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Background: Olfactory stimulation with mastic resin, derived from the Pistacia lentiscus tree, demonstrated a bona fide sialagogic effect in healthy volunteers [1]. Its main volatile compound, α-pinene, also showed this effect. The current study aimed to validate the effect of mastic resin volatiles in chronic dry mouth patients with confirmed decreased saliva secretion. Methods: 41 chronic dry mouth patients with decreased unstimulated saliva secretion (

Background Gingivitis in response to biofilm formation may exhibit different trajectories. The purposes of the present study were to characterize the composition of the supragingival microbiota and salivary cytokine and protein levels in healthy individuals with different gingivitis patterns, to test the hypothesis that manifestations of gingivitis associate with specific profiles in terms of supragingival microbiota, salivary cytokines, and proteins.Methods Forty orally and systemically healthy individuals refrained from all oral hygiene procedures for a period of 14 days, followed by a resolution period of 14 days with regular oral care. Supragingival plaque level and bleeding on probing (BOP) were recorded, and supragingival plaque as well as saliva samples were collected at baseline, day 14, and day 28. Based on change in BOP% from baseline to day 14, rapid (n = 15), moderate (n = 10), and slow (n = 15) responders were identified. Supragingival microbiota composition, salivary cytokine, and protein levels were compared between groups at baseline, day 14, and day 28.Results A significantly higher baseline abundance of Capnocytophaga, Eikenella, and Campylobacter species were recorded in rapid responders, whereas a significantly higher baseline abundance of Streptococcus species were detected in slow responders. Slow responders expressed a high degree of resilience, with minimal difference in microbial composition at baseline and after 14 days of resolution (day 28). On the contrary, significant differences in relative abundance of members of the core microbiota, Streptococcus, Actinomyces, and Rothia species, was noted in baseline samples versus day 28 samples in rapid responders. Comparable baseline cytokine and protein levels were recorded in all groups.Conclusion Supragingival microbiota composition, but not saliva cytokine and protein profiles, seems to influence the extent of the inflammatory response during development of gingivitis in systemically healthy individuals.

In the oral cavity Candida albicans interacts with many oral bacteria, including Porphyromonas gingivalis, both physically and metabolically. The aim of this in vitro study was to characterize these interactions and study their effects on the survival of P. gingivalis. First, metabolic interactions were evaluated by counting the colony forming units (CFU) after co-culturing. The results indicated that the anaerobic bacterium P. gingivalis survives under aerobic conditions when co-cultured with C. albicans. This is due to the oxygen consumption by C. albicans as determined by a reduction in survival upon the addition of Antimycin A. By measuring the protease activity, it was found that the presence of C. albicans induced gingipain activity by P. gingivalis, which is an important virulence factor. Adherence of P. gingivalis to hyphae of C. albicans was observed with a dynamic flow system. Using various C. albicans mutants, it was shown that the mechanism of adhesion was mediated by the cell wall adhesins, members of the agglutinin-like sequence (Als) family: Als3 and Als1. Furthermore, the two microorganisms could be co-cultured into forming a biofilm in which P. gingivalis can survive under aerobic culturing conditions, which was imaged using scanning electron microscopy. This study has further elucidated mechanisms of interaction, virulence acquisition and survival of P. gingivalis when co-cultured with C. albicans. Such survival could be essential for the pathogenicity of P. gingivalis in the oxygen-rich niches of the oral cavity. This study has emphasized the importance of interaction between different microbes in promoting survival, virulence and attachment of pathogens, which could be essential in facilitating penetration into the environment of the host.

Copper is routinely supplemented to weanling pig diets at concentrations above nutritional requirements to enhance growth performance. We hypothesised that this effect depends on the source of Cu and its dietary concentration. We tested this in weaned pigs (26 d of age) over a 35-d period using a 2 × 3 factorial arrangement with two Cu-sources (CuSO4 and Cu2O, monovalent copper oxide, CoRouge®) and three supplementary dietary Cu-levels (15, 80 and 160 mg Cu/kg) as respective factors. Increasing Cu level linearly increased (P

Background: Xerostomia, often associated with decreased saliva quality, poses challenges due to limited treatment efficacy. This study aimed to investigate alternative approaches to enhance saliva secretion through olfactory volatile stimulation with mastic resin and its main compound α-pinene, known for inhibiting acetylcholinesterase in vitro. Methods: The inhibitory effects of freshly prepared mastic resin extract oil and α-pinene oil on acetylcholinesterase (AChE) activity were measured in vitro. Eighty healthy participants were recruited and divided into two groups: exposed to mastic resin volatiles (n = 40) or α-pinene volatiles (n = 40). Saliva samples were collected pre, during and post exposure to analyze saliva flow rate, spinnbarkeit, ion composition and MUC5B levels. Results: Mastic resin extract oil and α-pinene oil inhibited AChE activity by 207 % and 22 %, respectively. Olfactory stimulation with these volatiles significantly increased saliva secretion rate without altering spinnbarkeit and ion composition. Salivary MUC5B concentration rose after exposure to mastic resin volatiles. Conclusions: Olfactory stimulation with mastic resin and α-pinene volatiles demonstrated a bona fide in vivo effect on saliva secretion, confirming their sialagogic capability, potentially as a result of local glandular AChE inhibition. These findings highlight the therapeutic potential of both volatile compounds in treating patients with xerostomia and hyposalivation through olfactory exposure.

ABSTRACTIntroduction The oral pathogen Porphyromonas gingivalis is not only associated with periodontitis but also with systemic diseases elsewhere in the body. The mechanisms by which P. gingivalis travels from the oral cavity to other organs in the body are largely unknown. This review describes the four putative mechanisms supported by experimental evidence, which enable translocation of P. gingivalis over the oral mucosa, endothelial barriers and subsequent dissemination into the bloodstream.Mechanisms The first mechanism: proteolytic enzymes secreted by P. gingivalis degrade adhesion molecules between tissue cells, and the extracellular matrix. This weakens the structural integrity of the mucosa and allows P. gingivalis to penetrate the tissue. The second is transcytosis: bacteria actively enter tissue cells and transfer to the next layer or the extracellular space. By travelling from cell to cell, P. gingivalis reaches deeper structures. Thirdly, professional phagocytes take up P. gingivalis and travel to the bloodstream where P. gingivalis is released. Lastly, P. gingivalis can adhere to the hyphae forming Candida albicans. These hyphae can penetrate the mucosal tissue, which may allow P. gingivalis to reach deeper structures.Conclusion More research could elucidate targets to inhibit P. gingivalis dissemination and prevent the onset of various systemic diseases.

Abstract Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)—a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Food production has a major impact on environmental emissions, climate change and land-use. To reduce this impact, the circularity of future food production systems is expected to become increasingly important. In a circular food system, crop land is primarily used for plant-based food production, while low-opportunity cost feed materials (LCF), i.e. crop residues, co-products of the food industry, grass from marginal land and food waste form the basis of future, animal feeds. Animal diets thus contain much less cereals and soybean meal and include a higher proportion of diverse co-products, residues and novel human-inedible ingredients. These diets are characterised by a lower starch content, and a higher content of fibre, protein, fat, and phytate compared to present diets. In this review, possible consequences of the development towards a more circular food system for the type, volume and nutritional characteristics of feed materials and complete feeds are addressed and related research questions in the area of animal nutrition, physiology and metabolism are discussed. Additional attention is given to possible effects on intestinal health and gut functionality and to (bio)technological processing of LCF to improve their suitability for feeding farm animals, with a focus on the effects in pigs and poultry. It is concluded that an increased use of LCF may limit the use of presently used criteria for the efficiency of animal production and nutrient utilisation. Development of characteristics that reflect the efficacy and efficiency of the net contribution of animal production in a circular food system is required. Animal scientists can have an important role in the development of more circular food production systems by focussing on the optimal use of LCF in animal diets for the production of animal-source food, while minimising the use of human-edible food in animal feed.

Petria Noble, along with Annelies van Loon and Jonathan Bikker, examined the paint application and composition in Rembrandt's The Standard Bearer and concluded that some pigments used for the costume have lost their colour; however, the white flag is as intended. It is also now clear how the artwork was restored in 1958.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

The present study aims to test whether probiotics protect against experimental gingivitis incited by 14 days of oral hygiene neglect and/or subsequently support the restoration of oral homeostasis. Eighty systemically and orally healthy participants refrained from oral hygiene procedures for 14 days, followed by 14 days with regular oral hygiene procedures. Additionally, participants consumed either probiotics (n = 40) or placebo (n = 40) throughout the trial. At baseline, day 14, and day 28, supragingival plaque score and bleeding-on-probing percentage (BOP %) were registered, and supragingival plaque and saliva samples were collected. The supragingival microbiota was characterized using 16S sequencing, and saliva samples were analyzed for levels of pro-inflammatory cytokines and proteases. At day 28, the relative abundance of Lautropia (p = 0.014), Prevotella (p = 0.046), Fusobacterium (p = 0.033), and Selenomonas (p = 0.0078) genera were significantly higher in the placebo group compared to the probiotics group, while the relative abundance of Rothia (p = 0.047) species was associated with the probiotics group. Streptococcus sanguinis was associated with the probiotics group, while Campylobacter gracilis was associated with the placebo group. No difference was observed in salivary cytokines, albumin, or any enzyme activity. The present study suggests that probiotics support the resilience of the oral microbiota in the resolution period after gingivitis.

The aim was to test if probiotics counteract oral dysbiosis during 14 days of sugar stress and subsequently help restore oral homeostasis. Eighty healthy individuals received either probiotics (n = 40) or placebo lozenges (n = 40) for 28 days and rinsed with a 10% sucrose solution 6–8 times during the initial 14 days of the trial. Saliva and supragingival samples were collected at baseline, day 14, and day 28. Saliva samples were analyzed for levels of pro-inflammatory cytokines, albumin, and salivary enzyme activity. The supragingival microbiota was characterized according to the Human Oral Microbiome Database. After 14 days of sugar stress, the relative abundance of Porphyromonas species was significantly higher (p = 0.03) and remained significantly elevated at day 28 in the probiotic group compared to the placebo group (p = 0.004). At day 28, the relative abundance of Kingella species was significantly higher in the probiotic group (p = 0.03). Streptococcus gordinii and Neisseria elongata were associated with the probiotic group on day 28, while Streptococcus sobrinus was associated with the placebo group on day 14 and day 28. On day 28, the salivary albumin level was significantly lower in the probiotic group. The present study demonstrates a potential stabilizing effect on the supragingival microbiota mediated by consumption of probiotics during short-term sugar stress.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

Food-derived carbon quantum dots (CQDs) can relatively easily be synthesized and chemically manipulated for a broad spectrum of biomedical applications. However, their toxicity may hinder their actual use. Here, Spinacia oleracea-derived CQDs i.e., CQD-1 and CQD-2, were synthesized by means of different shredding methods and followed by a microwave-assisted hydrothermal approach. Subsequently, these CQDs were analyzed in vitro and in an in vivo mice model to test their biocompatibility and potential use as bioimaging agents and for activation of osteogenic differentiation.When comparing CQD-1 and CQD-2, it was found that CQD-1 exhibited 7.6 times higher photoluminescent (PL) emission intensity around 411 nm compared to CQD-2. Besides, it was found that the size distribution of CQD-1 was 2.05 ± 0.08 nm, compared with 2.14 ± 0.04 nm for CQD-2. Upon exposure to human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro, CQD-1 was endocytosed into the cytoplasm and significantly increased the differentiation of hBMSCs up to 10 μg mL−1 after 7 and 14 days. Apparently, the presence of relatively low doses of CQD-1 showed virtually no toxic or histological effects in the major organs in vivo. In contrast, high doses of CQD-1 (1 mg mL-1) caused cell death in vitro ranging from 35% on day 1 to 80% on day 3 post-exposure, and activated the apoptotic machinery and increased lymphocyte aggregates in the liver tissue. In conclusion, S. oleracea-derived CQDs have the potential for biomedical applications in bioimaging and activation of stem cells osteogenic differentiation. Therefore, it is postulated that CQD-1 from S. oleracea remains potential prospective material at appropriate doses and specifications.

Microbiome modulation, aiming to restore a health-compatible microbiota, is a novel strategy to treat periodontitis. This study evaluated the modulation effects of antimicrobial peptide LL-31 and its D-enantiomer (D-LL-31) on saliva-derived microcosm biofilms, spiked with or without Porphyromonas gingivalis. To this end, one-day-old biofilms were incubated for 24 h with biofilm medium alone, or medium containing 40 µM LL-31 or D-LL-31, after which biofilms were grown for 5 days. Biofilms were assessed at 1 day and 5 days after intervention for the total viable cell counts, dipeptidyl peptidase IV (DPP4) activity, P. gingivalis amount (by qPCR) and microbial composition (by sequencing). The results showed that D-LL-31, not LL-31, significantly reduced the total viable cell counts, the P. gingivalis amount, and the DPP4 activity of the biofilms spiked with P. gingivalis, but only at 1 day after intervention. In the biofilms spiked with P. gingivalis, D-LL-31 tended to reduce the α-diversity and the compositional shift of the biofilms in time as compared to the control and LL-31 groups. In conclusion, D-LL-31 showed a better performance than LL-31 in biofilm modulation. The biofilm modulation function of the peptides could be impaired when the biofilms were in a severely dysbiotic state.

Plant products have been employed in medicine for centuries. As the world becomes more health-conscious, there is a growing interest in natural and minimally processed products for oral health care. This has led to an increase in research into the bioactive compounds found in plant products, particularly monoterpenes. Monoterpenes are known to have beneficial biological properties, but the specific mechanisms by which they exert their effects are not yet fully understood. Despite this, some monoterpenes are already being used in oral health care. For example, thymol, which has antibacterial properties, is an ingredient in varnish used for caries prevention. In addition to this, monoterpenes have also demonstrated antifungal, antiviral, and anti-inflammatory properties, making them versatile for various applications. As research continues, there is potential for even more discoveries regarding the benefits of monoterpenes in oral health care. This narrative literature review gives an overview of the biological properties and current and potential applications of selected monoterpenes and their derivatives in oral health care. These compounds demonstrate promising potential for future medical development, and their applications in future research are expected to expand.

The binding of Host Defense Peptides (HDPs) to the endotoxin of Gram-negative bacteria has important unsolved aspects. For most HDPs, it is unclear if binding is part of the antibacterial mechanism or whether LPS actually provides a protective layer against HDP killing. In addition, HDP binding to LPS can block the subsequent TLR4-mediated activation of the immune system. This dual activity is important, considering that HDPs are thought of as an alternative to conventional antibiotics, which do not provide this dual activity. In this study, we systematically determine, for the first time, the influence of the O-antigen and Lipid A composition on both the antibacterial and anti-endotoxin activity of four HDPs (CATH-2, PR-39, PMAP-23, and PMAP36). The presence of the O-antigen did not affect the antibacterial activity of any of the tested HDPs. Similarly, modification of the lipid A phosphate (MCR-1 phenotype) also did not affect the activity of the HDPs. Furthermore, assessment of inner and outer membrane damage revealed that CATH-2 and PMAP-36 are profoundly membrane-active and disrupt the inner and outer membrane of Escherichia coli simultaneously, suggesting that crossing the outer membrane is the rate-limiting step in the bactericidal activity of these HDPs but is independent of the presence of an O-antigen. In contrast to killing, larger differences were observed for the anti-endotoxin properties of HDPs. CATH-2 and PMAP-36 were much stronger at suppressing LPS-induced activation of macrophages compared to PR-39 and PMAP-23. In addition, the presence of only one phosphate group in the lipid A moiety reduced the immunomodulating activity of these HDPs. Overall, the data strongly suggest that LPS composition has little effect on bacterial killing but that Lipid A modification can affect the immunomodulatory role of HDPs. This dual activity should be considered when HDPs are considered for application purposes in the treatment of infectious diseases.

A systematic review and meta-analysis were conducted to investigate the SARS-CoV-2 viral load in human saliva and compared it with the loads in oropharyngeal swabs, nasopharyngeal swabs, and sputum. In addition, the salivary viral loads of symptomatic and asymptomatic COVID-19 patients were compared. Searches were conducted using four electronic databases: PubMed, Embase, Scopus, and Web of Science, for studies published on SARS-CoV-2 loads expressed by CT values or copies/mL RNA. Three reviewers evaluated the included studies to confirm eligibility and assessed the risk of bias. A total of 37 studies were included. Mean CT values in saliva ranged from 21.5 to 39.6 and mean copies/mL RNA ranged from 1.91 × 101 to 6.98 × 1011. Meta-analysis revealed no significant differences in SARS-CoV-2 load in saliva compared to oropharyngeal swabs, nasopharyngeal swabs, and sputum. In addition, no significant differences were observed in the salivary viral load of symptomatic and asymptomatic COVID-19 patients. We conclude that saliva specimen can be used as an alternative for SARS-CoV-2 detection in oropharyngeal swabs, nasopharyngeal swabs, and sputum.

ABCC8 and KCJN11 mutations cause the most severe diazoxide-resistant forms of congenital hyperinsulinism (CHI). Somatostatin analogues are considered as secondline treatment in diazoxide-unresponsive cases. Current treatment protocols include the first-generation somatostatin analogue octreotide, although pasireotide, a second-generation somatostatin analogue, might be more effective in reducing insulin secretion. Herein we report the first off-label use of pasireotide in a boy with a severe therapy-resistant form of CHI due to a homozygous ABCC8 mutation. After partial pancreatectomy, hyperinsulinism persisted; in an attempt to prevent further surgery, off-label treatment with pasireotide was initiated. Short-acting pasireotide treatment caused high blood glucose level shortly after injection. Long-acting pasireotide treatment resulted in more stable glycemic control. No side effects (e.g., central adrenal insufficiency) were noticed during a 2-month treatment period. Because of recurrent hypoglycemia despite a rather high carbohydrate intake, the boy underwent near-total pancreatectomy at the age of 11 months. In conclusion, pasireotide treatment slightly improved glycemic control without side effects in a boy with severe CHI. However, the effect of pasireotide was not sufficient to prevent near-total pancreatectomy in this case of severe CHI.

The fluorescent carbon dots (CDs) have found their extensive applications in sensing, bioimaging, and photoelectronic devices. In general terms, the synthesis of CDs is straight-forward, though their subsequent purification can be laborious. Therefore, there is a need for easier ways to generate solid CDs with a high conversion yield. Herein, we used collagen waste as a carbon source in producing solid CDs through a calcination procedure without additional chemical decomposition treatment of the raw material. Considering a mass of acid has destroyed the original protein macromolecules into the assembled structure with amino acids and peptide chains in the commercial extraction procedure of collagen product. The residual tissues were assembled with weak intermolecular interactions, which would easily undergo dehydration, polymerization, and carbonization during the heat treatment to produce solid CDs directly. The calcination parameters were surveyed to give the highest conversion yield at 78%, which occurred at 300°C for 2 h. N and S atomic doping CDs (N-CDs and S-CDs) were synthesized at a similar process except for immersion of the collagen waste in sulfuric acid or nitric acid in advance. Further experiments suggested the prepared CDs can serve as an excellent sensor platform for Fe3+ in an acid medium with high anti-interference. The cytotoxicity assays confirmed the biosafety and biocompatibility of the CDs, suggesting potential applications in bioimaging. This work provides a new avenue for preparing solid CDs with high conversion yield.

Abstract Background More people are surviving a first primary cancer and experiencing a second, different cancer. However, little is known about the diagnostic journeys of patients with second primary cancer (SPC). This study explores the views of patients and general practitioners (GPs) on their experiences of pathways to diagnosis of SPC, including the influence of a previous diagnosis of cancer on symptom appraisal, help-seeking and referral decisions. Methods Qualitative interviews with patients with a SPC diagnosis and case-linked GP interviews in a Scottish primary care setting. In-depth face to face or telephone interviews were conducted, underpinned by a social constructionist approach. Interviews were transcribed and Braun and Clarke’s thematic analysis undertaken. Three analysts from the research team read transcripts and developed the coding framework using QSR NVivo version 10, with input from a fourth researcher. Themes were developed from refined codes and interpreted in the context of existing literature and theory. Results Interviews were conducted with 23 patients (aged 43–84 years) with a SPC diagnosis, and 7 GPs. Five patient themes were identified: Awareness of SPC, symptom appraisal and help-seeking, pathways to diagnosis, navigating the healthcare system, and impact of SPC. GPs interviews identified: experience and knowledge of SPC and referrals and decision-making. Conclusions Insights into the pathway to diagnosis of SPC highlights the need for increased awareness of and vigilance for SPC among patients and healthcare providers (HCPs), and emotional support to manage the psychosocial burden.

Bastian Welz,* Rolf Bikker,* Leonie Hoffmeister, Mareike Diekmann, Martin Christmann, Korbinian Brand,* René Huber* Institute of Clinical Chemistry, Hannover Medical School, Hannover, 30625, Germany*These authors contributed equally to this workCorrespondence: Korbinian BrandInstitute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, 30625, GermanyTel +49 511 532 6614Fax +49 511 532 8614Email brand.korbinian@mh-hannover.deBackground: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.Keywords: TNF, GSK3, PKC, staurosporine, IL-8, NF-κB, termination of TNF-induced signaling, termination of inflammation

Introduction Changing attitudes and behaviour regarding mental health and help-seeking is a complex process, especially in poorer areas where access to mental health services is relatively new. Data from the Indonesian national health survey indicate that after the introduction of the Universal Health Coverage a large number of people suffering from mental illness remain untreated Objectives This study aims to address this issue by seeking the views of community leaders (i.e. village and banjars leaders) on ways to raise community awareness to improve access to mental health services, increase service utilisation rates and reduce the duration of untreated mental illness in Bali. Methods This is a qualitative study with community leaders (i.e. village and banjar leaders) in communities in Bali on barriers and facilitators of accessing mental health services for people. Results In Bali they still have faith in the traditional healer so if they see one and the mentally ill patient is getting better then they don’t think they need to go to the hospital. According to the community leaders the determinants for non-uptake of mental health services were mental health awareness should be integrated systematically starting at primary care and must be complemented by secondary care, and have linkages to informal community-based services and self-care. The community leaders can play a role in awareness-raising by empowerment the community and other logics in community care setting. Conclusions Community awareness can improve access to mental health services, increase service utilisation rates and reduce the duration of untreated mental illness in Bali Disclosure No significant relationships.

ABSTRACT: The novel chelator, L-glutamic acid, N,N-diacetic acid (GLDA) can be used as a dietary ingredient to safely reduce Zn supplementation in complete feed, without compromising the Zn status of farm animals. The objective of this study was to study dietary tolerance, bioaccumulation, and evaluate the safety of GLDA when supplemented in broiler diets at 0, 100, 300, 1000, 3,000, and 10,000 mg/kg. A total of 480 one-day-old Ross 308 male broilers were randomly allocated to 48 pens and fed one of the 6 experimental diets. Production performance was used to assess tolerance to the additive. At trial end, toxicity was evaluated using hematology, plasma biochemistry (n = 144) and gross necropsy (n = 48). Residue levels of GLDA were assessed in liver, kidney and breast tissue of birds used for necropsy. Performance showed an increase (P < 0.05) in body weight for GLDA inclusion at 300 mg/kg. A decrease on the measured performance parameters was found for the 10,000 mg/kg GLDA inclusion level (P < 0.05). The additive was added as a tetra-sodium salt, leading to sodium levels being 2.5 times higher in the latter treatment compared to the control diet which may have led to impaired intestinal barrier function. Mortality was not different between treatments. Residue levels for GLDA at the highest inclusion indicate that 0.0005% of total GLDA consumption is accumulated in breast tissue. Higher values of GLDA were found in kidney and liver at the highest inclusion level, potentially confirming that the small fraction of GLDA absorbed was readily excreted by the animal. At 100 and 300 mg/kg GLDA inclusion there were negligible amounts of GLDA present in all tissues measured. The present experiment demonstrated a high dietary tolerance to GLDA in broilers and indicated that GLDA does not pose a significant risk to food safety when supplemented below 3,000 mg/kg.