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Oscillatory increase in pancreatic beta cell cytoplasmic free ca (super 2,+) concentration is studied. Since it is an important feature in glucose-induced insulin release, subunit of beta cell may constitute the basis of novel diabetes therapy.

Background: The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated Ca V 1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca 2+ influx. In cardiomyocytes, the core subunit of Ca V 1.2, Ca V 1.2α 1 , exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to Ca V 1.2α 1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of Ca V 1.2 in heterologous models, and their role in cardiomyocytes also remains unclear., Results: We show that PKAC interacts with Ca V 1.2α 1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in Ca V 1.2α 1 -CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of Ca V 1.2α 1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte., Conclusions: We discover direct interactions between PKAC and two domains in Ca V 1.2α 1 . We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes., Competing Interests: Declarations. Ethics approval and consent to participate: Experiments were approved by Tel Aviv University Institutional Animal Care and Use Committee (IACUC permit # 01–20-083). Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Analysis of amatoxins is of great importance as these cyclic peptides contribute to a high number of fatalities each year. Development of analytical approaches needs to focus on rapid, sensitive, and reliable methods. By establishing an affinity column chromatography-based assay using the monoclonal amanitin antibody AMA9G3 and liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the trace detection of α-, β-, and γ-amanitin in human urine samples to confirm ingestion, we report the first approach that extents the current status of amatoxin analysis. The presented procedure allows detection of amatoxins in human urine down to 1 ng/mL. The method was successfully validated qualitatively for α- and γ-amanitin according to international recommendations. A proof of concept was performed by analyzing 37 urine samples after suspected amatoxin consumption submitted for regular clinical toxicological analysis. Using this antibody-based enrichment strategy, acute amatoxin intoxications can be determined within 90 min and due to the high sensitivity and selectivity, a comparable approach using target specific antibodies may also be used for other toxicological relevant peptides., (© 2024. The Author(s).)

Background: Temporary hemiepiphysiodesis with tension band plates or eight-plates is a common surgical procedure to treat malalignment of the lower limb axis in skeletally immature patients. The objective of this study was to compare a new minimally invasive surgical procedure with the conventional procedure and evaluate its safety and effectiveness in order to reduce the risk of hypertrophic scarring, which may cause functional impairment as well as cosmetic issues. Methods: Sixty-five growth plates of either the femur or the tibia were evaluated in 33 patients treated for genu valgum or varum between 2010 and 2017. Each growth plate was considered an individual case. The modified procedure was used in 17 cases and the conventional procedure in 48 cases. The modified surgical procedure is characterized by an 8 mm incision and preparation of the epi-periosteal layer, in which the eight-plate is positioned via a guide-wire. Positioning and implantation are controlled via fluoroscopy. Skin incision length, duration of surgery, revision rate, achievement of a defined correction goal, and correction rate were analyzed. Results: Using the minimally invasive procedure, the mean skin incision length (23.94 ± 10.18 mm vs. 8.75 ± 2.14 mm, p < 0.001) could be significantly reduced. No significant difference was found in regard to the duration of surgery, revision rate, achievement of the correction goal or correction rate. Conclusions: The minimally invasive procedure results in a reduction in incision length without significant impact on the duration of surgery, revision rate, achievement of correction goal or correction rate. Consequently, the modified procedure can be regarded as equally as effective and safe as the conventional procedure.

Background: Tight control of cytoplasmic Ca 2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca 2+ (Cav) channels, in modulating Ca 2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier., Methods: We investigated the function of Cavβ3 in BMECs by Ca 2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3 -/- (Cavβ3-deficient) mice as controls., Results: We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3 -/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3 -/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca 2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca 2+ -dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3 -/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3 -/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins., Conclusions: Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca 2+ concentration and Ca 2+ -dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease., Competing Interests: None.

Purpose: Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring. In advanced stage AAK, typically, conjunctival epithelial cells slowly replace the corneal epithelium. Based on previous results we hypothesize that alterations of the conjunctival cells in congenital aniridia may also support the corneal conjunctivalization process. The aim of this study was to identify deregulated proteins in conjunctival impression cytology samples of congenital aniridia subjects., Methods: Conjunctival impression cytology samples of eight patients with congenital aniridia [age 34.5 ± 9.9 (17-51) years, 50% female] and eight healthy subjects [age 34.1 ± 11.9 (15-54) years, 50% female] were collected and analysed using mass spectrometry. Proteomic profiles were analysed in terms of molecular functions, biological processes, cellular components and pathway enrichment using the protein annotation of the evolutionary relationship (PANTHER) classification system., Results: In total, 3323 proteins could be verified and there were 127 deregulated proteins (p < 0.01) in congenital aniridia. From the 127 deregulated proteins (DEPs), 82 altered biological processes, 63 deregulated cellular components, 27 significantly altered molecular functions and 31 enriched signalling pathways were identified. Pathological alteration of the biological processes and molecular functions of retinol binding and retinoic acid biosynthesis, as well as lipid metabolism and apoptosis related pathways could be demonstrated., Conclusions: Protein profile of conjunctival impression cytology samples of aniridia subjects identifies alterations of retinol binding, retinoic acid biosynthesis, lipid metabolism and apoptosis related pathways. Whether these changes are directly related to PAX6 haploinsufficiency, must be investigated in further studies. These new findings offer the possibility to identify potential new drug targets., (© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA-target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson's disease (PD). After performing 13,536 transfections, we validated 442 of the 705 putative MTIs (62,7%) through dual luciferase reporter assays. These analyses increased the number of validated MTIs by at least 2.1-fold for miR-133b and by a maximum of 24.3-fold for miR-873-5p. Our study contributes to the experimental capture of miRNA targetomes by addressing i) the ratio of experimentally verified MTIs to predicted MTIs, ii) the sizes of disease-related miRNA targetomes, and iii) the density of MTI networks. A web service to support the analyses on the MTI level is available online ( https://ccb-web.cs.uni-saarland.de/utr-seremato ), and all the data have been added to the miRATBase database ( https://ccb-web.cs.uni-saarland.de/miratbase )., (© 2024. The Author(s).)

Gonadotropes in the anterior pituitary gland are essential for fertility and provide a functional link between the brain and the gonads. To trigger ovulation, gonadotrope cells release massive amounts of luteinizing hormone (LH). The mechanism underlying this remains unclear. Here, we utilize a mouse model expressing a genetically encoded Ca 2+ indicator exclusively in gonadotropes to dissect this mechanism in intact pituitaries. We demonstrate that female gonadotropes exclusively exhibit a state of hyperexcitability during the LH surge, resulting in spontaneous [Ca 2+ ] i transients in these cells, which persist in the absence of any in vivo hormonal signals. L-type Ca 2+ channels and transient receptor potential channel A1 (TRPA1) together with intracellular reactive oxygen species (ROS) levels ensure this state of hyperexcitability. Consistent with this, virus-assisted triple knockout of Trpa1 and L-type Ca 2+ subunits in gonadotropes leads to vaginal closure in cycling females. Our data provide insight into molecular mechanisms required for ovulation and reproductive success in mammals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to investigate the effects and consequences of surgical treatment of patients with musculoskeletal malignancies on everyday life., Methods: A modified form of grounded theory was used for data collection and analysis. Data collection was systematic and analyzed simultaneously and 16 interviews were conducted: 2 narrative, 11 guided and 3 expert interviews (surgeon, physical therapist, support group). Data collection and analysis alternated until no new codes could be found. Once theoretical saturation was achieved, the main category was formed and described using the literature., Results: The main category results from the combination of all categories and leads to the core category. In the center is the affected person and in the immediate environment are the patient's relatives/partners. In the next instance the primary care physician is necessary to establish a sense of normalcy. This depends on the individuality of the person and the restored possibilities of movement., Conclusion: Based on the results, the necessity of implementing psychosocial care involving the social environment is shown. The importance of relatives/partners for recovery is emphasized. Furthermore, the communication between the specialists and family physicians should be simplified., (© 2023. The Author(s).)

Purpose: Little is known about the effect of sports activity levels on health-related quality of life (HRQOL) in long-term survivors of lower-extremity sarcoma., Methods: Eighty-three long-term survivors of bone and soft tissue sarcoma of the lower extremities with a median follow-up of 14 (range: 5-35) years completed the University of California and Los Angeles (UCLA) activity scores before tumor resection, 1 year after surgery and at the latest follow-up, as well as a Short Form 36 (SF-36) health survey at the latest follow-up. Simple linear regression models as well as stepwise variable selection with Akaike information criterion (AIC) were undertaken., Results: The preoperative UCLA activity level (median: 9, range: 2-10) dropped to a median of 4 (range: 1-10) 1 year after surgery before increasing to a score of 6 (range: 2-10) 5 years after surgery. The long-term SF-36 physical health component summary score (PCS) was 49 (SD: 9), and the mental health component summary score (MCS) was 54 (SD: 7). A linear model with stepwise variable selection identified a negative correlation of PCS with age at surgery (estimate: -0.2; p = 0.02), UCLA score at the last follow-up (estimate: 1.4; p = 0.02) and UCLA score 1 year after surgery (estimate: 1.0; p = 0.02)., Conclusion: As not only the final activity levels but also the status immediately after surgery affect the PCS, higher early activity levels should be a goal of modern rehabilitation after sarcoma treatment. Further studies are needed to weigh the potential postoperative risks of higher sport activity levels against the benefits described in this study., Level of Evidence: Level 4., (© 2023. The Author(s).)

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca 2+ -selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca 2+ ] i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca 2+ ] i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6 VL -eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6 VL -bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca 2+ transport., (Copyright © 2022. Published by Elsevier Ltd.)

Background: It is well-known that exercise through sports and physical activity has beneficial effects on health. After lower extremity amputation, low rates of participation in sports activities have been shown. So far, there is only limited scientific long-term information concerning sports activity and feasible types of sports in patients with lower extremity amputations due to malignant tumors. This study sought to provide very long-term information about the following questions: (1) What sports activity levels can be achieved after lower extremity amputation due to a malignant tumor? (2) What are feasible types of sports? (3) Is the University of California Los Angeles (UCLA) Activity Score influenced by patient's age, the age at the time of surgery and the amount of prosthesis sockets used to date?, Hypothesis: Patients with lower extremity amputation due to a malignant tumor cannot regularly participate in moderate sports activities (UCLA Activity Score≥6) in the very long-term., Materials and Methods: In a retrospective questionnaire survey, 21 former patients (seven females, 14 males; median age: 60.5years [range: 33-74]) who underwent an amputation of the lower extremity (14 transfemoral, four knee disarticulations, three transtibial) from 1961 to 1993 due to a malignant tumor were interviewed. The median follow-up was 41.1years (range: 23-55). The patients were interviewed about types of sports, frequencies and durations of sports sessions at the time of the last follow-up, as well as retrospectively. Sports activity levels were then assessed based on the UCLA Activity Score. Demographic background, data on amputation level, histological results, prosthetic devices and revision surgeries were collected., Results: Seven patients (33%) were still regularly exercising at least moderate sports activities at the last follow-up, including one patient regularly participating in impact sports. The preoperative median UCLA Activity Score of 9 (range: 2-10) decreased to 5 (range: 2-10) at the last follow-up (p<0.001). Swimming (13 patients, seven on a regular basis), cycling (seven), downhill skiing (five) and hiking (four) were among the most common feasible types of sports. Increasing age had a negative influence on the UCLA Activity Score [95% CI (-0.034, -0.006); R B =-0.020; SD=0.0074; p=0.007]., Conclusion: After a major amputation in the lower extremity due to a malignant tumor, high activity levels by practicing types of sports such as downhill skiing, are achievable. In the very long-term, a regular participation in at least moderate activities, such as swimming, is possible. Programs for physical and sports activities, based on recommendations drawn from long-term experience, are necessary and should be individually tailored to amputees and their limitations., Level of Evidence: IV., (Copyright © 2022. Published by Elsevier Masson SAS.)

Purpose: The use of dental restorative materials is a routine task in clinical dentistry. Upon exposure to the oral cavity, continuous adsorption of salivary proteins and other macromolecules to all surfaces occurs, representing the first step in dental biofilm formation. Different physico-chemical properties of substrate materials potentially influence the composition of the initial biofilm, termed pellicle. This study aimed at characterizing and comparing the individual proteomic composition of the 3-min pellicle formed on bovine enamel and six restorative materials., Experimental Design: After chemical elution, pellicle proteins were identified by nano-LC-HR-MS/MS. Proteomic profiles were analyzed in terms of molecular weights, isoelectric points, molecular functions and compared to saliva to reveal substrate material-specific adsorption patterns., Results: A total of 1348 different pellicle proteins were identified, with 187-686 proteins in individual 3-min pellicles. Unexpectedly, this yielded quite similar distribution patterns independent of the substrate materials. Furthermore, overall similar fold changes were obtained for the major part of commonly enriched or depleted proteins in the pellicles., Conclusions and Clinical Relevance: The current results point to a minor role of the substrate material on the proteomic composition of the 3-min pellicle and represent core data for understanding the complex surface interactions in the oral cavity., (© 2022 The Authors. Proteomics - Clinical Applications published by Wiley-VCH GmbH.)

To identify TRPV6 expression in the whole mouse with a cellular resolution we took advantage of TRPV6-IRES-Cre knock-in mice crossed with the enhanced ROSA26-τGFP reporter line. In the resulting TRPV6-IC/eR26-τGFP animals, TRPV6-expressing cells are labeled with τGFP. Data were collected from organs prepared from fixed experimental adult and juvenile TRPV6-IC/eR26τGFP and Cre-negative eR26-τGFP control animals of both sexes. Organ cryosections from each age and sex were stained for GFP and imaged with a slide scanner. Here, we describe reporter gene expression in a large number of tissues. We also document the absence of τGFP signal in the corresponding Cre-negative control tissues, including controls for the TRPV6 expression data described in [1]. The data reported here and in [1] constitute the TRPV6 expression atlas for the mouse. Our data offer a wealth of information to enable investigation of the functional role of TRPV6 channels in different tissues., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier Inc.)

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using - in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Staudt, Ratai, Bouzouina, Fecher-Trost, Shaaban, Bzeih, Horn, Shaib, Klose, Flockerzi, Lauterbach, Rettig and Becherer.)