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The purpose of this study was to gain insight into histamine's role in the exercise inflammatory response and recovery from exercise. To explore this, young healthy participants (n = 12) performed 300 eccentric leg extensions under control (Placebo) versus histamine H 1 and H 2 receptor antagonism (Blockade) in a randomized cross-over study. Circulating leukocytes and cytokines were measured for 72 h after exercise. Circulating leukocytes were elevated at 6 and 12 h after exercise (p < 0.05) with the peak response being a 44.1 ± 11.7% increase with Blockade versus 13.7 ± 6.6% with Placebo (both p < 0.05 vs. baseline, but also p < 0.05 between Blockade and Placebo). Of the cytokines that were measured, only MCP-1 was elevated following exercise. The response at 6 h post-exercise was a 104.0 ± 72.5% increase with Blockade versus 93.1 ± 41.9% with Placebo (both p < 0.05 vs. baseline, p = 0.82 between Blockade and Placebo). The main findings of the present investigation were that taking combined histamine H 1 and H 2 receptor antagonists augmented the magnitude but not the duration of the increase of circulating immune cells following exercise. This suggests histamine is not only exerting a local influence within the skeletal muscle but that it may influence the systemic inflammatory patterns., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Purpose of the Review: Compare pathophysiology for infectious and noninfectious demineralization disease relative to mineral maintenance, physiologic fluoride levels, and mechanical degradation., Recent Findings: Environmental acidity, biomechanics, and intercrystalline percolation of endemic fluoride regulate resistance to demineralization relative to osteopenia, noncarious cervical lesions, and dental caries. Demineralization is the most prevalent chronic disease in the world: osteoporosis (OP) >10%, dental caries ~100%. OP is severely debilitating while caries is potentially fatal. Mineralized tissues have a common physiology: cell-mediated apposition, protein matrix, fluid logistics (blood, saliva), intercrystalline ion percolation, cyclic demineralization/remineralization, and acid-based degradation (microbes, clastic cells). Etiology of demineralization involves fluid percolation, metabolism, homeostasis, biomechanics, mechanical wear (attrition or abrasion), and biofilm-related infections. Bone mineral density measurement assesses skeletal mass. Attrition, abrasion, erosion, and abfraction are diagnosed visually, but invisible subsurface caries <400μm cannot be detected. Controlling demineralization at all levels is an important horizon for cost-effective wellness worldwide., (© 2022. The Author(s).)

Aerobic exercise induces mast cell degranulation and increases histamine formation by histidine decarboxylase, resulting in an ∼150% increase in intramuscular histamine. The purpose of this study was to determine if the increase in skeletal muscle temperature associated with exercise is sufficient to explain this histamine response. Specifically, we hypothesized that local passive heating that mimics the magnitude and time course of changes in skeletal muscle temperature observed during exercise would result in increased intramuscular histamine concentrations comparable to exercising values. Seven subjects participated in the main study in which pulsed short-wave diathermy was used to passively raise the temperature of the vastus lateralis over 60 min. Heating increased intramuscular temperature from 32.6°C [95% confidence interval (CI) 32.0°C to 33.2°C] to 38.9°C (38.7°C to 39.2°C) ( P < 0.05) and increased intramuscular histamine concentration from 2.14 ng/mL (1.92 to 2.36 ng/mL) to 2.97 ng/mL (2.57 to 3.36 ng/mL) ( P < 0.05), an increase of 41%. In a follow-up in vitro experiment using human-derived cultured mast cells, heating to comparable temperatures did not activate mast cell degranulation. Therefore, it appears that exercise-associated changes in skeletal muscle temperature are sufficient to generate elevations in intramuscular histamine concentration. However, this thermal effect is most likely due to changes in de novo histamine formation via histidine decarboxylase and not due to degranulation of mast cells. In conclusion, physiologically relevant increases in skeletal muscle temperature explain part, but not all, of the histamine response to aerobic exercise. This thermal effect may be important in generating positive adaptations to exercise training. NEW & NOTEWORTHY The "exercise signal" that triggers histamine release within active skeletal muscle during aerobic exercise is unknown. By mimicking the magnitude and time course of increasing skeletal muscle temperature observed during aerobic exercise, we demonstrate that part of the exercise-induced rise in histamine is explained by a thermal effect, with in vitro experiments suggesting this is most likely via de novo histamine formation. This thermal effect may be important in generating positive adaptations to exercise training.

Purpose of Review: Compare noninfectious (part I) to infectious (part II) demineralization of bones and teeth. Evaluate similarities and differences in the expression of hard tissue degradation for the two most common chronic demineralization diseases: osteoporosis and dental caries., Recent Findings: The physiology of demineralization is similar for the sterile skeleton compared to the septic dentition. Superimposing the pathologic variable of infection reveals a unique pathophysiology for dental caries. Mineralized tissues are compromised by microdamage, demineralization, and infection. Osseous tissues remodel (turnover) to maintain structural integrity, but the heavily loaded dentition does not turnover so it is ultimately at risk of collapse. A carious tooth is a potential vector for periapical infection that may be life-threatening. Insipient caries is initiated as a subsurface decalcification in enamel that is not detectable until a depth of ~400μm when it becomes visible as a white spot. Reliable detection and remineralization of invisible caries would advance cost-effective wellness worldwide., (© 2022. The Author(s).)

Popularly known as "chalky teeth", molar hypomineralisation (MH) affects over 1-in-5 children worldwide, triggering massive amounts of suffering from toothache and rapid decay. MH stems from childhood illness and so offers a medical-prevention avenue for improving oral and paediatric health. With a cross-sector translational research and education network (The D3 Group; thed3group.org ) now highlighting this global health opportunity, aetiological understanding is urgently needed to enable better awareness, management and eventual prevention of MH. Causation and pathogenesis of "chalky enamel spots" (i.e., demarcated opacities, the defining pathology of MH) remain unclear despite 100 years of investigation. However, recent biochemical studies provided a pathomechanistic breakthrough by explaining several hallmarks of chalky opacities for the first time. This article outlines these findings in context of previous understanding and provides a working model for future investigations. The proposed pathomechanism, termed "mineralisation poisoning", involves localised exposure of immature enamel to serum albumin. Albumin binds to enamel-mineral crystals and blocks their growth, leading to chalky opacities with distinct borders. Being centred on extracellular fluid rather than enamel-forming cells as held by dogma, this localising pathomechanism invokes a new type of connection with childhood illness. These advances open a novel direction for research into pathogenesis and causation of MH, and offer prospects for better clinical management. Future research will require wide-ranging inputs that ideally should be coordinated through a worldwide translational network. We hope this breakthrough will ultimately lead to medical prevention of MH, prompting global health benefits including major reductions in childhood tooth decay., Competing Interests: MH is founder/director of The D3 Group for developmental dental defects (www.thed3group.org), a charitable network. The remaining 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 © 2021 Hubbard, Mangum, Perez and Williams.)

Introduction: The purpose of this study was to quantify and qualify the 3-dimensional (3D) condylar changes using mandibular 3D regional superimposition techniques in adolescent patients with Class II Division 1 malocclusions treated with either a 2-phase or single-phase approach., Methods: Twenty patients with Herbst appliances who met the inclusion criteria and had cone-beam computed tomography (CBCT) images taken before, 8 weeks after Herbst removal, and after the completion of multibracket appliance treatment constituted the Herbst group. They were compared with 11 subjects with Class II malocclusion who were treated with elastics and multibracket appliances and who had CBCT images taken before and after treatment. Three-dimensional models generated from the CBCT images were registered on the mandible using 3D voxel-based superimposition techniques and analyzed using semitransparent overlays and point-to-point measurements., Results: The magnitude of lateral condylar growth during the orthodontic phase (T2-T3) was greater than that during the orthopedic phase (T1-T2) for all condylar fiducials with the exception of the superior condyle (P <0.05). Conversely, posterior condylar growth was greater during the orthopedic phase than the subsequent orthodontic phase for all condylar fiducials (P <0.05). The magnitude of vertical condylar development was similar during both the orthopedic (T1-T2) and orthodontic phases (T2-T3) across all condylar fiducials (P <0.05). Posterior condylar growth during the orthodontic phase (T2-T3) of the 2-phase approach decreased for all condylar fiducials with the exception of the posterior condylar fiducial (P <0.05) when compared with the single-phase approach., Conclusions: Two-phase treatment using a Herbst appliance accelerates condylar growth when compared with a single-phase regime with Class II elastics. Whereas the posterior condylar growth manifested primarily during the orthopedic phase, the vertical condylar gains occurred in equal magnitude throughout both phases of the 2-phase treatment regime., (Copyright © 2020 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Molar hypomineralisation (MH) is becoming globally recognised as a significant public health problem linked to childhood tooth decay. However, with causation and pathogenesis unclear after 100 years of investigation, better pathological understanding is needed if MH is to become preventable. Our studies have implicated serum albumin in an extracellular pathomechanism for chalky enamel, opposing longheld dogma about systemic injury to enamel-forming cells. Hypothesising that chalky enamel arises through developmental exposure to serum albumin, this study used biochemical approaches to characterise demarcated opacities from 6-year molars. Addressing contradictory literature, normal enamel was found to completely lack albumin subject to removal of surface contamination. Querying surface permeability, intact opacities were found to lack salivary amylase, indicating that "enamel albumin" had become entrapped before tooth eruption. Thirdly, comparative profiling of chalky and hard-white enamel supported a dose-response relationship between albumin and clinical hardness of opacities. Moreover, albumin abundance delineated chalky enamel from white transitional enamel at opacity borders. Finally, addressing the corollary that enamel albumin had been entrapped for several years, clear signs of molecular ageing (oxidative aggregation and fragmentation) were identified. By establishing aged albumin as a biomarker for chalky enamel, these findings hold methodological, clinical, and aetiological significance. Foremost, direct inhibition of enamel-crystal growth by albumin (here termed "mineralisation poisoning") at last provides a cogent explanation for the clinical presentation of demarcated opacities. Together, these findings justify pursuit of an extracellular paradigm for the pathogenesis of MH and offer exciting new prospects for alleviating childhood tooth decay through medical prevention of MH., (Copyright © 2020 Perez, Mangum and Hubbard.)

Molar Hypomineralisation (MH) is gaining cross-sector attention as a global health problem, making deeper enquiry into its prevention a research priority. However, causation and pathogenesis of MH remain unclear despite 100 years of investigation into "chalky" dental enamel. Contradicting aetiological dogma involving disrupted enamel-forming cells (ameloblasts), our earlier biochemical analysis of chalky enamel opacities implicated extracellular serum albumin in enamel hypomineralisation. This study sought evidence that the albumin found in chalky enamel reflected causal events during enamel development rather than later association with pre-existing enamel porosity. Hypothesising that blood-derived albumin infiltrates immature enamel and directly blocks its hardening, we developed a "molecular timestamping" method that quantifies the adult and fetal isoforms of serum albumin ratiometrically. Applying this novel approach to 6-year molars, both isoforms of albumin were detectable in 6 of 8 chalky opacities examined (corresponding to 4 of 5 cases), indicating developmental acquisition during early infancy. Addressing protein survival, in vitro analysis showed that, like adult albumin, the fetal isoform (alpha-fetoprotein) bound hydroxyapatite avidly and was resistant to kallikrein-4, the pivotal protease involved in enamel hardening. These results shift primary attention from ameloblast injury and indicate instead that an extracellular mechanism involving localised exposure of immature enamel to serum albumin constitutes the crux of MH pathogenesis. Together, our pathomechanistic findings plus the biomarker approach for onset timing open a new direction for aetiological investigations into the medical prevention of MH., (Copyright © 2020 Williams, Perez, Mangum and Hubbard.)

Delayed cerebral ischaemia (DCI) after aneurysmal subarachnoid haemorrhage (aSAH) is a major contributor to morbidity and mortality. It is currently not possible to reliably predict patients at risk of DCI after aSAH. The aim of this study was to quantify cerebrospinal fluid (CSF) D-Dimer and plasminogen levels and to investigate any association with development of DCI. Cerebrospinal fluid (CSF) samples collected from 30 patients within 72 h post-aSAH (n = 13 DCI and n = 17 non-DCI patients) were analysed. DCI was diagnosed when angiographic vasospasm was detected in the presence of new onset neurological deficit. Enzyme-linked immunosorbent assays were used to quantify D-dimer concentrations while western blotting was used to quantify plasminogen levels. Significant differences in CSF proteins between DCI and non-DCI cohorts were verified using Mann-Whitney test. Sensitivity and specificity of these proteins for detecting DCI was examined using a ROC curve and verified with a Fischer's exact test. CSF levels of D-dimer within 72 h post aSAH were significantly elevated in DCI patients (54.29 ng/ml, 25.35-105.88 ng/ml) compared to non-DCI patients (26.75 ng/ml, 6.9-45.08 ng/ml) [p = 0.03]. In our sample population, D-dimer levels above 41.1 ng/ml had a sensitivity of 69.2% and specificity of 75% for predicting DCI. CSF levels of plasminogen (DCI: 0.50 signal-intensity/μl, 0.20-0.73 signal-intensity/μl, non-DCI: 0.28 signal-intensity/μl, 0.22-0.54 signal-intensity/μl) did not differ between the DCI and non-DCI cohort (p > 0.05). Our study suggests that elevated D-dimer in the first 72 h after aSAH may be a potential predictive biomarker for DCI., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Histamine is released within skeletal muscle during exercise. In humans, antihistamines have no effect on speed, power output, or time-to-completion of short-duration high-intensity exercise. In mice, blocking histamine's actions decreases speed and duration of endurance tasks. It is unknown if these opposing outcomes are the result of differences in histamine's actions between species or are related to duration and/or intensity of exercise, as blocking histamine during endurance exercise has not been examined in humans., Purpose: Determine the effects of histamine-receptor antagonism on cycling time trial performance in humans, with and without a preceding bout of sustained steady-state exercise., Methods: Eleven (3F) competitive cyclists performed six 10-km time trials on separate days. The first two time trials served as familiarization. The next four time trials were performed in randomized-block order, where two were preceded by 120 min of seated rest (rest) and two by 120 min of cycling exercise (Exercise) at 50% V˙O2peak. Within each block, subjects consumed either combined histamine H1 and H2 receptor antagonists (Blockade) or Placebo, before the start of the 120-min Rest/Exercise., Results: Blockade had no discernible effects on hemodynamic or metabolic variables during Rest or Exercise. However, Blockade increased time-to-completion of the 10-km time trial compared with Placebo (+10.5 ± 3.7 s, P < 0.05). Slowing from placebo to blockade was not different between rest (+8.7 ± 5.2 s) and Exercise (+12.3 ± 5.8 s, P = 0.716)., Conclusions: Exercise-related histaminergic signaling appears inherent to endurance exercise and may play a role in facilitating optimal function during high-intensity endurance exercise.

Introduction: Previous studies observed diurnal variation in hemodynamic responses during recovery from whole-body exercise, with vasodilation appearing greater after evening versus morning sessions. It is unclear what mechanism(s) underlie this response. Since small muscle-mass exercise can isolate peripheral effects related to postexercise vasodilation, it may provide insight into possible mechanisms behind this diurnal variation., Methods: The study was conducted in ten healthy (5F, 5M) young individuals, following single-leg dynamic knee-extension exercise performed in the Morning (7:30-11:30 am) or the Evening (5-9 pm) on two different days, in random order. Arterial pressure (automated auscultation) and leg blood flow (femoral artery Doppler ultrasound) were measured pre-exercise and during 120 min postexercise. Net effect for each session was calculated as percent change in blood flow (or vascular conductance) between the Active Leg and the Inactive Leg., Results: Following Morning exercise, blood flow was 34.9 ± 8.9% higher in the Active Leg versus the Inactive Leg ( p < 0.05) across recovery. Following Evening exercise, blood flow was 35.0 ± 8.8% higher in the Active Leg versus the Inactive Leg ( p < 0.05). Likewise, vascular conductance was higher in the Active Leg versus the Inactive Leg (Morning: +35.1 ± 9.0%, p < 0.05; Evening: +33.2 ± 8.2%, p < 0.05). Morning and Evening blood flow ( p = 0.66) and vascular conductance ( p = 0.64) did not differ., Conclusion: These data suggest previous studies which identified diurnal variations in postexercise vasodilation responses are likely reflecting central rather than peripheral modulation of cardiovascular responses.

Impaired therapeutic responses to anti-inflammatory glucocorticoids (GC) in chronic respiratory diseases are partly attributable to interleukins and transforming growth factor β1 (TGF-β1). However, previous efforts to prevent induction of GC insensitivity by targeting established canonical and non-canonical TGF-β1 pathways have been unsuccessful. Here we elucidate a TGF-β1 signaling pathway modulating GC activity that involves LIM domain kinase 2-mediated phosphorylation of cofilin1. Severe, steroid-resistant asthmatic airway epithelium showed increased levels of immunoreactive phospho-cofilin1. Phospho-cofilin1 was implicated in the activation of phospholipase D (PLD) to generate the effector(s) (lyso)phosphatidic acid, which mimics the TGF-β1-induced GC insensitivity. TGF-β1 induction of the nuclear hormone receptor corepressor, SMRT (NCOR2), was dependent on cofilin1 and PLD activities. Depletion of SMRT prevented GC insensitivity. This pathway for GC insensitivity offers several promising drug targets that potentially enable a safer approach to the modulation of TGF-β1 in chronic inflammatory diseases than is afforded by global TGF-β1 inhibition., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The protease kallikrein 4 (KLK4) plays a pivotal role during dental enamel formation by degrading the major enamel protein, amelogenin, prior to the final steps of enamel hardening. KLK4 dysfunction is known to cause some types of developmental defect in enamel but the mechanisms responsible for transient retention of KLK4 in semi-hardened enamel matrix remain unclear. To address contradictory reports about the affinity of KLK4 for enamel hydroxyapatite-like mineral, we used pure components in quasi-physiological conditions and found that KLK4 binds hydroxyapatite directly. Hypothesising KLK4 self-destructs once amelogenin is degraded, biochemical analyses revealed that KLK4 progressively lost activity, became aggregated, and autofragmented when incubated without substrate in both the presence and absence of reducer. However, with non-ionic detergent present as proxy substrate, KLK4 remained active and intact throughout. These findings prompt a new mechanistic model and line of enquiry into the role of KLK4 in enamel hardening and malformation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Developmental dental defects (DDDs, hereafter "D3s") hold significance for scientists and practitioners from both medicine and dentistry. Although, attention has classically dwelt on three other D3s (amelogenesis imperfecta, dental fluorosis, and enamel hypoplasia), dental interest has recently swung toward Molar Hypomineralisation (MH), a prevalent condition characterised by well-delineated ("demarcated") opacities in enamel. MH imposes a significant burden on global health and has potential to become medically preventable, being linked to infantile illness. Yet even in medico-dental research communities there is only narrow awareness of this childhood problem and its link to tooth decay, and of allied research opportunities. Major knowledge gaps exist at population, case and tooth levels and salient information from enamel researchers has sometimes been omitted from clinically-oriented conclusions. From our perspective, a cross-sector translational approach is required to address these complex inadequacies effectively, with the ultimate aim of prevention. Drawing on experience with a translational research network spanning Australia and New Zealand (The D3 Group; www.thed3group.org ), we firstly depict MH as a silent public health problem that is generally more concerning than the three classical D3s. Second, we argue that diverse research inputs are needed to undertake a multi-faceted attack on this problem, and outline demarcated opacities as the central research target. Third, we suggest that, given past victories studying other dental conditions, enamel researchers stand to make crucial contributions to the understanding and prevention of MH. Finally, to focus geographically diverse research interests onto this nascent field, further internationalisation of The D3 Group is warranted.

Histamine contributes to elevations in skeletal muscle blood flow following exercise, which raises the possibility that histamine is an important mediator of the inflammatory response to exercise. We examined the influence of antihistamines on postexercise blood flow, inflammation, muscle damage, and delayed-onset muscle soreness (DOMS) in a model of moderate exercise-induced muscle damage. Subjects consumed either a combination of fexofenadine and ranitidine (blockade, n = 12) or nothing (control, n = 12) before 45 min of downhill running (-10% grade). Blood flow to the leg was measured before and throughout 120 min of exercise recovery. Markers of inflammation, muscle damage, and DOMS were obtained before and at 0, 6, 12, 24, 48, and 72 h postexercise. At 60 min postexercise, blood flow was reduced ~29% with blockade compared with control ( P < 0.05). Markers of inflammation were elevated after exercise (TNF-ɑ, IL-6), but did not differ between control and blockade. Creatine kinase concentrations peaked 12 h after exercise, and the overall response was greater with blockade (18.3 ± 3.2 kU·l -1 ·h -1 ) compared with control (11.6 ± 2.0 kU·l -1 ·h -1 ; P < 0.05). Reductions in muscle strength in control (-19.3 ± 4.3% at 24 h) were greater than blockade (-7.8 ± 4.8%; P < 0.05) and corresponded with greater perceptions of pain/discomfort in control compared with blockade. In conclusion, histamine-receptor blockade reduced postexercise blood flow, had no effect on the pattern of inflammatory markers, increased serum creatine kinase concentrations, attenuated muscle strength loss, and reduced pain perception following muscle-damaging exercise. NEW & NOTEWORTHY Histamine appears to be intimately involved with skeletal muscle during and following exercise. Blocking histamine's actions during muscle-damaging exercise, via common over-the-counter antihistamines, resulted in increased serum creatine kinase, an indirect marker of muscle damage. Paradoxically, blocking histamine's actions attenuated muscle strength loss and reduced perceptions of muscle pain for 72 h following muscle-damaging exercise. These results indicate that exercise-induced histamine release may have a broad impact on protecting muscle from exercise-induced damage., (Copyright © 2017 the American Physiological Society.)

Improved understanding of dental enamel development will benefit not only dentistry but also biomedicine more generally. Rat and mouse models of enamel development are relatively well characterized and experimentally powerful. However, the diminutive size of murine teeth makes them difficult to study using standard proteomics approaches. Here, we describe gel-based proteomic methods that enable parallel quantification, identification, and functional characterization of proteins from developing rat and mouse teeth. These refined methods are applicable to other scarce samples including human enamel defects.

We investigated how students performed on weekly two-page laboratory reports based on whether the grading rubric was provided to the student electronically or in paper form and the inclusion of one- to two-sentence targeted comments. Subjects were registered for a 289-student, third-year human physiology class with laboratory and were randomized into four groups related to rubric delivery and targeted comments. All students received feedback via the same detailed grading rubric. At the end of the term, subjects provided consent and a self-assessment of their rubric viewing rate and preferences. There were no differences in laboratory report scores between groups (P = 0.86), although scores did improve over time (P < 0.01). Students receiving targeted comments self-reported viewing their rubric more often than students that received no comments (P = 0.02), but the viewing rate was independent of the rubric delivery method (P = 0.15). Subjects with high rubric viewing rates did not have higher laboratory report grades than subjects with low viewing rates (P = 0.64). When asked about their preference for the future, 43% of respondents preferred the same method again (electronic or paper rubric) and 25% had no preference. We conclude that although student laboratory report grades improved over time, the rate and degree of improvement were not related to rubric delivery method or to the inclusion of targeted comments., (Copyright © 2016 The American Physiological Society.)

Key Points: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors, in a variety of pathways that probably predate its more recent role in innate and adaptive immunity. Although histamine is normally associated with pathological conditions or allergic and anaphylactic reactions, it may contribute beneficially to the normal changes that occur within skeletal muscle during the recovery from exercise. We show that the human response to exercise includes an altered expression of thousands of protein-coding genes, and much of this response appears to be driven by histamine. Histamine may be an important molecular transducer contributing to many of the adaptations that accompany chronic exercise training., Abstract: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors. In humans, aerobic exercise is followed by a post-exercise activation of histamine H1 and H2 receptors localized to the previously exercised muscle. This could trigger a broad range of cellular adaptations in response to exercise. Thus, we exploited RNA sequencing to explore the effects of H1 and H2 receptor blockade on the exercise transcriptome in human skeletal muscle tissue harvested from the vastus lateralis. We found that exercise exerts a profound influence on the human transcriptome, causing the differential expression of more than 3000 protein-coding genes. The influence of histamine blockade post-exercise was notable for 795 genes that were differentially expressed between the control and blockade condition, which represents >25% of the number responding to exercise. The broad histamine footprint on the human exercise transcriptome crosses many cellular functions, including inflammation, vascular function, metabolism, and cellular maintenance., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Despite its critical role in maintaining glucose homeostasis, surprisingly little is known about proinsulin folding in the endoplasmic reticulum. In this study we aimed to understand the chaperones involved in the maturation and degradation of proinsulin. We generated pancreatic beta cell lines expressing FLAG-tagged proinsulin. Several chaperones (including BiP, PDIA6, calnexin, calreticulin, GRP170, Erdj3 and ribophorin II) co-immunoprecipitated with proinsulin suggesting a role for these proteins in folding. To investigate the chaperones responsible for targeting misfolded proinsulin for degradation, we also created a beta cell line expressing FLAG-tagged proinsulin carrying the Akita mutation (Cys96Tyr). All chaperones found to be associated with wild type proinsulin also co-immunoprecipitated with Akita proinsulin. However, one additional protein, namely P58(IPK), specifically precipitated with Akita proinsulin and approximately ten fold more PDIA6, but not other PDI family members, was bound to Akita proinsulin. The latter suggests that PDIA6 may act as a key reductase and target misfolded proinsulin to the ER-degradation pathway. The preferential association of PDIA6 to Akita proinsulin was also confirmed in another beta cell line (βTC-6). Furthermore, for the first time, a physiologically relevant substrate for PDIA6 has been evidenced. Thus, this study has identified several chaperones/foldases that associated with wild type proinsulin and has also provided a comprehensive interactome for Akita misfolded proinsulin., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

Cancer cachexia is characterized by the progressive loss of skeletal muscle mass. While mouse skeletal muscle's response to an acute bout of stimulated low-frequency concentric muscle contractions is disrupted by cachexia, gaps remain in our understanding of cachexia's effects on eccentric contraction-induced muscle growth. The purpose of this study was to determine whether repeated bouts of stimulated high-frequency eccentric muscle contractions [high-frequency electrical muscle stimulation (HFES)] could stimulate myofiber growth during cancer cachexia progression, and whether this training disrupted muscle signaling associated with wasting. Male Apc(Min/+) mice initiating cachexia (N = 9) performed seven bouts of HFES-induced eccentric contractions of the left tibialis anterior muscle over 2 wk. The right tibialis anterior served as the control, and mice were killed 48 h after the last stimulation. Age-matched C57BL/6 mice (N = 9) served as wild-type controls. Apc(Min/+) mice lost body weight, muscle mass, and type IIA, IIX, and IIB myofiber cross-sectional area. HFES increased myofiber cross-sectional area of all fiber types, regardless of cachexia. Cachexia increased muscle noncontractile tissue, which was attenuated by HFES. Cachexia decreased the percentage of high succinate dehydrogenase activity myofibers, which was increased by HFES, regardless of cachexia. While cachexia activated AMP kinase, STAT3, and ERK1/2 signaling, HFES decreased AMP kinase phosphorylation, independent of the suppression of STAT3. These results demonstrate that cachectic skeletal muscle can initiate a growth response to repeated eccentric muscle contractions, despite the presence of a systemic cachectic environment., (Copyright © 2016 the American Physiological Society.)

While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

It is widely accepted that healthy enamel formation depends on a steady supply of calcium, yet only fragmentary understanding exists about the mechanisms underlying transepithelial calcium transport. Several lines of evidence indicate that calcium principally follows a transcellular route, which classically is thought to be facilitated by cytosolic calcium-binding proteins termed calbindins. In enamel cells, however, this 'calcium-ferry' dogma appears to fail as we previously found that the major calbindin in murine enamel cells (calbindin-28 kDa) was down-regulated during the peak period of calcium transport and enamel was formed normally in mice lacking calbindin-28 kDa. It remains to be clarified whether the two other known calbindins could function as calcium ferries instead. This study used biochemical and proteomic approaches to obtain definitive identification and quantification of the 30-kDa calbindin (calretinin) and calbindin-9 kDa (S100-G) in enamel epithelium from rat. By establishing that both of these calbindins contribute insufficient calcium capacities in molars and incisors, our results render the calcium-ferry dogma untenable. Of significance to enamel defects and dental bioengineering, these findings support other evidence for an alternative organelle-based mode of calcium transport (calcium transcytosis) and also implicate S100-G/calbindin-9 kDa, but not calretinin, in a calcium-signaling role during enamel maturation., (© 2011 Eur J Oral Sci.)

Growing interest in the treatment and prevention of Molar/Incisor Hypomineralization (MIH) warrants investigation into the protein composition of hypomineralized enamel. Hypothesizing abnormality akin to amelogenesis imperfecta, we profiled proteins in hypomineralized enamel from human permanent first molars using a biochemical approach. Hypomineralized enamel was found to have from 3- to 15-fold higher protein content than normal, but a near-normal level of residual amelogenins. This distinguished MIH from hypomaturation defects with high residual amelogenins (amelogenesis imperfecta, fluorosis) and so typified it as a hypocalcification defect. Second, hypomineralized enamel was found to have accumulated various proteins from oral fluid and blood, with differential incorporation depending on integrity of the enamel surface. Pathogenically, these results point to a pre-eruptive disturbance of mineralization involving albumin and, in cases with post-eruptive breakdown, subsequent protein adsorption on the exposed hydroxyapatite matrix. These insights into the pathogenesis and properties of hypomineralized enamel hold significance for prevention and treatment of MIH.

Improved understanding of dental enamel development will benefit not only dentistry but also biomedicine more generally. Rat and mouse models of enamel development are relatively well characterized and experimentally powerful. However, the diminutive size of murine teeth makes them difficult to study using standard proteomic approaches. Here we describe gel-based proteomic methods that enable parallel quantification, identification, and functional characterization of proteins from developing rat and mouse teeth. These refined methods are also likely to be applicable to other scarce samples.

The biomedical need for streamlined approaches to monitor proteome dynamics is growing rapidly. This study examined the ability of a knowledge-based triplex-profiling strategy (i.e., three functionally distinct chaperones, ERp29/PDI/BiP) to clarify uncertainties about how cancer affects the endoplasmic reticulum (ER) proteome. Investigating a wide range of samples at the tissue and cellular levels (>114 samples from 9 tissues of origin), we obtained consistent evidence that the ER proteome undergoes a major but variable expansion in cancer. Three factors having a strong influence on the ER proteome were identified (cancer-cell type, growth rate, culture mode), and the functionally enigmatic chaperone ERp29 was linked distinctively to histogenetic aspects of tumorigenesis. These findings justify pursuit of the ER-proteome as a medical target in cancer, validate ERp29/PDI/BiP profiling as a streamlined yet powerful measure of ER-proteome dynamics, and suggest that biomarker sets based on distinct functionalities could have broader biomedical utility.

Proteome analysis of rat enamel-forming cells, initiated over a decade ago, has provided valuable insights to enamel biology. In preparation for a more comprehensive, second-generation proteomic exploration, we evaluated an updated microsample-profiling strategy that comprises sequential extraction of enamel epithelium, parallel one- and two-dimensional gel electrophoresis, and mass spectrometric sequence analysis. The results indicated that several hundred proteins, representing various cellular compartments (including membranes), are amenable to identification with a starting tissue volume of <10 microl. With its increased proteomic depth and breadth, this straightforward approach constitutes a major advance from the first-generation work (10-fold increased proteome coverage), although care was needed to ensure a comparably high stringency of protein identification. Expression proteomics has an exciting potential to elucidate the inner workings of murine enamel epithelial cells, leading to an improved understanding of enamel in health and disease.

Craniofacial disorders are associated with one-third of human birth defects but the underlying molecular and cellular causes remain poorly understood. Proteomics seems well-placed to benefit this medically important area but the scarcity of embryonic tissues poses a major challenge. In this study, we applied a microsample proteomics strategy to investigate the first branchial arch, an embryonic structure crucial for facial development, and found that proteome analysis is both practicable and informative despite the scarcity of tissue. Exploiting the embryonic chick as a tractable source of accurately staged tissue, we developed a sequential extraction procedure to interface with one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and 2-D PAGE. In 2-D gels, about 8% of the visible proteome changed between embryonic days 3 and 5, and the identities determined for 21 proteins accorded with the rapid growth during this period. These results led to the first molecular identification of chicken alpha-fetoprotein, and an unusual localisation of vimentin to endoderm. With over 470 protein spots accessible, this comparative proteomics approach has good prospects for providing new markers, functional hypotheses and genes to target in functional tests. A broader value of extending these approaches to facial development in other species and to other areas in embryology can be anticipated.

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (M(r)monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.