporter was functionally expressed and responsive to changes in clamped Ca2+ concentration.Because the relationship between anisotropy values and Cae and pH , the Ca2 at 35°C B. For intration . We founin vitro   were significantly lower compared to anisotropies obtained during the trough (0.228 ± 0.002), indicating that more Ca2+ was bound to the sensor during the peak of the circadian cycle (C). This corresponded to an estimated Ca2+ concentration that was 1.3-fold higher at the peak  compared to the trough  (D). These data show that a circadian rhythm in intracellular Ca2+ can be detected from SCN neurons with the Venus FLARE-Cameleon sensor, and to our knowledge, provides a new method to track circadian changes in cytosolic Ca2+.The in situ calibration values were next used to calculate Caan cycle C. This –365 nM) D. These2+ signaling under these in situ conditions, 50 mM KCl was applied to a subset of SCN slices at the peak and trough. Volumetric images were acquired in 10-s intervals for a 2-min baseline period before KCl was applied to the bath chamber and up to 10 min after KCl application. Consistent with prior studies . The maximal KCl-evoked responses were compared with the average baseline anisotropies and Ca2+ concentrations at the circadian peak and trough. KCl produced a transient increase in Ca2+ levels of 3.66-fold during the peak  and 3.52-fold during the trough . Thus, Venus FLARE-Cameleon detects changes in Ca2+ evoked during maximal Ca2+ signaling at both the peak and trough.To define the response of the biosensor to acute changes in Ca studies , SCN sli2+ channel subtypes to neuronal Ca2+ in the SCN, we measured the effects of Ca2+ channel inhibitors on anisotropy  and estimated Ca2+ concentration (A–D) during the peak and trough of the circadian cycle. For Ca2+ channel pharmacology experiments, volumetric images were captured in 30-s intervals for 2 min to obtain baseline anisotropy values before drugs or vehicle controls were applied. The effects of each drug were analyzed in slices imaged in 30-s intervals for 2 min of baseline and for 10 min after the application of a drug or vehicle control , which were added to the bath solution just before time 0. The average change in anisotropy values  and corresponding change in Ca2+ concentrations (A and B) relative to the baseline average of each slice were plotted as a function of time. Anisotropy values were stable, with no significant change for the duration of the 2-min baseline recordings in each condition  . As a control for neuronal health after drug treatments, 50 mM KCl was applied to some slices after drug effects were obtained. KCl produced decreases in anisotropy values corresponding to increases in Ca2+ concentration that were 2–20 times higher than baseline Ca2+ levels (data not shown). The duration and magnitudes of these transient KCl-evoked responses were similar to those observed for slices in control conditions. These KCl responses obtained at the end of the experimental protocol verify that SCN slices are able respond to stimuli after Ca2+ channel inhibitors were applied, demonstrating that drug exposure did not affect slice viability.Next, to test the contributions of the different voltage-gated and intracellular Caisotropy , A–D andntration , A–D dury values , A and Btrations , A and Bondition , A and BμM Nim to target L-type Ca2+ channels and a cocktail containing voltage-gated channel inhibitors (VGCs) targeting N-type (3 μM ConoGVIA), P/Q-type (200 nM AgaIVA), R-type (30 μM nickel), and T-type (1 μM TTA-P2) Ca2+ channels. For each slice, paired comparisons were made between baseline, and the Ca2+ concentration averaged from 9 to 10 min after drugs were applied (C–F). Application of vehicle control  during the peak or the trough did not significantly affect anisotropy values (C and D) or Ca2+ concentrations (E and F) compared to baseline. In contrast, voltage-gated Ca2+ channel inhibitors produced an increase in anisotropy values (C and D), which corresponded to a reduction in the Ca2+ levels in paired comparison to baseline values (E and F). These data implicate voltage-gated Ca2+ channels as contributors to the cytosolic Ca2+ levels during the peak and the trough.First, to probe the contributions of voltage-gated channels, we used 10  applied , C–F. Apy values , C and Dtrations , E and Fy values , C and De values , E and F2+ channels. Previous studies have shown that inhibiting RyR-mediated Ca2+ release from the ER produced a decrease in cytosolic Ca2+ levels . These results suggest that intracellular channels also contribute to cytosolic Ca2+ levels at both the peak and the trough.We then tested the contributions of intracellular Ca+ levels ,41, but  release . In a thch slice , E and F2+ channels across experiments requires accounting for the variation in baseline Ca2+ levels in each SCN slice. To make this comparison, the average Ca2+ concentration from 2 min of baseline was subtracted from the average Ca2+ levels 9–10 min after drug application to obtain the change in Ca2+ (ΔCa2+) within each slice. First, we focused on the ΔCa2+ produced by each drug during the peak (A and C). Starting with the vehicle controls, the ΔCa2+ was negligible at −2 ± 7 nM (−2 ± 5% change) (C). The voltage-gated channel inhibitors produced a ΔCa2+ of −35 ± 10 nM (−20 ± 4% change) (Nim) and −40 ± 13 nM (−19 ± 4%) (VGC); however, these decreases were not statistically different than the vehicle control. This may be partly explained by the variability in the ΔCa2+ responses of individual slices, which ranged from −6 to −78 nM (Nim) and −5 to −79 nM (VGC) (C). In contrast, intracellular Ca2+ channel inhibitors produced decreases in peak Ca2+ that were significantly larger than the vehicle controls. Dantrolene produced the largest decrease, with a ΔCa2+ of −50 ± 6 nM (−36 ± 6% change). Similarly, the SERCA inhibitor CPA produced a ΔCa2+ of −59 ± 3 nM (−24 ± 5%), and the mixture of VGC inhibitors along with nimodipine and dantrolene added together (X cocktail) produced a ΔCa2+ of −67 ± 21 nM (−22 ± 6%) (C). The responses of individual slices to intracellular Ca2+ channel inhibitors were less variable, with the range of −38 to −73 nM for dantrolene, −52 to −71 nM for CPA, and −27 to −114 nM for cocktail X. Taken together, these results suggest that peak intracellular Ca2+ is predominantly set by RyR channel contribution.Determination of the relative contributions for VGCCs and intracellular Cathe peak , A and C change) C. The vnM (VGC) C. In co22 ± 6%) C. The r2+ during the trough of the circadian cycle (D). Vehicle control had little effect, −5 ± 4 nM (−7 ± 5% change). Nimodipine decreased Ca2+ by −28 ± 8 nM (−17 ± 5%) overall with a range of responses between −2 and −56 nM. Similarly, VGC decreased Ca2+ by −20 ± 6 nM (−16 ± 3%), with changes in Ca2+ ranging from −3.5 to −37 nM. The decreases in trough Ca2+ were significantly larger for dantrolene, −53 ± 12 nM (−28 ± 6%); CPA, −43 ± 8 nM (−32 ± 7%); and cocktail X, −51 ± 11 nM (−23 ± 5%). These overall decreases were accompanied by a larger range in ΔCa2+ responses, which were −14 to −106 nM for dantrolene, −16 to −65 nM for CPA, and −15 to −91 nM for cocktail X (D). Thus, at both peak and trough time points, the largest decrease in Ca2+ was produced by dantrolene. In contrast, the contribution of voltage-gated channels appears less significant and in some cases has more variability.These inhibitors had similar effects on Caan cycle D. Vehiccktail X D. Thus,2+ in live organotypic brain tissue. The Venus FLARE-Cameleon Ca2+ sensor captured the collective signal in basal cytosolic Ca2+ averaged from multiple neurons within a region of the SCN without integrating shorter-timescale Ca2+ signals, such as action-potential-evoked Ca2+ transients. Using these techniques, Ca2+ concentrations  were refined over prior absolute and relativistic estimates  and a state of decreased excitability at night (trough of the cycle), during which voltage-gated Ca2+ channel activity is reduced  (2+-ATPases (SERCA)  ,75 and e (SERCA) , which mrial NCX  and Ca2+lic Ca2+ , 78, 79.channels . Anotherproteins , which cansients ,82, leadansients . SCN neulretinin ,83. In Slretinin . This ev2+ regulatory mechanisms by subregion, but the phase of the Ca2+ rhythm exhibits regional differences. It has not yet been addressed whether the Ca2+ channels themselves differ by subregion as the basis. The shell region of the SCN, defined by expression of the neuropeptide arginine vasopressin, exhibits a rhythmic Ca2+ peak 3–5 h before the core, defined by vasoactive intestinal polypeptide (VIP) expression  failed to show a regional difference in its effects on Ca2+ levels  knockdown of intracellular Ca2+ release via IP3Rs or SERCA, but only one set of neurons was sensitive to VGCC knockdown (This study did not focus on defining the Capression ,30,33. S neurons ,85, in wnockdown .A.E.P. and A.L.M. designed the experiments and wrote the manuscript. A.E.P. performed the experiments and analyzed the data, V.P.R. wrote the computer code and assisted with image analysis, and M.A.R. designed and built the piSPIM microscope.


They are assigned specific functions within organelles, but share many transport mechanisms to cross organellar membranes. Despite their points of interaction, those elements are usually investigated and reviewed separately. This review takes them out of this isolation. It highlights our current mechanistic understanding and points to open questions of their functions, their transport, and their interplay in the endoplasmic reticulum (ER), vesicular compartments , vacuoles, chloroplasts, mitochondria, and peroxisomes. Complex processes demanding these cations, such as Mn2+-dependent glycosylation or systemic Ca2+ signaling, are covered in some detail if they have not been reviewed recently or if recent findings add to current models. The function of Ca2+ as signaling agent released from organelles into the cytosol and within the organelles themselves is a recurrent theme of this review, again keeping the interference by Mn2+ in mind. The involvement of organellar channels , transporters , Ca2+ exchangers (CAX), metal tolerance proteins (MTP), and bivalent cation transporters (BICAT)], and pumps [autoinhibited Ca2+-ATPases (ACA) and ER Ca2+-ATPases (ECA)] in the import and export of organellar Ca2+ and Mn2+ is scrutinized, whereby current controversial issues are pointed out. Mechanisms in animals and yeast are taken into account where they may provide a blueprint for processes in plants, in particular, with respect to tunable molecular mechanisms of Ca2+ versus Mn2+ selectivity.Calcium (Ca Calcium and manganese play essential roles in organellar compartments, yet they share many transport mechanisms, implying synergistic, and antagonistic interactions of both cations. It aims to show that a more integrative view of both elements is overdue, since classical, as well as more recent work, points to an interaction of those two cations, owing to their similar coordination geometry and ionic radii. For instance, induction of Ca2+ deficiency symptoms and diminished Ca2+ translocation by excess Mn2+ in bean (Phaseolus vulgaris) plants has already been observed in the 1970s -, IP3 receptor (IP3R)-, and voltage-gated Ca2+ channel (VGCC)-type also conduct Mn2+  family  family ; the phyignaling .2+ in plants has been the subject of a number of comprehensive reviews (2+ considered as most important in plants is that as a redox-active constituent of the oxygen (O2)-evolving complex (OEC) in photosystem II (PSII), where Mn2+ and Ca2+ form a Mn4CaO cluster to catalyze the splitting of water (H2O). The chloroplast that harbors this machinery, hence, needs to accumulate large amounts of Mn2+, in parallel with dynamically modulating free Ca2+ as regulatory element, as discussed below. Further roles of Mn2+ are distributed amongst different compartments, such as reactive O2 scavenging in the mitochondria by Mn2+-Superoxide Dismutase (SOD), phytohormone deconjugation in the endoplasmic reticulum (ER), and glycosylation in the Golgi apparatus (2+/Mn2+ transporter Gcr1-dependent translation factor1 (GDT1), high-Ca2+ media cause glycosylation defects which could be rescued by the addition of Mn2+  and Autoinhibited Ca2+-ATPases (ACAs), respectively. The former also transport Mn2+, and the latter are feedback-regulated by a Ca2+/calmodulin-binding autoinhibitory domain. Those pumps have recently been reviewed by 2+ and Mn2+ homeostasis. The Ca2+ exchanger (CAX) family, reviewed by 2+-specific transporters and those transporting Ca2+ and Mn2+; both are energized by H+ antiport  family  family, called metal tolerance proteins (MTPs) in plants, mediate the Hganelles . Transpo) family . Natural cytosol . The ZRTembranes . For manal basis . Within ing Mn2+ . All pla2+, and potentially also Mn2+, from organelles along their electrochemical gradient is mediated by channel proteins. Although pharmacological analyses have frequently pointed to a contribution of organellar Ca2+ release to the generation of Ca2+ signals, the respective Ca2+ stores and the identity of the channels have remained mostly undefined , glutamate receptor-likes (GLRs), mitochondrial conductivity units (MCUs), and possibly annexins have been uncovered , which engulfs the nucleus. The steady-state cyt. Further complexity is added by a likely interference of cADPR synthesis and poly-ADP(ribosyl)ation due to their common precursor, NAD+  channel. A large gating ring structure of the protein contains Ca2+-binding sites that biphasically stimulate or inhibit channel activity , employ Ca2+ or Mn2+ for their catalytic activity (Nicotiana tabacum), Ca2+ serves as cofactor for a Golgi-localized calreticulin, likely required for correct protein folding similarly to ER-located calreticulin , a formidable challenge lies in the pH dependence of most of those fluorescence-based reporters , and the PVC. In plants, vacuolar sorting receptors (VSRs) are involved in cargo transport to the vacuole  to VSRs occurs in a Ca2+-dependent manner  in a sphingomyelin-dependent manner  uptake transporter IRT1 in a Ca2+-dependent way, albeit in that study, EHB1 was localized to the plasma membrane , which contain two cytosolic C2 domains, are also predestined targets of such local [Ca2+]cyt hotspots. In animals, SYTs regulate membrane trafficking and fusion in a Ca2+-dependent manner , Mnpparatus . GTs arepparatus . Most ofatus , N-acetylglucosaminyltransferase I (GNTI), Golgi α-mannosidase II (GMII), N-acetylglucosaminyltransferase II (GNTII), α-1,3-fucosyltransferase (FUT11 and FUT12), β-1,2-xylosyltransferase (XYLT), β-1,3-galactosyltransferase (GALT1), and α-1,4-fucosyltransferase , some further Arabidopsis GTs involved in O-glycosylation are predicted to contain a DxD motif or to bind Mn2+ by sequence similarity (Extensins (EXTs) and arabinogalactan proteins (AGPs) are two major activity . Mutantsactivity , 2015b. milarity .2+-dependent manner  sphingolipids and hence was renamed to inositol phosphorylceramide glucuronosyltransferase1  takes place exclusively in the Golgi. Some of the Arabidopsis GTs involved in this process have been described to work in a Mnt manner . There aactivity . Another mutants . These m2+-dependent, which has yet to be confirmed experimentally. In this respect, it is interesting to note that 2+ supply. This mutant contains a nonsynonymous point mutation which, however, is not close to the Mn2+-binding site of the protein, but which may cause a partial loss in enzymatic activity, leading to the Mn2+-dependent growth defect. It is unclear if the mutant protein requires a higher [Mn2+]Golgi to function, or if Mn2+ may even have a regulatory role.Based on its homology to GUX1, PGSIP6/IPUT1/MOCA1 is bound to be Mn2+ with Ca2+ signaling. Intriguingly, a mutant called2+]i increases1 monocation-induced cyt signals in response to salt (Na+) stress  to the cytosol  nanoprobe with particle-induced X-ray emission (PIXE) microanalysis and backscattering spectrometry, 2+ within the Golgi of PC12 dopaminergic cells at physiological concentrations, and a further increase of the Golgi-allocated fraction when cells were exposed to 100 µM MnCl2, a subcytotoxic level. Upon exposure to toxic concentrations, Mn2+ was also detected in cytoplasm and nucleus. Based on monitoring the subcellular distribution of Mn2+ in living HEK293T cells by a fluorescent Mn2+ sensor and by nano-SXRF imaging, 2+ storage organelle under physiological conditions, has an additional trafficking role under subcytotoxic Mn2+ conditions. No direct measurement of compartmental Mn2+concentrations has been conducted in plants yet. However, a crucial role of the Golgi in Mn2+ detoxification has been inferred from the Mn2+-hypersensitive phenotype of the Golgi-localized Mn2+ transporter MTP11  and rice (OsMTP11) also complemented a Mn species . This isor lines . In a pansporter . As for ns-Golgi  and the ntration . Across 2+ transporter accompanying the pump is not known yet. Also, the transport proteins identified so far have been associated with Mn2+ detoxification, whereas the Mn2+ supply of the complex Golgi-localized glycosylation processes is completely obscure. A role of ECA3 in glycosylation remains to be elucidated, and further transport proteins to support this function remain to be identified, whereby homologs of GDT1/TMEM165 represent promising candidates.Unlike in animals and fungi, a Ca2+ from the Golgi is mediated by RyRs and IP3Rs . CBL proteins together with CBL-interacting protein kinases (CIPKs) form modules to transduce Ca2+ signal information into phosphorylation events. In Arabidopsis, four CBLs  localize to the tonoplast accumulating species that store vacuolar Mn2+ mainly in complex with carboxylates, such as malate, citrate, or oxalate  reduction mediated by ferric chelate reductase (FRO2), hence triggering iron chlorosis  across the tonoplast, and by ACAs, of which ACA11 resides in the membrane of lytic vacuoles . Auxin responses were also affected in cax1 and cax3 mutants, causing a defective regulation of stomatal guard cells  family  family , which afamilies , the modaffinity . Howeverd to IP6 . We are TPC1, has emerged as one of the regulators of [Ca2+]cyt. Identified electrophysiologically in the 1980s  channel, encoded by he 1980s , it reprhe 1980s . Its molhe 1980s . Studiesproteins . These f2+ release from lysosomes and endosomes P2 , which would allow them to contribute to a ROS wave-driven Ca2+ wave , a thylakoid membrane-associated Ca2+-binding protein that has been identified as a central regulator of cellular Ca2+ homeostasis and signaling. CAS modulates chloroplast stromal Ca2+ signals induced by darkness, flg22, and heat, and, surprisingly, it governs external-Ca2+-induced Ca2+ transients in the cytosol (2+-activated K+ channel TPK3 (see above), has also been described to localize to thylakoid membranes and to be vital for the generation of the pmf  and abiotic  stresses as well as environmental stimuli  binuclear center, which unravels a crucial step in state transitions between PSI and PSII  divide the chloroplast into three distinct internal compartments, consisting of the intermembrane space between the two chloroplast envelope membranes, the stroma, and the thylakoid lumen. To transport ions and metabolites through these three membranes, the chloroplast harbors transport proteins in each of them .2+ uptake from the cytosol into the chloroplast is a light-dependent process , with a second member named PAM71-homolog  based on one of its substrates and its localization  and BICAT2, based on the substrate spectrum of all known eukaryotic homologs , a Ca2+ channel localized in the chloroplast inner envelope as well as in root mitochondria was identified  stress responses, signaling events downstream of the Ca2+ signal were altered, and mutants were drought-tolerant  cycle, that is, pyruvate dehydrogenase (PDH), NAD+-isocitrate dehydrogenase (NAD-ICDH), and 2-oxoglutarate dehydrogenase  or Ca2+-dependent dephosphorylation H+ and ATP synthesis. Such a role of [Ca2+]mito is less clear in plants.Compared to chloroplasts, the role of Ca subject . In animtabolism . This rese OGDH; . Regulation PDH; . The ATP2+]mito in animals is to trigger apoptotic cell death. Thereby the mitochondrial permeability transition pore, a membrane-spanning multi-protein complex, that likely originates from the F-ATP synthase, is activated by mitochondrial Ca2+ overaccumulation, causing membrane rupture and the release of apoptotic signals, such as cytochrome c -dependent glutamate dehydrogenase2 (GDH2), but not GDH1  in mitochondria are the NAD-dependent malic enzymes (NAD-MEs), which catalyze the oxidative decarboxylation of malate to yield pyruvate and COatalysis . Intrigusubunits , as wellsubunits . Their anits , MCUR1, MICU1, and MICU2 , which is due to the activity of the electron transport chain, Cachanisms . The out+ import . Convers the MCU . The chaore loop . Its strore loop . Notablynd MICU2 . In prinEF-hands . At low bits MCU . This invate MCU . In cons to Mn2+ .2+ influx by MCU, animal mitochondria are able to accumulate Ca2+ with high affinity. A protein localized to the mitochondrial inner membrane, leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), has been identified to mediate this uptake of Ca2+ in exchange of H+ at [Ca2+]cyt < 1 µM  and mitoferrin 1 (Mfrn1). There is evidence that DMT1 transports Mn2+ from the cytosol across the OMM to the intermembrane space  occupy Ca2+-binding domains. Both cations also cooperate, as in the OEC. Such functions and interactions of Ca2+ and Mn2+ are determined by their free concentrations in the different compartments, which might be examined by genetically encoded indicators, that need yet to be developed for Mn2+. This issue may also be approached by direct subcellular imaging of Ca2+ and Mn2+, for example, by synchrotron radiation, but such techniques with subcellular resolution are not widely available, extraordinarily complex, and unable to show dynamic changes. Both approaches need to be pushed forward (see Advances and Outstanding Questions).Ca2+ and Mn2+ into chloroplasts and mitochondria are now known, albeit not fully understood, and further candidates are lining up to be tested. The plant ER/NE has turned out as a hub of Ca2+ signaling in different circumstances, and channel proteins in this compartment have been found: The identification of CNGCs in the NE solved the long-standing question how nuclear Ca2+ signals are generated in symbiotic signaling, which has very recently taken another turn with the characterization of CASTOR as Ca2+ channel. The finding that GLRs essential for systemic Ca2+ signaling may also reside in ER and vacuole, besides their operation in the plasma membrane, demands a scrutiny of their roles in those endomembranes. In this respect, the function and regulation of the most-studied plant channel, TPC1, is also far from clear. Some major gaps in the array of organellar transporters still need to be filled. For example, in the plant Golgi, no transporters associated with Mn2+ import for glycosylation and no Ca2+ release channels are known; peroxisomal Ca2+ and Mn2+ transport is a black box.Membrane transport proteins are the backbone of cellular cation (re-)distribution, and in the last 5 years, a number of new players have shown up on the pitch. Notably, routes for Ca2+ and Mn2+. Here, tunable discrimination mechanisms have become apparent from nonplant work in the form of EF-hands, either in accessory subunits  or in the transport protein itself (as in yeast PMR1 or human SPCA1). Conversely, for the chloroplastic BICAT proteins that supply the organelle with Mn2+ and shape its Ca2+ signals, mechanisms of tell Mn2+ from Ca2+ are unknown, and even the mode of transport of this family is debated. The search for interactors, transport assays on proteoliposomes, and eventually the solving of a protein structure will lead toward a better understanding.Mechanisms on protein, organellar, and cellular levels ought to be in place that define the distribution and ratio of Ca2+ and Mn2+ are allocated to organelles is far from clear. How do organelles get prioritized? Are there hard-wired logics? To test the simplest assumption that substrate allocation is determined by different affinities of the transport proteins, their kinetics need to be determined, which is not trivial. However, a more sophisticated coordination and communication between compartments and subcompartments seem inevitable for an efficient utilization of the cations and the optimum supply of target proteins. An impressive example of coordinated ion concentrations is the animal secretory pathway with its subcompartmental [Ca2+] gradient. Yet there is some evidence for specific supply chains in plants. Mn2+ delivery to chloroplasts, for example, is believed to require sequential Mn2+ release from the TGN and the vacuole by NRAMP2 and NRAMP3/4, respectively. This bears some similarity to yeast, where the NRAMP2 homolog SMF2 supplies the mitochondria by endosomal Mn2+ release.Finally, the way Ca2+ or Mn2+ are passed from one organelle to the next, are likely to play a major role that has yet to be unveiled in plants. Examples from yeast and animals are in support of this, for instance between ER and mitochondria, where Ca2+ transport proteins of different organelles even physically interact.What guarantees that these intraorganellar fluxes reach the correct recipient? Besides spatial proximity, membrane contact sites, at which Ca2+ and Mn2+ transport, the regulation of those proteins and their interaction within and between organelles needs to be resolved to reach an understanding of Ca2+ and Mn2+ handling by the plant cell. Sneaking a peek at animals and yeast has proven fruitful in the past and will yield more insights in the future.After completing the toolbox of Ca2+ and Mn2+ transport activities across organellar membranes has been revealed. In particular, transporters for Ca2+ and Mn2+, and a Ca2+ channel, have recently been identified in the chloroplast.The molecular basis of many Ca2+ signaling in plants has become evident.The contribution of organelles, in particular the ER and the vacuole, to cytosolic Ca2+ have been identified in the Golgi and the vacuole.Transporters that detoxify Mn2+ distribution has become apparent.A role of the TGN in cellular Mn2+ from the Golgi, or to move Ca2+ and Mn2+ across peroxisomal membranes? Which transport proteins supply the Golgi and ER with Ca2+ and Mn2+ for glycosylation processes?What is the identity of the missing transport proteins, e.g. to release Ca2+ and Mn2+? Is there a gradient of Ca2+ along the secretory pathway, as there is in animals?What are the (sub)organellar concentrations of total and free Ca2+ and Ca2+ homeostasis controlled individually if transport proteins are unspecific?How are Mn2+ signals?What is the contribution of different organelles to specific Ca2+ signaling?How is the vacuolar cation channel TPC1 integrated in systemic Ca2+ interfere with Ca2+-dependent signaling processes, and does it have a signaling function on its own?Does Mn2+ and Mn2+ allocated to different organelles, especially under limiting conditions?How are Ca


The influence of scaler tip design on root surface roughness, tooth substance loss and patients’ pain perception is investigated.This article was divided into the following parts: Part 1 Surface roughness and substance loss: an in vitro study, which involves intact extracted teeth sectioned and treated using a piezoelectric ultrasonic device  with a conventional scaler tip (FS-407) and a Perio Slim (PS) scaler tip (Perio Slim DS-016A). All sectioned samples for tooth surface roughness (n = 20) and tooth substance loss (n = 46) analyses were measured and compared using a 3D surface texture analyser and scanning electron microscope (SEM) respectively, at baseline and following scaling. Part 2 Pain Perception: a clinical study, which was a split mouth study design including 30 participants with gingivitis and/or mild chronic periodontitis; treated with supra-gingival scaling from teeth #13 to #23. Subjects were randomised to group A or group B. Group A was treated first with PS scaler tips, whereas group B was treated first with conventional scaler tips. Pain perception was recorded using the visual analogue scale (VAS).p < 0.05), but no significant difference between the two scaler tips (p > 0.05) was observed. The PS scaler tip caused statistically significantly less root substance loss (p < 0.05) when the initial thickness of the tooth was < 1000 µm. Clinical study: the participants reported significantly lesser pain score during scaling using the PS scaler tip (median: 3) than when using the conventional scaler tip (median: 5) (p < 0.05).In vitro study: both scaler tips caused significant reduction in root substance roughness after scaling  scaler tip design. The effective removal of calculus and prevention of the recolonization of periodontal bacteria to periodontal pockets are common strategies used in periodontal therapy . Hand inThe performance of an ultrasonic scaler increases with displacement amplitude, and the magnitude of displacement amplitude increases when the power setting is increased, working load is reduced, and a wide scaler tip is used , 5. DispTooth substance loss as a result of a scaling procedure with an ultrasonic scaler produces a rough surface that facilitates the recolonization of bacteria on the root surface . ExcessiA new-generation piezo ultrasonic scaler designed for gentle biofilm removal, Vector ™ , was introduced in year 2000. Vector produces smoother root surfaces than conventional hand scaler . This scThe analysis of tooth substance loss after scaling suggests that ultrasonic scalers remove lesser amount of tooth substance than hand instruments , 11, 12.Patient’s compliance to dental treatment is affected by several factors, including socioeconomic status, dental fear and dentist’s behaviour . AccordiThis section describes the methodology in two parts: Part 1 Surface roughness and substance loss—an in vitro study that investigated the influence of scaler tip design on tooth surface roughness and tooth substance loss, and Part 2 Pain perception—a clinical study that investigated patients’ pain perception.Single-rooted human permanent teeth extracted within the last 6 months were obtained from a government dental clinic in Bangsar, Kuala Lumpur. The teeth were disinfected using 0.5% chloramine-T trihydrate  for a week. All teeth are then stored in distilled water, and placed in a fridge at 4 °C before analysis. The inclusion criteria for tooth selection were as follows: (1) sound teeth and (2) minimal calculus. The exclusion criteria were (1) teeth with crack lines; (2) teeth with signs of abrasion and/or erosion; and (3) hypomineralised teeth, or teeth with signs of amelogenesis imperfecta and dentinogenesis imperfecta.Before the experiment, the teeth were prepared as follows: Approximately 3 mm of the apical portion of each tooth was embedded in a clear cold-curing epoxy resin  for subsequent cutting. The tooth was sectioned in the apico-coronal direction with a slow-speed precision cutter . Apical to the CEJ by 1 mm, an area of 3 mm (width) × 5 mm (height) was marked using a permanent marker   Fig. , 4. A meScaling was performed according to a standard protocol as follows. The scaler tip was placed parallel to the tooth surface and at zero-degree angle during scaling. The ultrasonic machine was activated prior to scaling to avoid the heaviest vertical movements. Once activated, the scaling was carried out in three continuous strokes with light force. The light force used was practiced without activating the tool, by probing under a fingernail with the amount of force that causes no pain . Before a), defined as the average peaks and valley distances from the centre line of an area. The magnification was set at × 200 and 80 µm working length. Measurements were done in triplicate for each sample before and after scaling. The mean Sa values were then calculated.Scaled tooth specimens were dried and mounted on a glass slab by means of utility wax. The specimens were observed under a 3D optical surface texture analyser . Surface roughness was determined as mean roughness  on the cross-section surface, which included upper and lower reference points. The shortest distance between reference points and outer tooth surface was measured in micrometres (µm) and referred to as ‘tooth thickness’. Tooth thickness was measured in triplicate, and the mean was obtained. Tooth substance loss was the difference in tooth thickness before and after scaling. Magnification was set at 50 × and working distance of 10.0 mm.This study is a single-centre, single-blinded, and randomised controlled clinical study conducted at a teaching institution. Ethical approval was obtained from the Medical Ethics Committee Faculty of Dentistry, University of Malaya DF RD1705/0022(U).Trial registration This clinical study followed the Consolidation Standards of Reporting Trials Statement and was retrospectively registered on 10/11/2020 at ClinicalTrials.gov (No. NCT04623723).The protocol of this study was previously described in detail elsewhere . The parSupragingival scaling was carried out from teeth #13 to #23 with a portable ultrasonic scaler device (EMS) with either a conventional  or PS  scaler tips. A medium power setting (power: 4) and maximum water coolant level were used as recommended by the manufacturer. The scaler tips were held parallel to the long axis of the tooth during scaling. Light force was used for scaling as described in the in vitro part, for a-2 min duration.The primary outcome for this study was pain perception.Pain perception was assessed using the Visual Analog Scale (VAS), which consists of a line numbered from 0 to 10. Scale ‘0’ indicates no pain, whereas scale ‘10’ indicates the worst possible pain. Detailed information about the procedure and how to score VAS were explained to all participants before the treatment. Participants were asked to complete the VAS score themselves by mark on the line the score that they feel represents their pain they experienced during treatment Fig. . A new VData on socio-demography status, oral hygiene habits, education and lifestyle habits were collected using self-administered questionnaires. Periodontal parameters were recorded from teeth #13 to #23 by using a William’s probe . The periodontal parameters measured were probing pocket depth (PPD), visible plaque index (VPI) (Ainamo & Bay 1975) and gingival bleeding index (GBI) (Ainamo & Bay 1975). The clinical examination was performed by a periodontics resident (NAAH) who was calibrated and trained by a periodontist. Figure Data was analysed using G Power version 3 statistical software . The samParticipants were recruited among individuals who were seeking periodontal treatment and matched the eligibility criteria. Randomisation was done with SPSS  by a co-researcher (RS). For the removal of the ordering effect, predetermined recruitment numbers (1–30) were randomly allocated to groups A or B through generated randomisation list. The participants in group A received scaling with PS scaler tip first and then with a conventional scaler tip, whereas the participants in group B received scaling with a conventional scaler tip first and then with a PS scaler tip. The allocated groups were concealed in envelops.This study was a single-blinded study where each participant was blinded to the scaler tip design used. The double-blinded approach was not possible in this study because of some limitations: a single operator performed the intervention.In the Primary Care Unit patients seeking scaling treatment were given appointments at the Periodontology Specialist Clinic, Faculty of Dentistry, University of Malaya. Appointment date was set, and screening for inclusion and exclusion criteria was performed. Clinical examination was performed. From 40 patients screened, 30 patients fulfilled the inclusion and exclusion criteria and were invited to participate in the study. Each participant was asked to fill out a questionnaire and was instructed to answer the VAS immediately after each scaling treatment procedure. The group allocation of the participants was only revealed to the operator after the clinical examination was done.Each participant experienced scaling procedure using both scaler tips. The teeth were split into two sections: section 1 comprised teeth # 11 to # 13, and section 2 comprised teeth # 21 to #23. One of the sections was for the PS, and the other was for conventional scaler tips. The first round of scaling procedure for both groups started off with section 1 (teeth #13 to #11) using their respective scaler tips. The scaling procedure was performed for 2 min.The second round of scaling was performed after an hour. Scaling was done on section 2 (teeth #21 to # 23) using the respective scaler tip. The same procedure was performed.Immediately after each scaling procedure, the participants were asked to score their pain perception with the VAS. Full mouth scaling (if necessary) was given to subjects at the end of the study.p < 0.05. For the in vitro study, the normality of data was analysed using Shapiro–Wilk test. The intra-group comparison of tooth surface roughness (before and after) was analysed using Wilcoxon signed-rank test. Independent t test was used for inter-group comparison. For tooth substance loss, paired t test and independent t test were used in analysing data with normal distribution. For the clinical study, the sociodemographic comparison between groups was analysed using Mann–Whitney test. The baseline clinical parameters were analysed using paired sample t test. Based on Shapiro–Wilk test, VAS data distribution was not normal. Therefore, differences between the VAS scores after therapy of the two groups were compared using Wilcoxon signed-rank test.Data were analysed using SPSS statistical program . Level of significance was set at p < 0.05). The reduction of surface roughness after scaling was 3.1 (± 2.5) µm and 4.7 (± 3.3) µm for PS and conventional group, respectively. However, the difference between the groups was non-significant (p > 0.05).The mean surface roughness (Sa) value before scaling was 9.8 (± 4.7) µm and 10.0 (± 3.2) µm in PS and conventional groups, respectively (Table The distance (D) between the outer tooth surface (T) and upper reference point (UR) or lower reference point (LR) is considered the ‘tooth thickness’. Tooth thickness before scaling is referred to as ‘thickness before,’ and tooth thickness after scaling is referred to as ‘thickness after’. Figure Mean (SD) of tooth thickness (µm) before and after scaling at upper and lower reference points are shown in Tables At the upper reference point, the mean thickness before scaling was 790.9 (130) µm and 745.0 (± 197) µm in the PS and conventional group, respectively (Table p < 0.05).ly Table . After sAt the lower reference point, the mean thickness before scaling was 811.9 (± 179) µm and 802.4 (± 269) µm in the PS and conventional group, respectively. After scaling, thickness decreased to796.0 (± 178) and 780.5 (± 280) µm in the PS and conventional groups, respectively. The mean difference in tooth thickness after scaling was significantly lower in the PS group than that in the conventional group when the initial thickness was < 1000 µm (p < 0.05).At the upper reference point, the mean thickness before scaling was 1280.1 (± 190) and 1267.9 (± 121) µm in the PS and conventional groups, respectively (Table p > 0.05).ly Table . After sAt the lower reference point, the mean thickness before scaling was 1131.0 (± 25) and 1218.0 (± 200) in the PS and conventional groups, respectively. After scaling, the thickness decreased to 1113.0 (± 254) and 1177.0 (± 191) µm in the PS and conventional groups, respectively. The mean difference in tooth thickness after scaling between the PS and conventional groups was not significant (p > 0.05).p > 0.05).Table p > 0.05).The baseline periodontal parameters are summarised in Table Figure p < 0.05).Table Over the last few years, new scaler tip designs have been introduced for painless and less aggressive scaling. Given that periodontal treatment success is largely dependent on continuous periodontal visits, repeated instrumentation may expose tooth structures to risks of tooth substance loss and tooth surface roughness. Moreover, patients undergoing life-long supportive periodontal therapy may have been affected in view of their compliance if treatment was perceived as painful.To the best of our knowledge, no previous study compared tooth surface roughness produced by piezoelectric ultrasonic scalers using either slim (Perio Slim) or conventional scaler tips. An earlier study investigated surface roughness outcomes on restorative materials; the conventional scaler tip caused rougher surfaces than the slim scaler tip . In the The study also reported that when the initial tooth thickness was < 1000 µm, tooth substance loss was lower when scaling was carried out with a PS scaler tip than when a conventional scaler tip was used. A thinner tooth structure may comprise less calcified dentine, which constitutes the hard part of the subgingival portion of a tooth structure. The middle layer of root dentine was harder than the cementum and other layers of root dentine . In caseThe limitation of the study includes lack of information on the history of the extracted teeth whether the teeth were exposed to repeated scaling which causes root cementum loss. Besides, identification of cementum and dentine layers was not performed due to source and time constraint. In a clinical setting, the layers of root substance cannot be determined. Given that periodontal patients are exposed to risks of tooth substance loss after repeated instrumentation, using a slim scaler tip design is an advantage.Slimmer scaler tip design was subjected to the dampening effect that produces flattened oscillation pattern by which displacement amplitude is reduced . The flaThe reduced tooth substance loss after the use of PS scaler tip compared with conventional scaler tip was not due to the differences in scaling procedure. Standardised scaling procedure was practised in this study under a medium power setting, light force, parallel technique and standardised duration of 2 min. Increased tooth substance loss is associated with increasing tip angulation, power setting and force , 10, 25.The findings of this study were in agreement with Jepsen et al. , where sIn the current study, the operator was not blinded to the scaler tip design. Thus, risk of bias in terms of pressure exerted during scaling. Flemmig et al.  reportedThe findings from the clinical part of the present study demonstrated that PS scaler tip design caused less pain during scaling than a conventional scaler tip. These findings are in agreement with a previous study by Braun et al. . Braun ePain during scaling could be due to the oscillation pattern produced by the vibration of the scaler tip. Less pain following scaling using PS scaler tip could be attributed to the flattened oscillation pattern . ResearcIn order to reduce the differences in pain threshold between participants, a split-mouth study design was used in this study and it was commonly reported in other studies , 27, 28.The study indicates that for periodontal patients that have had repeated scaling and left with minimal and/or delicate tooth structure, the use of slim design ultrasonic scaler tips is patient-friendly. This will be in line with the overall aim to deliver dental care with a minimum patient discomfort. Thus, increasing a patient’s compliance during dental treatment may be possible.Other limitation in the in vitro study includes small sample size. On the other hand, for the clinical study, other limitations includes clinician was not blinded and no correlation between pain and treatment time was carried out.Within the limitation of the study, it was demonstrated that in the in vitro study, using a slim scaler tip design causes less tooth substance loss compared to a wide  scaler tip design. In the clinical study, less pain was observed compared than a wide  scaler tip design.


In clinically euthyroid patients presenting with urticaria, a trial period of withholding antithyroid medications can be exercised. In clinically hyperthyroid patients, antithyroid medications may be stopped with close observation of response. In clinically euthyroid patients presenting with urticaria, a trial period of withholding antithyroid medications can be exercised. In clinically hyperthyroid patients, antithyroid medications may be stopped with close observation of response. In this paper, we present a case of acute urticaria secondary to hyperthyroidism. This link appears to be due to the autoimmune thyroid cascade (particularly IgE antithyroid peroxidase [TPO] antibodies) sensitizing mast cells for activation and histamine release. Although antihistamines and steroids are first‐line treatment, identifying the “causative” factor is not easy in hyperthyroid patients due to the independent links urticaria shares with both carbimazole and thyrotoxicosis.Urticaria consists of blanchable, erythematous “wheals” secondary to vasoactive mediators, predominantly histamine release from mast cells. Wheals are usually transient, disappearing from one aspect of the body and reappearing in new areas, and almost always pruritic. Acute urticaria refers to a presentation of <6 weeks; otherwise, the term chronic urticaria is used. Most cases are idiopathic, but known triggers include drugs, infections, foods, and even certain physical factors .Hyperthyroidism results from an excess state of thyroid hormones caused by excess release and increased synthesis from the thyroid gland, or less commonly, from extrathyroidal sources. Excess thyroid release is often stimulated by specific antibodies, including antithyroid peroxidase (TPO), antithyroglobulin (Tg), and antithyroid‐stimulating hormone receptor (TSHR) antibodies.Urticaria and autoimmune hyperthyroidism appear to share an aetiological relationship, although the exact mechanism remains ambiguous.2A 23‐year‐old female patient with a recent diagnosis of Graves' disease presented to her GP with an urticarial rash localized to her neck. She was prescribed a course of cetirizine and prednisolone. Shortly after, she presented to the emergency department with a spreading urticarial rash extending to her neck, stomach, chest, back, genitals, upper and lower legs, consisting of “extensive excoriations and dermatographia, with blanching and wheals” Figures  and 2. S9/L), and her renal and liver function tests all remained within normal limits. No clinical findings were evident on examination of her chest and abdomen, and no focal neurology was elicited. Her FT4 2 weeks prior to admission was 38.4 pmol/L, with a TSH of 0.01 mU/L, although her FT4 on the day of admission was normal at 10.7 pmol/L. She was treated with intravenous chlorphenamine in the emergency department then admitted for observation and referred to the endocrinologist.Investigations revealed normal inflammatory markers , topical menthol 1% cream and her steroid dose was gradually reduced. After 5 days of treatment, her urticaria was limited to her legs and scalp, and she had symptomatically improved.3Once the patient's symptoms had improved, she was discharged having stayed in hospital for a total of 1 week. She had an ultrasound of her thyroid 2 weeks after discharge which showed diffuse thyroiditis along with a right U2 thyroid nodule. She was discharged without any further carbimazole and followed up in endocrine clinics to monitor thyroid function and to potentially commence propylthiouracil.4The lifetime prevalence of acute urticaria is estimated to be 15%‐23% in adults and up to 14.5% in children.In rare circumstances, urticaria can be the presenting feature of hyperthyroidism. RothfieldThe mechanism linking urticaria and hyperthyroidism is not clearly understood. Chronic urticaria is thought to be triggered by activation and degranulation of mast cells. Although IgG antithyroid auto‐antibodies do not directly activate mast cells, they are postulated to make them more susceptible to activation by other mediators.Treatment for hyperthyroidism consists of medications, surgery, or radioiodine. The currently licensed medications for hyperthyroidism include carbimazole (converted to methimazole) and propylthiouracil, both of which reduce the conversion of thyroxine (T4) to its active form, triiodothyronine (T3).Carbimazole itself is also a known cause of rash and urticaria.In patients that are otherwise clinically euthyroid, a suitable strategy would be to withhold carbimazole (or propylthiouracil) when an urticarial rash first develops. But the correct action in patients with uncontrolled hyperthyroidism is less clear, as withholding antithyroid drugs could result in an exacerbation of thyrotoxicosis and consequently intensify the urticaria. In such patients, a trial period of carbimazole‐free therapy in order to closely assess urticarial response would be justified. Talapatra et alIn our patient, the hyperthyroidism developed shortly before urticaria, although she was clinically euthyroid when she presented to hospital. But despite the unremarkable FT4 level, it is likely that the IgE anti‐TPO cascade was still rampant as evidenced by the high anti‐TPO levels. Interestingly, the decision to stop carbimazole did result in symptomatic relief; however, this coincided with high dose antihistamines.Overall, urticaria is a complication of both autoimmune hyperthyroid disease and the medications used to treat hyperthyroidism. Differentiating between the two is both vital and challenging. In the absence of other clues, it is worth attempting a trial period of withholding antithyroid medications with close assessment of the urticarial rash  This material is the authors' own original work, which has not been previously published elsewhere. (2) The paper is not currently being considered for publication elsewhere. (3) The paper reflects the authors' own research and analysis in a truthful and complete manner. (4) The paper properly credits the meaningful contributions of coauthors and coresearchers. (5) The results are appropriately placed in the context of prior and existing research. (6) All sources used are properly disclosed (correct citation). Literally copying of text must be indicated as such by using quotation marks and giving proper reference. (7) All authors have been personally and actively involved in substantial work leading to the paper, and will take public responsibility for its content.


We describe a middle-aged male presenting to the emergency department with bilateral shoulder pain, holding both arms in abduction after trauma. Radiographs demonstrated a bilateral inferior dislocation of the glenohumeral joints consistent with luxatio erecta humeri.We review the clinical presentation of luxatio erecta and its complications. We also describe the characteristic presentation on radiographs. Our case illustrates the hallmark findings of luxatio erecta of an abducted humeral shaft parallel to the scapular spine. A 53-year-old male with a history of diabetes presented to the emergency department (ED) with bilateral shoulder pain. He was the victim of a carjacking; he held on to his car as the perpetrator sped away, was subsequently dragged, and then run over by the vehicle. He presented with both arms fully abducted and fixed. Imaging revealed bilateral inferior dislocation of the glenohumeral joint consistent with bilateral luxatio erecta, which were reduced in the ED –3.Luxatio erecta humeri is the inferior dislocation of the glenohumeral joint.2Radiographs will demonstrate the humeral heads at the subglenoid region, with an abducted humeral shaft almost parallel to the scapular spine.3What do we already know about this clinical entity?Luxatio erecta humeri is a rare presentation of a shoulder dislocation, with a bilateral presentation being even more rare.What is the major impact of the image(s)?Patients have a hyperabducted arm, imaging reveals a subglenoid humeral head, and treatment involves reduction.How might this improve emergency medicine practice?Proper recognition allows for reduction, immobilization, and arrangement of Orthopedic follow-up, all of which can potentially reduce long-term sequelae.


This paper proposes a new vibration-based structural health monitoring method for the identification of delamination defects in composite bulkheads used in small-length fiber-based ships. The core of this work is to find out if the variations of vibration energy can be efficiently used as a key performance indicator for the detection and quantification of delamination defects in marine composite bulkheads. For this purpose, the changes of vibrational energy exerted by delamination defects in sandwich and monolithic composite panel bulkheads with different types of delamination phenomenon are investigated using a non-destructive test. Experiments show that the overall vibration energy of the bulkheads is directly dependent on the damage conditions of the specimens and therefore, the variations of this parameter are a good indicator of the incorporation of delamination defects in composite bulkheads. Additionally, the overall vibration energy changes also give interesting information about the severity of the delamination defect in the panels. Hence, this methodology based on vibratory energy can be used to accurately determine delamination defects in medium-sized composite bulkheads with the advantages of being a simple and cost-effective approach. The findings of this research possess important applications for the identification of delamination failures in composite components such as bulkheads, turbine blades, and aircraft structures, among others. Composite laminates have been extensively used in aerospace ,2, marinIn the last decade, a large number of non-destructive methods have been reported by the scientific and industrial community for the analysis of delamination defects in fiber-reinforced polymer (FRP) materials. Most of the reported papers suggested the techniques of ultrasounds ,10, therThe research focusing on how delamination defects influence the modal parameters of composite laminates started several decades ago and it has been widely reported by a large number of scientific studies ,21,22,23With regards to the natural frequencies, a vast number of the publications published in this field reported that the presence of delamination defects in composite laminates produces small percentual changes in the natural frequencies ,25,26. AIn parallel, the potential of the vibratory mode shape changes as a key performance indicator (KPI) of the damage state of composite laminates was also examined in a large number of research works ,28,29. TDespite numerous publications investigated the feasibility of the natural frequencies and mode shapes shifts as a diagnostic tool for the identification of delamination defects in composite laminar materials, the potential of the damping as a diagnostic parameter for the identification of delamination defects in fiber-reinforced polymer (FRP) panels has been scarcely addressed in the literature ,30. For In comparison to the modal parameters, the application of the vibration energy as a diagnostic parameter for the identification of delamination breakages in FRP lightweight components has not been explored by the scientific community. The primary focus of this paper is to prove that the variations in overall vibration energy can be used as a simple and cost-effective approach to evaluate the presence of internal delamination defects in sandwich and monolithic composite bulkheads used in small-length FRP ships (below 50 m). For this aim, a non-destructive test is carried out to determine the overall vibration energy of bulkhead panels without delamination (known as pristine) and with delamination (known as damaged) via modal analysis. From the application point of view, this technique might be potentially used to evaluate the structural integrity of lightweight composite bulkheads in aeronautic and marine structures with the advantages of being a low-cost and straightforward approach.The main contribution of this scientific study is to prove that the shift in the overall vibration energy of the composite specimens can be used as a simple and cost-effective approach to evaluate the structural integrity of FRP-based bulkheads used in the small-length ships. Apart from that, this study shed light on how the delamination defects affect the vibratory behavior of glass fiber epoxy composite bulkheads with and without internal defects, with a special emphasis on the damping and vibration mode shape changes. The rest of the paper is structured as follows. In s for a total number of ten layers. The core of the sandwich panels is made out of two layers of PVC with an overall thickness of approximately 15 mm.Three sandwich bulkhead panels with identical dimensions and three different damage levels were kindly manufactured by TUCO shipyards using the process of vacuum infusion procedure, which is a technique commonly applied for the manufacturing of composite components in the aeronautic and shipping industry . The dims with a total number of 24 layers. The stiffeners of the monolithic panels are based on six layers of glass fiber epoxy embedded in a foam core with an overall dimension of 110 × 100 mm. The stacking sequence of the stiffeners is composed of 2 layers of 600 g/m2 and 4 Layers of 450 g/m2 with ply orientations of 0/90 and +45/−45, respectively. The stiffeners of the panels are glued onto the monolithic laminate using Norpol FI-177 adhesive.Furthermore, three monolithic bulkhead panels with identical dimensions of 1800 × 750 × 10 mm were also fabricated using the same infusion procedure. The stacking sequence of the composite panels specified as monolithic panels is Delamination was introduced artificially by inserting a non-adhesive peel ply layer in interlaminar regions core-laminate and stiffener-laminate of the sandwich and monolithic panels, respectively. In other words, the 400 × 400 mm delamination defects of the sandwich panel are introduced in the interlaminar regions between the PVC core and the main laminate top section, as it can be seen from the frontal view of The configuration of the sandwich and monolithic panels  are shown in The material properties of the components from the sandwich and monolithic bulkhead panels used in this research study are detailed in In this paragraph, a non-destructive vibration-based methodology to identify the presence of interlaminar delamination defects in sandwich and monolithic FRP bulkheads is introduced. This strategy is based on the comparison of the vibration energy levels and modal parameters of intact and damaged composite bulkheads with different delamination scenarios.The experimental setup used to extract the vibration energy and modal parameters of the composite bulkheads tested in this experimental campaign is described hereafter in The measurement points for each specimen tested in this experimental campaign are represented on the surface of the composite panel shown in The random excitation was repeated 20 times to obtain multiple realizations for each measurement point. Furthermore, the vibratory responses for each panel configuration were tested in 40 different positions along the composite panel. The standard deviation for the natural frequencies from the 40 measurements is smaller than 1%, the values of the peaks did not change, so the experimental results obtained in this experiment are rather stable and can be considered reliable.In summary, the vibration-based method applied for the evaluation of the structural integrity of the panels is divided into seven stages that are defined in the flowchart shown in The recorded FFT spectrums shown in ix represents the amplitude for a certain frequency and N indicates the number of values analyzed in the range of frequencies. The vibration energy of the composite bulkheads was calculated in the range of frequencies between 0 Hz and 800 Hz, which is the frequency range recommended for this experimental case scenario. Therefore, an interesting aspect of this methodology is that the vibration energy levels of the composite bulkheads can be calculated easily without the need of complicated and time-consuming mathematical procedures.The overall vibration energy of the panels can be defined as the sum of the vibrational energy measured within a specified frequency range. The overall vibration energy for the composite bulkheads with and without internal delamination is directly calculated from a frequency response function see  through Another interesting question addressed in this study deals with the number of measurement points necessary to determine the vibration energy levels of the FRP-based bulkheads. In the author’s opinion, a good approach is to measure the vibration energy in three points of the FRP bulkhead far enough away from the excitation source. As a result, only a few measurement points are necessary to evaluate the damage integrity of composite bulkheads with an area of 1800 × 750 mm in a cost-effective way.w1, w2, and w3 are the frequencies associated with the first, second, and third points of the frequency response function (FRF) represented in w2 can be defined as the frequency of the resonant peak in the bulkheads and w1,3 stands for the points of the peak located 3 dB below the maximum amplitude.Damping can be defined as the energy dissipation properties of a material under cyclic stress. This parameter is an indicator of the amount of vibration energy that a material can dissipate, where higher damping values are associated with a greater capability for energy vibration dissipation. The damping of the first ten natural frequencies of the composite bulkheads is determined through the half-power method  through The calculation of the damping for ten frequencies of the panel was carried out using the software LMS Test Lab, which allows the simultaneous analysis of multiple measurements via the Modal Analysis module. This solution offers a powerful acquisition tool for modal parameter identification, enabling faster data interpretation and accurate measurement analysis.The vibration mode shapes of the first ten modes of the specimens are simulated using the modal analysis tool of the vibration analysis software Siemens LMS Test Lab. To carry out this task, the Fast Fourier Transform (FFT) spectrum characteristic for each measurement point is used to simulate the vibration mode shapes. The principal idea is to find out if the dynamic motions of the vibration mode shapes are affected by the delamination failures.The aim of this section is to analyze the feasibility of the shifts of vibration energy and modal parameters for the detection and quantification of delamination failures in FRP-based bulkheads. The section is divided into three subsections. This section looks at the influence of delamination defects on the vibration energy levels of composite bulkheads used in marine applications. To address this investigation, the levels of vibration energy for the identical sandwich and monolithic bulkheads with three different damage states are compared in order to find out how the delamination breakages produce changes in the overall vibration energy of the materials.The energy variations in percentage values for the sandwich/monolithic panels due to delamination defects are represented in The findings of this study support the idea that the overall vibration energy is a good key performance indicator for the detection and quantification of delamination defects. For example, it is observed that the overall vibration energy of the monolithic bulkheads decreases approximately 10% for the panel damaged with a delamination size of 100 × 400 mm and around 25% in the composite panel with the defect of 200 × 400 mm. From the authors’ point of view, the higher percentual decrement of vibration energy in the panel with the 200 × 400 mm delamination failure is induced by the larger size of the defect that increases the amount of energy dissipated. Thus, it can be concluded that delamination defects produce phenomena of energy dissipation that result in a decay of overall vibration energy, which is proportional to the size of the delamination defect.The feasibility of the overall vibration energy as a key performance indicator for the localization of delamination defects in composite laminates is not evident from the experimental data provided by this research study. The overall vibration energy of the two sandwich panels with identical delamination defects of 400 × 400 mm decreases by more than 20% in the majority of the measurement points. However, the shift of vibration energy recorded in the points around the delaminated regions of the bulkhead panels is similar to that of the areas in good conditions. In the author’s opinion, this behavior might be explained because the intensity of the energy dissipation phenomena generated by the frictions of the delaminated layers are barely affected by the defect localization. Thus, the overall vibration energy variations are not sufficient for the localization of delamination defects in the sandwich/monolithic panels.In spite of the fact that a large number of publications are focused on the variation of the modal parameters, the potential of the overall vibration energy for the detection of delamination defects in composite laminar materials has been not explored in the literature. Indeed, the majority of the research publications published in the field of vibration-based structural health monitoring (VSHM) are concentrated on the variation of the modal parameters. A couple of interesting contributions to the field were reported by K. Alnefaie et al.  and M. IOn the basis of these results, it is noticed that the changes in overall vibration energy show great potential for the detection of delamination defects in sandwich and monolithic composite bulkheads. Therefore, the results of this paper can be used as a proof-of-concept that the changes of vibration energy can be used as a diagnostic parameter for the detection of delamination defects in composite bulkheads. From the authors point of view, this technology has important implications for the detection of defects in a wide range of composite applications such as wind energy turbines, bulkheads, aircraft structures, etc. In any case, it is important to be cautious because these interesting results for the detection of delamination in composite bulkheads were obtained on a lab-scale and, therefore, the feasibility of this methodology still needs to be proven in an industrial case scenario with sea waves impacts, propeller excitations, and other external excitations that may disturb the vibration measurements.This paragraph analyzes how the delamination defects affect to the damping behavior of the composite panels. To carry out this investigation, the damping for three identical sandwich/monolithic composite bulkheads with different damage states are compared in order to evaluate the influence of this type of defects on this key performance indicator. The damping values of the first ten vibration modes of the sandwich and monolithic bulkheads with and without delamination defects are compared in The experimental data given in The damping percentual changes of the sandwich panels see  are quitThe findings of this paper are in line with the results reported in other research works published in the literature. For example, Z. Kiral et al.  carried It is well known the heterogeneous structure of the composite materials facilitates the phenomena of dissipation of energy due to their multiple numbers of layers and anisotropic structure. In conclusion, it can be said that the delamination defects tend to increase the damping because the delamination regions act as dampers of the laminates. However, these changes are quite dependent on the vibration mode as the ability of the frequencies to excite a particular defect is related to their wavelength. In the authors’ view, the changes of damping are an interesting parameter but are not recommended as a diagnostic tool for the detection of delamination defects in composite bulkheads because they are strongly dependent on the frequency mode shape and do not give consistent results for all the modes.Another diagnostic tool analyzed in this paper is based on tracking the local changes of the vibration mode shapes due to the presence of delamination defects in the composite panels. This strategy is based on the analysis of the relative displacements that the delamination defects exert on the vibration mode shapes of composite bulkheads. Therefore, the primary objective of this section is to identify the local changes, which may appear in certain mode shapes of the laminates due to the presence of delamination failures.Modes 4 and 5 of the sandwich panels with and without internal delamination are represented in Modes 4 and 5 of the monolithic panels with and without internal delamination are represented in The influence of delamination defects in the vibration mode shapes of composite laminates has been investigated previously by other researchers ,28,29. AIn summary, the findings of the current study reported that there are small deviations in the relative displacements of some mode shapes, which might be induced by the dissipation phenomena generated by the delamination defects of the bulkheads. However, the authors of this paper discarded the variation of the mode shapes as a potential tool for the detection of delamination failures due to the large number of measurement points required for the calculation of the modes  and the long time required to post-process and analyze the experimental data.This paper has considered the problem for the identification, quantification, and localization of delamination defects in marine composite bulkheads, which are used for the design and construction of small-length ships (less than 500 GT). To evaluate the integrity of the panels, the overall vibration energy, damping and vibration mode shapes of six sandwich and monolithic composite panels with different levels of damage are considered. On the basis of the experimental results, the following conclusions may be drawn.The variation of vibration energy in composites with and without internal delamination is considered as the best key performance indicator. This study proves that the overall vibration energy of laminates with delamination is inferior with respect to the neat specimens. This can be explained due to the fact that composites with delamination defects dissipate higher energy due to multiple interlaminar frictions among the damaged layers. As a result, it is concluded that the shift of vibration energy is a reliable and economic parameter for the detection of delamination in composite bulkhead panels.In comparison to the natural frequencies, the damping of the damaged composite specimens is more sensitive to the presence of delamination phenomena. In general, the delamination defects tend to increase the damping of the composite bulkheads because the delamination regions act as dampers. However, these changes are quite dependent on the vibration mode as the ability of the frequencies to excite the defect is related to their wavelength. Additionally, the paper reveals that the sandwich bulkheads dissipate more vibratory energy than the monolithic panels, and therefore the selection of these type of composite materials is more appropriated for applications where vibrations are a source of problems.Apart from that, this paper investigates the changes of the vibration mode shapes due to the incorporation of delamination regions in composite laminates. The findings of this research highlighted that the relative displacements of certain vibration mode shapes are slightly affected by the delamination failures. However, the authors of this paper discarded the variation of the mode shapes as a potential tool for the detection of delamination failures because the reported displacement changes are small for the modes investigates in this research study.In summary, this paper introduces an innovative non-destructive method for the detection and characterization of internal delamination defects in composite panels based on the changes of the overall vibration energy. Since vibration measurements can be easily acquired, this approach provides a cost-effective approach to identify the existence of delamination defects in composite laminar materials. It is important to highlight that it is only required one single measurement point to calculate the overall vibration energy of panels with an area of 1800 × 750 mm , and therefore it is not necessary to carry out a large experimental testing campaign to evaluate the damage integrity of the bulkhead panels. The findings of this research possess important industrial applications for monitoring destructive delamination failures in the FRP-based bulkheads used in aircrafts, wind turbines, vessels, and other engineering composite structures in which this approach is an excellent structural assessment tool and an evident advantage for their extension of life-cycle.