FGFR1, FGFR2, FGFR3, and TWIST1 genes.Craniosynostosis is one of the major genetic disorders affecting 1 in 2,100–2,500 live newborn children. Environmental and genetic factors are involved in the manifestation of this disease. The suggested genetic causes of craniosynostosis are pathogenic variants in In order to describe their major clinical characteristics and the presence of pathogenic variants, a sample of 36 Mexican patients with craniosynostosis diagnosed as: Crouzon , Pfeiffer , Apert , Saethre‐Chotzen , and Muenke  was analyzed.FGFR1 , p.Ser252Trp, p.Pro253Arg FGFR2 , p.Pro250Arg, FGFR3 , and p.Gln119Pro TWIST1 , PCR amplification and restriction enzyme digestion were performed. Four and two patients with Apert presented the pathogenic variants p.Ser252Trp and p.Pro253Arg in FGFR2, respectively (with a frequency of 11.1% and 5.5%). The p.Pro250Arg pathogenic variant of FGFR3 was found in a patient with Muenke (with a frequency of 2.8%). The above percentages were calculated with the total number of patients.In addition to craniosynostosis, most of the patients presented hypertelorism, midface hypoplasia, and abnormalities in hands and feet. To detect the pathogenic variants p.Pro252Arg FGFR1, FGFR2, FGFR3, and TWIST1 genes, to determine different pathogenic variants. On the other hand, it would be important to include other genes, such as TCF12 , MSX2 , RAB23 , and EFNB1 , to determine their participation in craniosynostosis in the Mexican population.The contribution of this work is discreet, since only 4 genes were analyzed and sample size is small. However, this strategy could be improved by sequencing the  FGFR1, FGFR2, FGFR3, and TWIST1 genes revealed a higher frequency of variants in Apert.We describe the major clinical characteristics of Mexican patients with craniosynostosis. Analysis of  Pathogenic variants in FGFR2 have been related to the Apert (AS) , Pfeiffer (PS) , Crouzon , and Antley‐Bixler  syndromes, and FGFR3 have been related to the Muenke syndrome (MS) , however, pathogenic variants in other FGFR genes may overlap between different syndromes.It is known that FGFR1, FGFR2, and FGFR3 belong to the family of fibroblast growth factor receptors (FGFR)  motif, whose gene product acts as a transcription factor. The helix‐loop‐helix (HLH) region of this motif is important for homo‐ or heterodimerization, whereas the basic domain is essential for binding of the dimer complex to a target DNA‐binding sequence(s)  pathogenic variants related to Saethre‐Chotzen syndrome  (SCS) have been described , in which the author performs the karyotype and SNP array analysis in one JBS case. The patient presented developmental delay, craniosynostosis , craniofacial dysmorphism, and a normal count and size of thrombocytes  exon IIIa (NM_015850.3), FGFR2  exon IIIa (NM_000141.4), FGFR3  exon 7 (NM_000142.4), and TWIST1  exon 1 (NM_001002926.1) genes were selected, since they are important pathogenic variants in syndromic craniosynostosis: P252R, NM_023110.2(FGFR1):c.755C>G (p.Pro252Arg), rs121909627; S252W, NM_022970.3(FGFR2):c.755C>G (p.Ser252Trp), rs79184941; P253R, NM_000141.4(FGFR2):c.758C>G (p.Pro253Arg), rs77543610; P250R, NM_000142.4(FGFR3):c.749C>G (p.Pro250Arg), rs4647924; and Q119P, NM_000474.3(TWIST1):c.356A>C (p.Gln119Pro), rs104894057. The latter pathogenic variant was chosen because it alters the DNA‐binding domain modifying the affinity of TWIST to bind DNA . The PCR carried out under the following conditions: 35 cycles of denaturation at 95°C for 1 minute, annealing at 60–62°C for 1 minute, and extension at 72°C for 1 minute. Amplicons were verified by 2% agarose gel electrophoresis.The optimized PCR conditions consisted of 200 ng of DNA, 0.5 mmol/L of each primer, 0.2 mmol/L dNTPs, PCR buffer , 1.5 mmol/L MgClPathogenic variants were detected by specific restriction enzyme digestion of each amplicon, performing the reaction according to the manufacturer's instructions , and subsequently analyzed on a 6.0% polyacrylamide gel.We used the HGVS (Human Genome Variation Society) recommendations for the description of the pathogenic variants  and 35.1 years (from 25 to 55 years) at the time of the patient's birth, respectively. Seven Crouzon had a family history of craniosynostosis: one with father and brother with Crouzon; one with mother and cousin with Crouzon; one with mother and sister with Crouzon; one with grandmother and aunt with Crouzon; one with grandfather with ocular proptosis; one with a brother and cousin referred only as craniosynostosis; and one with a daughter with Crouzon.The main clinical features that the patients presented, in addition to craniosynostosis, were the following: hypertelorism, midface hypoplasia, and abnormalities in hands and feet; different skull forms were observed.Table n = 18), all had ocular proptosis; midface hypoplasia 14; malocclusion 13; strabismus 9, prognathism 8; short neck 8; ears low implantation 7; hypertelorism 5; beaked nose 5; palpebral ptosis 3; geno valgus 3; broad first toe in feet 2; hearing loss 1; and broad first finger in the hand 1 , in addition to craniosynostosis, are acrocephaly, hypertelorism, midface hypoplasia, ocular proptosis, hand syndactyly, and otitis media; 5 cases presented rhizomelic shortening and hyperhidrosis; 3 elbow ankylosis, heart disease, and glenohumeral dysplasia; 3 agenesis of the corpus callosum; 2 strabismus; 2 choanal stenosis or choanal atresia; 2 hydrocephaly and developmental delay; 2 short neck; and 1 palpebral ptosis, speech disorders, and cleft/lip palate; and 1 malocclusion, hearing loss, and intellectual disability. In FGFR2, four patients presented the pathogenic variant p.Ser252Trp and two the pathogenic variant p.Pro253Arg, with a frequency of 11.1% and 5.5%, respectively , the case had brachycephaly, ocular proptosis, midface hypoplasia, broad first toe in hands, geno valgus, elbow alteration, intellectual disability, and the p.Pro250Arg pathogenic variant in FGFR3. The frequency of the variant was 2.8%.In MS (n = 4) were detected, each with a different type of skull: plagiocephalus, brachycephalus, dolico/scaphocephalus, and turricephalus. Three cases presented midface hypoplasia ; 2 cases hypertelorism (patients 3 and 4); 2 ocular proptosis (patients 1 and 2); 2 broad first toe in feet (patients 1 and 4); 1 nistagmus, hearing loss and developmental delay (patient 2); 1 prognathism and cleft lip palate (patient 3); 1 high ogival palate (patient 1); and 1 mal occlusion (patient 4) (Table Four cases of type I PS (n = 4), 3 cases showed plagiocephaly ; 2 cases midface hypoplasia (patients 2 and 4); 2 ocular proptosis (patients 1 and 4); 2 palpebral ptosis and short neck (patients 1 and 3); 1 strabismus, syndactyly in feet, and broad first toe in feet (patient 3); 1 retrognathia (patient 4); 1 cleft/lip palate (patient 2); and 1 ears with low implantation (patient 1) , maternal age 40 years and paternal age 43 years, mother had a previous abortion (6 weeks), is a product of pregnancy 3, pregnancy with threatened abortion at 2 months, for which the mother was treated and the delivery was premature, at 7 months of gestation, and delivery with fetal distress. In addition to acrobrachycephaly, midface hypoplasia, hypertelorism, ocular proptosis, strabismus, high ogival/palate, otitis media, hearing loss, and syndactyly of the hands and feet of AS, it also presents vertebral fusion at cervical level and lordosis, heart problems with diastolic murmur, dilation of the right ventricle, tricuspid insufficiency, and aortic insufficiency; pulmonary stenosis, tetralogy of Fallot, hydrocephalus, symptomatic epilepsy, and seizures were present. He presented complete syndactyly type II in hands and type IV in the feet. The patient exhibited moderate intellectual disability, with developmental delay. This patient showed the p.Pro 253Arg pathogenic variant in a Figure .FGFR2 exon IIIa was present , grade I chronic malnutrition was also diagnosed. The patient had the p.Ser252Trp pathogenic variant in FGFR2 exon IIIa gene.AS‐4: Male patient of 1 year, family history is unknown; maternal age 20 years, without data of the father. With heart disease, type I syndactyly in hands, and the patient had the p.Ser252Trp pathogenic variant in FGFR2 exon IIIa , up to 5 different types in Crouzon: acrocephalus, brachycephalus, dolico/scaphocephalus, turricephalus, and acrobrachyturricephalus.Multiple hypotheses have been proposed to explain the pathogenesis of abnormal fusion of sutures. Both environmental factors  and genetic factors  predispose to craniosynostosis.When the skull has an elongated and narrow shape, similar to a boat is called scaphocephaly , and atrophy of the tympanic membranes (Crouzon), to name a few , MSX2 , RAB23 , and EFNB1 , using different methodologies such as multiplex ligation‐dependent probe amplification (MPLA), array comparative genomic hybridization (aCGH), and next‐generation sequencing (NGS).Given the complexity that occurs in syndromic craniosynostosis, we decided to elaborate Figure In conclusion, although enormous advances have been made in recent years in the detection and analysis of the genes responsible for craniosynostosis, the great heterogeneity represented by this disorder continues to be a complex challenge, which requires long‐term studies to identify others disease‐related genes, as well as the inclusion of different populations.5The small size of the sample and the number of pathogenic variants studied.Two patients, who had many clinical features that pointed to a diagnosis of Crouzon, were misdiagnosed, since they presented alterations of hands and feet, but the specific pathogenic variant was not performed. In the same way, a patient, who was thought to have MS, was also ruled out as such, since he did not present the characteristic pathogenic variant.Another limitation was that we did not have MRI or CT studies of all patients.The molecular strategy used did not include all the pathogenic variants necessary for the analysis of patients with syndromic craniosynostosis (see Figure However, despite these limitations, we consider that our study could be a contribution to the knowledge of these pathologies in Mexicans.6The Authors declare no conflict of interest.All authors prepared the bibliographic survey, manuscript preparation, and approved the final version of the manuscript.


Duchenne muscular dystrophy (DMD) is characterized by chronic inflammation and fibrotic tissue production by fibroblasts. The promyogenic factor nuclear factor of activated T-cells 5 (NFAT5) is virtually present in all cells, responding to hyperosmolar or pro-inflammatory stress. In embryogenic fibroblasts, absence of NFAT5 results in cell cycle arrest. Here, unaffected skeletal muscle fibroblasts from one healthy donor showed NFAT5 nuclear translocation upon hyperosmolar stress and normal cell viability. Absence of NFAT5 translocation under pro-inflammatory conditions resulted in decreased cell growth (Incucyte ZOOM). In DMD skeletal muscle fibroblasts from one DMD patient, NFAT5 was merely located in the nucleus. Exposure to hyperosmolar conditions or pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α had no influence on NFAT5 physiology . Hyperosmolarity resulted in decreased cell viability and pro-inflammatory stress in unaltered cell growth. These findings suggest that NFAT5 is vital to DMD fibroblast survival. Exposure to pro-inflammatory or hyperosmolar stress in DMD fibroblasts results in an unexpected NFAT5 response, where fibroblasts are not triggered by inflammatory cytokines and do not withstand hyperosmolarity. Chronic inflammation could be viewed as a non-restrictive factor in the formation of fibrosis in DMD. Abnormal NFAT5 physiology could provide a molecular explanation for permanent fibrotic matrix production by DMD fibroblasts. This lentration ,6. Pertuntration .The transcription factor of activated T cells 5 (NFAT5) is important for cell survival in mouse embryogenic fibroblasts. Absence of NFAT5 in this cell type leads to loss of function . In cellIn a previous study, the effect of hyperosmotic conditions on aldose reductase, regulated by NFAT5 in response to cellular hyperosmolarity, was investigated in DMD myoblasts. The expression levels of aldose reductase, which is a source of the cytoprotective osmolyte sorbitol, was observed to be no different between untreated cells and cells exposed to hyperosmolar stress, probably due to permanent intracellular increased ionic stress and subsequent permanent activation of NFAT5 in DMD myoblasts . With NFUnaffected fibroblasts (UFibro) showed a normal NFAT5 physiological reaction towards both pro-inflammatory cytokines and hyperosmolar stress. At rest, NFAT5 was mainly localized in the cytoplasm and could be discretely observed in the nuclei of UFibro A. ExposuFibroblasts donated by a DMD patient (DMDFibro) displayed unexpected NFAT5 behavior after exposure to pro-inflammatory cytokines and hyperosmolar stress. In unstimulated DMDFibro, NFAT5 was faintly present in the cytoplasm and more clearly present in nuclei compared to UFibro as indicated by the white arrow in IF and strong presence in the nuclear compartment of fractionated WB A. In frap < 0.006 . The following mechanism could explain this phenomenon: activation of p38-MAPK could induce its interaction with NFAT5, thereby avoiding NFAT5 nuclear translocation. With presence of NFAT5 in the nucleus being essential to fibroblast cell growth, this could explain senescence. This mechanism could be a safeguard as to avoid fibrosis during tissue repair. In this study, NFAT5 remains in the nucleus of fibroblasts issuing from DMD. This could imply that p38-MAPK and NFAT5 are not able to communicate with each other and that senescence cannot occur. This could explain the unaltered cell growth observed in this study in DMD fibroblasts. Indeed, no significant difference was observed between DMD fibroblast with or without exposure to pro-inflammatory cytokines.In muscle regeneration, the timely coordinated ballet of fibroblasts and myoblasts is essential to proper muscle reconstruction . The swiTNF-α was used at a dosage of 10 ng/mL in this study, whereas dosages of physiological TNF-α during myogenesis are estimated around several pictograms per mL. TNF-α dosages of 10 to 20 ng/mL, as seen in chronic inflammation, have been shown to inhibit myogenesis . ResultsTM AmpFLSTRTM IdentifilerTM Plus PCR Amplification Kit system  and an ABI capillary sequencer with STR markers for CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX and vWA and the gender marker Amelogenin. Certificates are included in Healthy and DMD fibroblasts were obtained from the Myobank Banque d’ADN   and idenn = 3)  and confocal microscope, both UFibro and DMDFibro were cultured in 8-well chamber slides .After local Ethic Committees approval, Myobank’s healthy skeletal muscle fibroblasts obtained from a healthy donor (“UFibro”) and DMD skeletal muscle fibroblasts donated by a DMD patient (“DMDFibro”) were grown each in three different passages (n = 3) . These fAt confluence, UFibro and DMDFibro were exposed to hyperosmolar or pro-inflammatory conditions. Briefly, fibroblasts were exposed to a mixture of pro-inflammatory cytokines IFN-γ with IL-1β and TNF-α diluted in DMEM (“DMEMCyto”) to respective concentrations of 300 U/mL, 20 ng/mL and 30ng/mL for 7 h and 24 h  .+ concentration measured in DMD patients’ fibers [23Na increase with 38.4 ± 6.8 mmol/L compared with 25.4 ± 2.1 mmol/L in healthy volunteers (p < 0.001). A maximum increase of 21.9 mM in Na+ was, thus, measured. A similar increase was measured in mdx mice, in the murine DMD model by Dunn et al. [+ concentration in blood (135–145 mM) in combination with increased salt intake; in 2006, Yoshida et al. described the benefit of such a diet in mdx mice. These mice expressed less muscle necrosis than mdx mice that did not receive this increase in salt uptake [Hyperosmolar conditions were obtained by adding 3.50 mg/mL NaCl to DMEM, obtaining a 60 mM increase of the solution (“DMEM60”), resulting in a final osmolarity of 170 mM (443 mOsm/L). Addition of NaCl was performed as to mimic Na’ fibers . DMEM con et al. . Therefot uptake . Both cen = 3) were seeded in a 24-NUNC well plate   and allowed to adhere overnight at 5% CO2 and 37°C. The plate was placed following the manufacturer’s guidelines in the IncuCyte ZOOM , allowing real-time monitoring of cell confluence over time. Cells were exposed to the cytokines TNF-α (30 ng/mL), IFN-γ (300 U/mL) and IL-1β (20 ng/mL). Relative cell confluence was obtained by normalizing values to 1, thereby allowing to compare cell lines.Fibroblasts were seeded at high densities: UFibro and DMDFibro  [www.qbaseplus.com)  [Total lysis of 75-cmMA, USA) . MinimumMA, USA)  were appBelgium) . geNorm After transfer to nitrocellulose membranes by electroblotting, proteins from total protein extracts were incubated with primary antibodies . Anti-GAStaining and specificity tests were performed as described previously . PrimaryConfocal images were captured with a Zeiss LSM880 confocal microscope . Images were taken using a 40×Plan-Apochromat/1.3 oil objective, with the pinhole set at 1 Airy Unit.p < 0.05, ** = p < 0.01, *** = p < 0.005.For all results, mean and standard deviation was calculated for each passage at each time point. This was performed for all conditions  using SPSS 26.0 . Means and standard deviations from three passages  resulted in one mean and one standard deviation by using a one-way ANOVA with Tukey’s multiple comparison test. Following significant values were considered: * =


HDAC3 siRNA was added to monocytes prior to incubation with IFNγ, which were then repeatedly challenged with LPS and analyzed by means of protein analyses and transcriptional profiling. Pan-HDACi and HDAC3i reduced cytokine secretion in monocytes and M1 macrophages, whereas HDAC6i yielded no such effect. Notably, neither pan-HDACi nor HDAC3i reduced cytokine secretion in M2 macrophages. In contrast to previous reports in mouse macrophages, HDAC3i did not affect macrophage polarization in human cells. Likewise, HDAC3 was not required for IFNγ signaling or IFNβ secretion. Cytokine and gene expression analyses confirmed that IFNγ-treated macrophages consistently develop a cytokine response after LPS repeated challenge, but pretreatment with HDAC3i or HDAC3 siRNA reinstates a state of tolerance reflected by general suppression of tolerizable genes, possibly through decreasing TLRs expression, and particularly TLR4/CD14. The development of endotoxin tolerance in macrophages is important to reduce exacerbated immune response and limit tissue damage. We conclude that HDAC3 is an attractive protein target to mediate macrophage reactivity and tolerance induction in inflammatory macrophages.Histone deacetylases (HDACs) are a group of enzymes that control histone deacetylation and bear potential to direct expression of large gene sets. We determined the effect of HDAC inhibitors (HDACi) on human monocytes and macrophages, with respect to their polarization, activation, and their capabilities of inducing endotoxin tolerance. To address the role for HDACs in macrophage polarization, we treated monocytes with HDAC3i, HDAC6i or pan-HDACi prior to polarization into M1 or M2 macrophages using IFNγ or IL-4 respectively. To study the HDAC inhibition effect on cytokine expression, macrophages were treated with HDACi prior to LPS-stimulation. TNFα, IL-6, and p40 were measured with ELISA, whereas modifications of Histone 3 and STAT1 were assessed using western blot. To address the role for HDAC3 in repeated LPS challenge induction, HDAC3i or  Histone acetylation controls chromatin remodeling, which in turn is thought to regulate gene transcription. Where histone acetyltransferases (HAT) add acetyl groups to lysine residues, thereby enabling transcription factor binding and subsequent gene expression, histone deacetylases (HDACs) remove histone residues leading to chromatin compaction, which generally results in gene repression , 2. Suchin vivo  , class IIa HDACs , class IIb HDACs HDACs 6 and 10), class III sirtuins (Sirt1–7) and class IV HDACs (HDAC11) , 13. Alt and 10, in vivo . In athein vivo . Furtherβ (TGFβ) .Given these outcomes, several studies have hypothesized a potential role for HDAC3 in developing tolerance towards endotoxins, such as lipopolysaccharides (LPS) , 26, 27.In the current study, we investigated the effect of HDAC3i on differentiation and activation of IFNγ-primed human peripheral blood-derived monocytes and macrophages. To this end, we utilized an inhibitor targeting HDAC3  as well as the nonselective pan-HDACi  . As ITF3Pan-HDAC inhibitor , HDACi with activity toward HDAC3   and HDAC6 alone  were provided by Italpharmaco and used at concentrations described previously , 24. Ant+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec).Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors  by Ficoll density gradient (Invitrogen). The human biological samples were sourced ethically, and their research use was in accordance with the terms of informed consent under an IRB/EC approved protocol. Written informed consent was obtained from donors, as approved by the UK East of England—Cambridgeshire and Hertfordshire Research Ethics Committee and Amsterdam UMC Institutional Review Board under project number B07.002-X. CD14+ monocytes were used in subsequent analyses or polarized immediately after isolation towards M1  macrophages using 50 ng/mL IFNγ (R&D systems/Peprotech), or M2  macrophages using 40 ng/mL interleukin (IL) 4 (R&D systems/Peprotech) in culture media for 72 h. Freshly isolated CD14+ monocytes and M1- or M2-polarized macrophages were pre-treated with an increasing concentration  of HDAC3i  = 144 nM; HDAC6 IC50 = 48 nM), HDAC6i , pan-HDACi  to 4 h (to assess protein expression). Macrophages that were treated first with 24 h medium then challenged with medium are designated as M/M (medium/medium), macrophages stimulated with one dose of LPS after 24 h medium pretreatment are designated as M/L (medium/LPS), macrophages stimulated with LPS for 24 h and challenged with LPS are designated as L/L (LPS/LPS).+ monocytes were pretreated with either DMSO or HDAC3i for 30 min or with non-targeting scrambled siRNA or HDAC3 siRNA for 48 h, after which they were polarized to M1 macrophages overnight with 50 ng/mL IFNγ and then M/M, M/L and L/L conditions were applied as described above.To study whether HDAC3 plays a role in regaining LPS tolerance in M1 macrophages, CD146 cells were used for each condition in all experiments.Isocove’s Modified Dulbecco’s Medium  supplemented with 10% fetal bovine serum (FBS) (Lonza), 2 mM l-glutamine (Lonza), 100 U/ml penicillin (Lonza) and 100 U/ml streptomycin (Lonza) was used as culture media. 2x10+ monocytes were transfected with siGENOME human smartpool HDAC3 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon).The CD14® ELISA Development Systems according the manufacturer’s protocol (R&D systems™).Secreted cytokine levels of IFNβ, tumor necrosis factor (TNF)α, IL-6, P40 and IL-10 were quantified in collected supernatant using the DuoSet® FastStart DNA MasterPLUS SYBR Green I (Roche) on LightCycler 480 . Transcript expression levels were analyzed using LinRegPCR  in accordance with the manufacturer’s instructions. Total RNA was transcribed into complementary DNA by qScript cDNA SuperMix (Quanta Biosciences) according to manufacturer’s instructions. Quantitative reverse transcriptase polymerase chain reaction (qPCR) was performed using a LightCyclerinRegPCR  and normCells were harvested and lysed in 50 μL ice-cold lysis buffer containing 150 mM NaCl, 0.5% Triton X-100, 5 mM Ethylenediaminetetraacetic acid (EDTA) and 0.1% SDS. Samples were mixed with sample buffer, denatured, and separated by SDS-PAGE before blotting onto polyvinyldifluoride membranes (Millipore). Membranes were blocked with non-fat dry milk (5% solution) and incubated overnight with the primary antibody. Membranes were then washed and incubated with HRP-conjugated secondary antibodies before visualizing the proteins using western blot substrate (Roche).2 3 mM), incubated for 15 min on ice, and 25 μL 10% NP40 was added and the solution was vortexed. The solution was then centrifuged for 10 min, and the supernatant was kept for analyses of the cytosolic fraction. The pellet was resuspended in 50 μL of cell extraction buffer , 0.1% SDS, 0.5% deoxycholate, 20 mM Na4P2O7) and kept on ice for 30 min with 10 min vortex intervals. This fraction was then centrifuged for 30 min at 14,000xg and the supernatant was used for nuclear fraction analyses.Cells were collected, transferred to a pre-chilled collection tube, and washed twice with cold PBS. Cells were resuspended in 500 μL hypotonic buffer  before flow cytometric analysis using the LSRFortessa and FACSCalibur (both BD Biosciences). FlowJo  was used for data analysis.Donor + Treatment + Tolerized + Treatment: Tolerized. Our research question specifically pertained the interaction between tolerization and HDAC3i treatment. Subsequent visualizations were done using ggplot2 (v3.2.0)  and transcribed into cDNA by qScript cDNA SuperMix (Quanta Biosciences) according to manufacturer’s instructions. Subsequently, cDNA was sequenced in a 50 bp single-ended fashion on the Illumina HiSeq4000 to a depth of 35 million reads at the Amsterdam UMC Core Facility Genomics. Quality control of the reads was done with FastQC (v0.11.8)  and summ(v3.2.0) . Enrichm(v3.2.0)  and visu(v3.2.0) .2 transformed and the mean was taken across samples. Gene symbols were translated between human and mice using the ortholog annotations available in biomaRt (v2.44.1)  .Data are shown as mean ± standard error of the mean (SEM) unless indicated differently. Statistical analysis was performed with GraphPad Prism v5.0a (GraphPad Software Inc.). For multi-experimental group analysis, data were subjected to one-way ANOVA or Student’s t-test followed by post-hoc test (Dunnett) for group differences. The two-tailed level of significance was set at p ≤ 0.05 (*), 0.01 (**) or 0.001 (***) for group differences.+ monocytes were polarized into M1 and M2 macrophages using IFNγ and IL-4, respectively, or left untreated in medium (M0 macrophages). We verified the polarization by measuring the gene expression of CD163, CD64, and CD200R, that mark M0, M1, and M2 phenotypes, respectively (+ monocytes with Control (DMSO) or with 500 nM of pan-HDACi, HDAC3i, or HDAC6i  overnight to induce LPS tolerance state, after which they were washed and re-stimulated with a higher concentration of LPS (100 ng/mL) , 59. As ). Taken together, HDAC3i overcomes the potential of IFNγ to block tolerance induction, via a mechanism independent of enhanced IFNγ or IFNβ production nor signaling.To investigate this further, we tested whether HDAC3i reinstated tolerance by reducing IFNβ secretion as was observed in IFNγ-primed mouse macrophages , 27. How+ monocytes were treated with HDAC3i or DMSO as a control prior to polarization to M1 macrophages using IFNγ and then treated as naïve control without any LPS stimulation (M/M), LPS repeated challenge (L/L) or only treated once with LPS (M/L) after which the transcriptome was profiled at the same time-point for all groups. Principal component (PC) analysis revealed that the first principal component (PC1) separated the M/M, M/L and L/L macrophages treated with DMSO , IL1β, MIP-1α and MIP-1β were downregulated, CD14 as well as the TLR genes themselves were upregulated  were downregulated in HDAC3i-treated L/L versus M/L macrophages relative to DMSO-treated L/L versus M/L macrophages   indicateHIVEP2, ELK3, IRF7, NFKB1, ARID5B, REL, KLF7, ETV3 and MXD1 were bound by HDAC3, with one peak associated to the intergenic region of HIVEP2 belonging to the top 2.5% strongest peaks  obtained from GSE106701 , 52. Comst peaks , 52, sugThis study suggests that HDAC3 is required for the organization of the inflammatory gene program and supporting mechanisms to evade tolerance induction in such a way that we are capable of partially recapitulating tolerance towards LPS in M1 macrophages by treatment with HDAC3i. We showed that HDAC3i resulted in the reduced expression of LPS-induced pro-inflammatory cytokines after a second exposure to LPS in human monocytes and M1 macrophages, but not in M2 macrophages, implying that the M1 macrophages have developed some degree of tolerance towards LPS.Nfkbiz and Nfkbia in mice, both of which were also upregulated in HDAC3i pre-treated macrophages  complex in tolerized cells  can be found in the article/Study design and conduction of the study, laboratory and writing of the manuscript: MG, JZ, and AL. Bioinformatics analyses: MG and AL. Study design, supervision and writing of the manuscript: NH, DT, PH, MW, WJ. Laboratory analyses: RS, JD, CV, TM, IB. Technical support: PPH. All authors contributed to the article and approved the submitted version.MG and AL are funded by European Union’s Horizon 2020 research and innovation program under Grant Agreement No. ITN-2014-EID-641665. This project has also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 814168. WJ is funded through the Dutch Ministery of Economic Affairs . GlaxoSmithKline provided support in the form of salary for authors AL, NH, and DT. GlaxoSmithKline had no additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding for open access charge will be covered by Amsterdam University Medical Centers, University of Amsterdam.MG, AL, NH, PH and DT were employed by GlaxoSmithKline.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.


Calanoides acutus (copepodite stage V (CV), females) and the more omnivorous Calanus propinquus  storing wax esters and triacylglycerols, respectively, which were collected in summer (end of December). Feeding carbon-labelled diatoms to these copepods, 13C elucidated assimilation and turnover rates of copepod total lipids as well as specific fatty acids and alcohols. The 13C incorporation was monitored by compound-specific stable isotope analysis (CSIA). CV stages of C. acutus exhibited an intense total lipid turnover and 55% of total lipids were labelled after 9 days of feeding. By contrast, total lipid assimilation of female C. acutus and C. propinquus was lower with 29% and 32%, respectively. The major dietary fatty acids 16:0, 16:1(n − 7) and 20:5(n − 3) had high turnover rates in all specimens. In C. acutus CV, the high rates of the de novo synthesized long-chain monounsaturated fatty acids and alcohols 20:1(n − 9) and 22:1(n − 11) indicate intense lipid deposition, whereas these rates were low in females. The differences in lipid assimilation and turnover clearly show that the copepod species exhibit a high variability and plasticity to adapt their lipid production to their various life phases.The study revealed species- and stage-specific differences in lipid accumulation of the dominant Antarctic copepods, the primarily herbivorous This article is part of the theme issue ‘The next horizons for lipids as ‘trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'. To cope with unfavourable conditions, i.e. periods of low food supply, herbivorous zooplankton, in particular calanoid copepods, have developed specific biochemical strategies –3. They Calanoides acutus and Calanus propinquus are dominant components of the zooplankton community. Together, they may comprise more than half of the mesozooplankton biomass [C. acutus is primarily herbivorous and survives the wintertime in a resting stage called diapause [C. acutus, C. propinquus has a broader diet, switching from a phytoplankton-based diet in summer to opportunistic feeding in winter. This is also reflected in its triacylglycerol storage, which is typical of species active year-round [The polar oceans especially are characterized by a short and intense period of primary production during summer and an extended period of food scarcity in winter due to light limitation and ice cover ,6. Here, biomass ,9. Thesediapause . Its extdiapause ,4. Compaar-round ,9–11.Calanus species showed that it takes two to three weeks to fully change their diatom-dominated fatty acid pattern to a signature typical of flagellates, and vice versa [The huge lipid deposits accumulated by herbivorous zooplankton species, in particular calanoid copepods, are partly biosynthesized de novo from the phytoplankton diet, but they also contain specific phytoplankton fatty acids incorporated without biochemical alteration. The latter fatty acids are used as so-called trophic markers. Examples are 16:1(n − 7) typically abundant in diatoms and 18:4(n − 3) characteristic of flagellates ,13. The ce versa . Omnivorce versa ,16.13C-labelled phytoplankton diet and compound-specific isotope analyses of lipids, however, allows following the assimilation of dietary carbon into specific lipid compounds of copepods and assessing uptake rates. When wax ester-storing Arctic Calanus species were fed with labelled diatoms, their labelled fatty acids were rapidly taken up and fatty acids, and notably alcohols, were synthesized de novo to finally produce wax esters [et al. used the same labelling technique on small Arctic copepods [Pseudocalanus minutus exhibited high lipid turnover rates and that the species incorporated fatty acids more rapidly from diatoms than from flagellates. The lipid turnover rates in Oithona similis were lower, but this species was able to ingest and/or assimilate lipids with the same intensity from both food sources, possibly owing to its more flexible feeding behaviour and thus being better adapted to a broader dietary spectrum. These studies indicate that tackling assimilation dynamics of lipids using labelled food has the potential to better understand lipid synthesis processes and how they reflect life-history traits. Such knowledge is still completely lacking for Antarctic copepods.Lipid biomarkers do not provide reliable rates of assimilation and turnover of specific fatty acids and alcohols. Using x esters ,17. Rececopepods . They shC. acutus and C. propinquus to assess the transfer and turnover rates of carbon into individual lipid compounds. We examined the hypothesis that wax esters can be more rapidly biosynthesized than triacylglycerols, since the reduction of fatty acids to alcohols diminishes a negative feedback mechanism, which occurs in the presence of surplus fatty acids [C. acutus and C. propinquus are ideal for comparing species with deviating lipid storage modes [For the first time, we therefore applied the compound-specific isotope analysis (CSIA) approach to elucidate the lipid dynamics of the dominant Antarctic copepods ty acids . It mighge modes .2.(a)Polarstern . Copepods were collected at Station 53  in the Antarctic Weddell Gyre on 28 December 2011 by vertical bongo net hauls down to 300 m depth. Specimens of C. acutus  and of C. propinquus  were gently sorted from the catch, maintained alive in filtered seawater at 0°C in a cooling container on board and transported to Germany at 0°C by airplane. The copepods arrived in excellent condition 9 days after the catch. Copepods are able to cope with starving conditions for longer periods [Samples were taken during the Antarctic expedition PS 79 (ANT XXVIII/2) with RV  periods . The fee(b)Conticribra weissflogii [13C by adding labelled sodium bicarbonate  at a concentration of 15 mg l−1. The labelled bicarbonate was added regularly to ensure high 13C concentrations. Extra silicate was added to the medium. The cultures were kept at constant light and 4–6°C and stirred twice a day to keep cells suspended. Algae were grown for several days and maintained in the exponential growth phase. Samples for lipid analyses were taken by filtrating 300 ml duplicates of the algal cultures on GF/F filters (0.7 µm pore diameter).To provide the copepods with the labelled diet, the diatom ssflogii  was cultC. acutus  and C. propinquus  were transferred in groups of 20–45 individuals each to 2 l glass bottles containing filtered seawater. Due to the limited number of specimens, only one sample for each group could be analysed per experimental day. To determine the starting conditions (day 0), samples with 17–32 specimens  were immediately frozen at −80°C for lipid analyses. During the nine days of the experiment, copepods were kept at continuous light to imitate polar day conditions. Every three days one third of the seawater was exchanged with freshly labelled algal suspension (max. 154 357 cells l−1). The green colour of the incubation water indicated that copepods were fed ad libitum (greater than 200 µg C l−1). At day 1, 3, 6 and 9 of the experiment, all specimens of one bottle containing each species and stage  were collected and frozen at −80°C for lipid analyses.Live and healthy-looking copepods of (c)et al. [Total lipid was extracted by homogenizing the copepods and the filters with algae in dichloromethane:methanol (2:1 v:v), modified after Folch et al. . As inteet al. . Unknown(d)13C isotopic enrichment in fatty acids and alcohols was measured using a Thermo GC-c-IRMS system, equipped with a Trace GC Ultra gas chromatograph, a GC Isolink operated in combustion mode at 1000°C and a Delta V Plus isotope ratio mass spectrometer connected via a Conflo IV interface . Fatty acid methyl esters and alcohols were analysed in splitless mode and separated on a DB-FFAP column [δ notation, but for this study, we used the atom per cent values, calculated by the Isodat 3.0 software, which is more appropriate than relative values to express isotope data in terms of isotope concentrations. The 13C enrichment that resulted from the assimilation of labelled food, the atom per cent excess, the proportion of carbon assimilated in the copepod fatty acids and alcohols, and the carbon assimilation as mass (µg Cassi ind−1) was calculated after Boissonnot et al. [13C/12C ratio [The P column . Isotopit et al. . The car2C ratio .(e)13C assimilation into fatty acids and alcohols presented as mass and percentage. Three regression types were tested to reflect the temporal trends: linear and polynomial degree 2 and 3. The best fit (R2) was found for linear regression for most compounds . One-way ANOVA followed by Tukey honest significant difference tests were performed on the slope of linear regressions.Statistical analyses were performed using the free software R 3.2.1 . Normal distribution was tested with the Shapiro–Wilk test with regard to 3.(a)Conticribra weissflogii were 16:0 , 16:1(n − 7) (12.3%), 16:3(n − 4) (19.5%) and 20:5(n − 3) (17.2%). When we exposed the diatoms to 13C-labelled bicarbonate prior to our feeding experiments, 13C uptake in the algae increased exponentially and reached on average 37.4 ± 9.0 atom% after four days; thereafter, 13C enrichment of the algae was slightly variable but remained high.Major fatty acids of the diatom (b)C. acutus, the total lipid content  was 15 ± 4.5 µg per CV and 63.8 ± 25 µg per female during the course of the experiment. Parallel analyses from the same station revealed similar low total lipid content of C. acutus CV (20 µg ind−1) comprising less than 10% of dry mass. 13C carbon assimilation was already detected after the first days of the feeding experiment. At the end of the experiment (day 9)  , lipid aC. propinquus females had a mean total lipid content of 34 ± 19 µg ind−1 which is also comparable to females at more southerly stations. They assimilated about 26% 13Cassi TL−1 into total lipids comparable to C. acutus females (i)C. acutus CV analysed by conventional GC-FID . The fatty acid 20:5(n − 3) showed maximum carbon assimilation with 2.2 µg Cassi ind−1 by day 9. By contrast, carbon assimilation into most other fatty acids, especially 18:1(n − 7) and 22:1(n − 11), was relatively low with less than 0.7 µg Cassi ind−1 during the entire experiment. Carbon uptake into the fatty alcohols of C. acutus CV ranged from 0.3 to 1.6 µg Cassi ind−1 with maximum uptake for 20:1(n − 9). Carbon assimilation into alcohols essentially started after day 3 . Assimilated carbon of the other fatty acids 14:0, 22:6(n − 3), 20:1(n − 9) and 22:1(n − 11) accounted for 25–35%. The polyunsaturated 16:3(n − 4) comprising almost 20% of the diatom fatty acids as well as 16:2(n − 4) showed nearly 100% assimilation, but increased only slightly in the copepods. The prevailing alcohols were also highly 13C enriched. After a lag phase of three days, the 16:0 alcohol was labelled up to 90% (10.4% 13Cassi day−1) and the alcohols 14:0, 20:1(n − 9) and 22:1(n − 11) up to 50–60% at the end of the experiment.On a percentage basis (assimilated fatty acids of fatty acid mass) the fatty acids 16:1(n − 7), 20:5(n − 3) and 16:0 showed the highest portions of assimilated carbon with more than 60%, which correspond to a mean daily assimilation of 10.4, 11.1 and 7.2% (ii)C. acutus females, the fatty acids 22:1(n − 11), 16:1(n − 7), 20:5(n − 3) and 22:6(n − 3) dominated with a portion greater than 10%, followed by 20:1(n − 9), 16:0, and 18:1(n − 9). Especially, 20:1(n − 9) showed a strong increase by 11.2%. The other fatty acids remained constant or decreased by 1.3% to 4.4%. The dominant alcohols 20:1(n − 9) and 22:1(n − 11) accounted for more than 80% of total fatty alcohols  and 16:0 was 3.5 and 1.4 µg Cassi ind−1, respectively. After a lag phase of six days, highest carbon incorporation occurred into 16:1(n − 7) (2.8 µg Cassi ind−1) and 20:1(n − 9) (1.8 µg Cassi ind−1). By contrast to the CV stages, the assimilation in fatty alcohols was clearly lower in the females ranging only from 0.04 to 0.07 µg Cassi ind−1 . Labelling of the other fatty acids 14:0, 22:6(n − 3), 20:1(n − 9) and 22:1(n − 11) ranged between 15% and 25%. The major difference between CV and females was the weak 13C assimilation into the alcohols with only up to 10% of 13C.Similar to the CV stages, the fatty acids 20:5(n − 3), 16:1(n − 7) and 16:0 were labelled with more than 60%, which corresponds to a rate of 10.8, 7.5 and 7.1% (iii)C. propinquus females remained rather constant throughout the experiment with maximum changes of only 5%. Principal fatty acids were 20:5(n − 3) (approx. 30%), followed by 16:0 and 22:6(n − 3) (approx. 20%). Contributions of 24:1(n − 9) and 16:1(n − 7) as well as 16:3(n − 4) and 16:2(n − 4) were clearly lower  and 22:1(n − 11) incorporation remained below 0.5 µg Cassi ind−1. Significant changes during the course of the experiment were only detected for the fatty acids 16:0 (p < 0.01), 20:5(n − 3) (p < 0.01) and 22:6(n − 3) , reaching 2.0, 3.5 and 1.0 µg Cassi ind−1, respectively, after 9 days . The long-chain monounsaturated fatty acids showed only minor labelling.The percentage of assimilated fatty acids was about 50% for 16:0 and 20:5(n − 3) and 20% for 22:6(n − 3). The value for 16:1(n − 7) was also about 50% at day 6, but it decreased towards the end of the experiment. The daily assimilation rates of 16:0, 16:1(n − 7) and 20:5(n − 3) were 5.1, 2.2 and 5.3% 4.a concentrations measured by a CTD mounted fluorometer. However, low lipid levels are not unusual in late December, especially for C. propinquus, since females may have invested dietary energy primarily in egg production instead of accumulating storage lipids. Analyses of lipid-poor winter specimens revealed that females of C. propinquus totally lacked 20:1 and 22:1 fatty acids. In C. acutus, these storage fatty acids also occurred only at lower levels, similar to our data. By contrast, lipid-rich C. acutus and C. propinquus contain high amounts of long-chain monounsaturated moieties with chain lengths ≥20 carbon atoms, which comprise 50% and even more of total fatty acids and alcohols in the wax ester and triacylglycerol fractions, respectively [The copepods used in our experiment were lipid-poor, although they had been collected in late December (summer), when phytoplankton food should still be available. Specimens can have very different amounts of storage lipid varying between 12% and 50% of total lipid per dry mass . Possiblectively ,26.C. acutus, as shown by Albers et al. [The initial fatty acid patterns of both copepod species reflected a typical diatom-dominated diet in the field. Since diatoms were also offered during the feeding experiment, a considerable change in the relative fatty acid composition was not expected. Phytoplankton was provided in excess and therefore copepods were not food-limited . They ass et al. , which sC. acutus, a considerable fraction of the 20:1(n − 9) fatty acid was converted to the corresponding alcohol. 13C labelling revealed that almost all carbon of this alcohol was replaced by dietary carbon. Correspondingly, the concentration of the 20:1(n − 9) fatty acid increased only slightly. There seems to be a delay of about 3 days in the production of all alcohols. This may be due to the fact that the precursor fatty acids have to be produced in advance, mainly de novo, and then reduced to alcohols, which most likely requires a higher energetic effort. The pronounced biosynthesis of alcohols by C. acutus CV stages during feeding also shows that they were actively accumulating lipid stores by biosynthesizing wax esters.In the CV stages of C. acutus, the biosynthesis of alcohols was generally low, although they still ingested dietary fatty acids and produced the 20:1(n − 9) fatty acid de novo after a lag phase of about 6 days. Although the portion of 20:1(n − 9) doubled in the fatty acid composition, the females did not convert this fatty acid to the corresponding alcohol, which is the major alcohol in this species [C. acutus. It is, however, possible that the females used the dietary fatty acids for gonad development and the production of eggs, but this has not been monitored. In the northern Calanus species, C. glacialis and C. finmarchicus, high egg production is related to food supply and egg-laying usually starts after several days of feeding. The eggs of these copepods contain only small amounts of wax esters and therefore wax esters may not be essential for egg production, besides offering energy [By contrast, in the females of  species . This wag energy . HoweverCalanus species, these fatty acids with 16 carbon atoms were clearly enriched during feeding [Polyunsaturated fatty acids with 16 carbon atoms, which belong to the major dietary fatty acids, were highly labelled in the copepods, but increased only slightly in mass. These fatty acids are not essential for membranes and our data suggest that they are not retained in storage lipids. However, in similar experiments with Arctic  feeding . AlthougC. acutus CV suggest that wax ester deposition is advantageous, perhaps not faster but more efficient compared to storing triacylglycerols. Unfortunately, a direct comparison with C. propinquus CV stages was not possible.Our results suggest that copepods, in particular lipid-poor specimens, start lipid accumulation with the uptake of fatty acids originating from the phytoplankton diet when food becomes available. After 3–6 days, the production of de novo synthesized lipids from proteins and carbohydrates as well as fatty acid precursors strongly increased. Potential fatty acid precursors are 14:0, 16:0 and 18:1(n − 9), although the latter only occurred in low amounts in the diatom 3.2%). The 16:1(n − 7) fatty acid, a major fatty acid of diatoms, is not elongated to long-chain monounsaturated fatty acids with 20 and 22 carbon atoms, because it would result in fatty acids with a double bond in n − 7 position %. The 16. Such fa13C-labelled phytoplankton to zooplankton, it has to be considered that only a small part, usually less than 10%, of dry mass of the phytoplankton diet consists of lipids, while the main portion comprises proteins with few carbohydrates. Therefore, only a minor part of the food is directly transferred as phytoplankton lipids to zooplankton. Hence, the majority of the labelled dietary carbon is used for de novo production and energetic requirements. Part of the ingested carbon is also lost via respiration and egestion. Therefore, future studies should include the analysis of faecal pellets, although this may be challenging.When feeding 5.C. acutus and C. propinquus to assimilate dietary carbon in variable amounts into different lipid compounds depends on their developmental stages and their life-cycle strategies. In our study, CV stages of C. acutus were lipid-poor. This does not reflect the usual situation at the end of the primary production period in late summer, when the CV stages are already prepared for overwintering, including a wax ester-rich oil sac. In our experiment, the lipid-poor CV stages of C. acutus assimilated carbon at high rates. They produced fatty acids and alcohols, hence wax esters, indicating that they were able to continue lipid biosynthesis when food became available. By contrast, the females of both species had a down-regulated storage lipid metabolism with weak carbon assimilation into their storage lipids. At this stage of their life cycle, females seem to preferentially assimilate dietary fatty acids, which is less energy-consuming than de novo synthesis, but important for maintaining membrane activity. The differences in carbon and lipid assimilation clearly show that copepods exhibit a high variability and plasticity to adjust their lipid metabolism to the various life phases, which depend on the species, stage and on spatiotemporal conditions.The capacity of


Therefore, based on these finds, ARHGAP21 may be an important target in Diabetes Mellitus (DM) treatment.ARHGAP21 is a RhoGAP protein implicated in the modulation of insulin secretion and energy metabolism. ARHGAP21 transient-inhibition increase glucose-stimulated insulin secretion (GSIS) in neonatal islets; however, ARHGAP21 heterozygote mice have a reduced insulin secretion. These discrepancies are not totally understood, and it might be related to functional maturation of beta cells and peripheral sensitivity. Here, we investigated the real ARHGAP21 role in the insulin secretion process using an adult mouse model of acute ARHGAP21 inhibition, induced by antisense. After ARHGAP21 knockdown induction by antisense injection in 60-day old male mice, we investigated glucose and insulin tolerance test, glucose-induced insulin secretion, glucose-induced intracellular calcium dynamics, and gene expression. Our results showed that ARHGAP21 acts negatively in the GSIS of adult islet. This effect seems to be due to the modulation of important points of insulin secretion process, such as the energy metabolism (PGC1α), Ca Diabetes Mellitus (DM) is a highly prevalent disease, and pancreatic islet dysfunction is crucial for its development –3. Thus,ob/JUnib) and hypo (endurance-trained mice) insulin secretion.We previously demonstrated that ARHGAP21 inhibition increases insulin secretion in neonatal isolated pancreatic islet . Howeverob/JUnib mice (Ob/Ob), an animal model known to secrets a huge amount of insulin, display reduced ARHGAP21 expression in their pancreatic islets in comparison to a lean mice. In contrast, an animal model known to secrets a low amount of insulin (endurance-trained mice) showed the opposite results, presenting high ARHGAP21 content compared to control group. These findings suggest that our target protein acts as a negative regulator of insulin secretion. This observed phenomenon was corroborated by the fact that adult islet from ARHGAP21 knockdown mice showed increased glucose-stimulated insulin secretion (GSIS). This is probably due to upregulation of proteins involved in the lipid metabolism (PGC1α), Ca2+ sensibility (SYTVII), granule extrusion (SNAP25) and cell-cell interactions (CX36).Here, we observed that B6.V-LepTaken together, these findings confirm the involvement of ARHGAP21 over insulin/glucose homeostasis. The discovered of this important role of ARHGAP21 as a negative regulator of insulin secretion may provide a target for therapies to DM management. However, further studies are necessary to better understand by which mechanism ARHGAP21 acts in metabolic diseases, such as obesity and diabetes.The oligonucleotides (antisense-ARHGAP21 and Mismatch) and primers were purchased from Sigma Adrich . Fura-2 acetoxymethyl ester was acquired from Life Technologies . Anti-ARHGAP21 and anti-GAPDH were acquired from Santa Cruz Biotechnology . The secondary antibody goat-anti-rabbit was obtained from Thermo Scientific .All experiments were approved by the Committee for Ethics in Animal Experimentation of the State University of Campinas (CEUA/IB/UNICAMP – 2507-1).mC*mU*mU*mU*C*C*T*C*C*T*C*T*G*T*mU*mU*mC*mC and Mismatch: mC*mU*mU*mU*C*T*A*C*C*T*C*A*G*T*mU*mU*mC*mC.For experiments involving acute inhibition of the ARHGAP21: C57BL/6, aged 60 days from the State University of Campinas animal facilities were used in the experiments. Mice were maintained in appropriate cages  on a 12 h light-dark cycle at 20–21°C with controlled humidity, with access to food and water ad libitum. They received daily intraperitoneally (i.p.) injections of 0.15 nmol/g of anti-ARHGAP21 oligonucleotide (AS) or Mismatch (CTL) in Tris-EDTA (Veichel) buffer for 3 consecutive days, and all experiments were performed 24 h after the last injection. Oligonucleotides utilized in this study: Anti-ARHGAP21: ad libitum. Mice were randomly assigned to a sedentary control group (CTL), which limited to typical movement inside the cages, and the endurance-training group (T). As previously described  and lean males C57BL/6J of matched age were acquired from the State University of Campinas, the mice were killed and the islets were isolated.For experiments involving hyper insulin-secreting mice: 60 days old male B6.V-Lep2 gas exposure and the pancreas was inflated with a Hanks buffer  containing 0.8 mg/ml of collagenase. After removal, the inflated pancreas was incubated for 17 min at 37°C. After the incubation, the solution was shaken gently to complete dissociation of the islets and washed with cold Hanks to stop the enzyme activity. To confirm the inhibition of ARHGAP21, pancreatic islets were lysed in an urea anti-protease/anti-phosphatase buffer , and ARHGAP21 expression was evaluated by western blotting. For GSIS analysis, the islets were collected one-by-one and pre-incubated in Krebs bicarbonate buffer  supplemented with 5.6 mM glucose and 0.2 g/L BSA for 1 h at 37°C, and were, subsequently, incubated in a similar buffer with 2.8, 5.6, 8.3, 11.1, 16.8, or 22.2 mM of glucose or 30 mM of KCl for 1 h at 37°C. After incubation, the supernatant fraction was collected, and the secreted insulin was measured by Radioimmunoassay (RIA). For total insulin content, after the incubation period, the islets were collected and transferred to 1.5 ml tubes. Deionized water (1 ml) was then added to the samples, followed by disruption of the pancreatic cells using a Polytron PT 1200 C homogenizer . The total insulin content was also measured by RIA. The insulin secretion was normalized by the total insulin content.Mice were killed by CO10 hours fasting mice received an i.p. glucose solution at a concentration of 1 g/kg in 0.9% w/v of NaCl solution. The blood glucose was measured at baseline  and after 15 and 60 min of the injection. Glucose was evaluated with glucose strips on an Accu-Chek Performa II instrument (Roche). Blood samples (100 µl) were collected from the tail immediately before the injection (0 min), and at 15 and 60 min following the injection to determine the concentrations of insulin that were measured by RIA.Fasted mice (8 h) were anesthetized  and received a catheter in the right carotid. After surgery, mice received an i.p. insulin-bolus injection (400 mU/Kg). During the test, it was infused continuously 30 mU/Kg/min of insulin and 5% glucose solution. This infusion was regulated until the mouse maintains normoglycemia for 30 min. Glucose was evaluated every 5 min with glucose strips on an Accu-Chek Performa II instrument (Roche). The glucose infusion rate was determinate by calculation 1000/bodyweight x infused glucose.2+-sensitive dye Fura-2 acetoxymethylesther (AM). Afterward, single islets were placed inside a thermostatically-regulated chamber (37°C) over poly-L-lysine-treated glass coverslips and perfused with a BSA-free KRB buffer containing 2.8 or 22.2 mM glucose or 30 mM KCl. Fura-2AM loaded islets were imaged using an inverted epifluorescence microscope . A ratio image was acquired every 3 s with a Cool One camera  using a dual filter wheel equipped with 340, 380, and 10 nm bandpass filters, and a range of neutral density filters . Data were acquired using the Image Master Version 5.0 software .Islets were incubated in KRB buffer containing 5.6 mM glucose at 37°C for 2 h. During the last hour of incubation, islets were loaded with 5 µM of the CaMus musculus genome (GenBank). Relative quantification was performed using the Step-one real-time PCR system (Applied Biosystems). The relative quantities of the target transcripts were calculated from duplicate samples (2ΔΔCT), the data were normalized against the endogenous control GAPDH. The studied genes were as follows: NKX6.1, PDX1, MAFA, HFN4α, INS1, INS2, CX 36, GLUT2, GCK, PGC1α, SYT VII, VAMP2, SYNTAXIN 1A, SNAP25, CYCLIN D2, and CDK4. The primer sequences are available in the Supplementary.Pancreatic islets mRNA was extracted by RNeasy kit , then 200 ng of purified mRNA was used to synthesize the cDNA . The primers were designed and tested against the Protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as standard. Fifty (50) μg of the lysate was boiled in SDS loading buffer, applied on 10% SDS-PAGE and transferred to nitrocellulose membranes, subsequently blocked in TBS buffer  containing 5% BSA powder for 1 h at room temperature. Membranes were then incubated with primary antibodies at 4°C, overnight, and secondary antibodies for 1 h at room temperature. The intensities of the protein bands were detected using a LAS-3000 CCD camera, and quantification was performed using densitometry . The densitometry values of target protein were normalized by GAPDH bands intensities. The antibodies used in this article can be found in Supplementary Material.The data are expressed as the mean ± SEM. Statistical analyses were performed using Student’s t-test and ANOVA two-away when necessary. P ≤0.05 was considered statistically significant.ob/JUnib), that secrete high amount of insulin  of islet ARHGAP21 expression, compared to CTL mice islet . We observed higher insulin secretion in islets from the AS group at 16.8 and 22.2 mM glucose, compared with CTL    and ARHG mM  and To confirm this observation, we investigate the ARHGAP21 expression in islets of animal models known to display increased - Ob/Ob mice;  - or decTo further explore the ARHGAP21 role in GSIS, we generated mice with 50% reduction of islet ARHGAP21 (AS) expression  and we observed that the insulin secretion was similar between AS and CTL islets  and glucose-induced intracellular Ca protein –18, was  protein , was alsAn increase in the transcript level of CX36 in islet from AS-treated mice, compared to CTL, was also observed , 21. Of Reduction in PGC1α gene expression with reduction in insulin secretion and development of type 2 diabetes was already noticed , 24. TheIn our previous studies, we observed a possible relationship between ARHGAP21 and PGC1α pathway genes. ARHGAP21 heterozygotes mice presented an increased expression of UCP1 in BAT (Brown Adipose Tissue) , and incIn conclusion, ARHGAP21 seems to be an important repressor of glucose-stimulated insulin secretion in islet from adult mice through modulation of gene expression such as SYTVII, SNAP25, CX36, and PGC1α. These effects are in line with the reduction (Ob/Ob mice) or augment (trained-mice) in ARHGAP21 expression, concomitantly with increase and decrease in GSIS in these models .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.


Many tumor cell types overexpress integrins, a glycoprotein, on their cell membranes. The tripeptide motif Arg-Gly-Asp (RGD) is well-known for being recognized by the integrin superfamily members and can thus be used to actively target nanoparticles containing cytotoxic drugs directly to the tumor cells. According to this strategy, the antitumor activity is boosted, and healthy organs are spared. In this paper, silk fibroin, a naturally derived protein, has been used to prepare nanoparticles (SFNs) functionalized on their surface with RGD. In vitro experiments revealed that functionalization of SFNs with RGD provided active internalization by tumor cells overexpressing integrin receptors. Therefore, RGD-SFNs may be used for tumor-specific delivery of anticancer drugs.Arg-Gly-Asp (RGD)-based cyclopentapeptides (cRGDs) have a high affinity towards integrin αvβ3 and αvβ5, which are overexpressed by many tumor cells. Here, curcumin-loaded silk fibroin nanoparticles (SFNs) have been functionalized on the surface with cRGD to provide active targeting towards tumor cells; a “click reaction” between the RGD-based cyclopentapeptide carrying an azide group and triple-bond-functionalized nanoparticles has been exploited. Both naked and functionalized SFNs were less than 200 nm in diameter and showed a round-shaped morphology but, after functionalization, SFNs increased in size and protein molecular weight. The functionalization of SFNs’ surfaces with cRGD provided active internalization by cells overexpressing integrin receptors. At the lowest concentration tested (0.01 mg/mL), functionalized SFNs showed more effective uptake with respect to the naked by tumor cells that overexpress integrin receptors (but not for non-overexpressing ones). In contrast, at higher concentrations, the non-specific cell membrane protein–particle interactions are promoted and coupled to specific and target mediated uptake. Visual observations by fluorescence microscopy suggested that SFNs bind to integrin receptors on the cell surface and are then internalized by endocytosis. Overall, SFN functionalization provided in vitro active targeting for site-specific delivery of anticancer drugs, boosting activity and sparing healthy organs. Cancer is one of the major disease burdens worldwide. Among the primary cancer treatment modalities, chemotherapy has been widely performed in the clinic because of its convenient and straightforward process  and stilAmong current drug delivery systems, nanoparticles have shown great potential in recent years ,5,6. SilInterestingly, the nanoparticle accumulation into the tumor can be increased by adopting active targeting strategies. An actively targeted nanoparticle is usually obtained by adding ligands’ surfaces for recognition by specific receptors/antigens on target cells . In thisThe coating of nanoparticles with integrin ligands has been explored to guide cytotoxic reagents directly to the cancer cell surface ,18. In tGiven these premises, this study aims to fabricate curcumin-loaded SF nanoparticles (SFNs), conjugated with cRGDs, to achieve active tumor targeting towards various cancer cell lines. Curcumin is here selected as a drug model and for its anticancer effects . In the ® Ultra filters were purchased from Sigma-Aldrich . Dialysis cellulose tubes were purchased from Visking, Medicell Membranes Ltd. . Moreover, 70 μm nylon meshes were purchased from Greiner Bio-One GmbH . Natriumazide and sodium sulphate were from Carlo Erba Reagenti . Caucasian colorectal adenocarcinoma cells (Caco-2), human cervix cancer cells (HeLa) and human urinary bladder (ECV) cancer cells were purchased from the European Collection of Authenticated Cell Cultures Cell Bank . All reagents used for cell cultures were purchased from Euroclone .Acetone, Azide-fluor 545, collagenase IA, curcumin, dimethylsulfoxide (DMSO), glutaraldehyde, ethanol, lithium bromide, methanol, potassium chloride, potassium dihydrogen phosphate, sodium carbonate, sodium hydroxide, 3--2,5-diphenyl-tetrazolium bromide (MTT) and Millipore’s AmiconBombyx mori cocoons according to previously reported procedures [2CO3) for 30 min. Then, the degummed fibers were washed in distilled water (37 °C) and dried at room temperature. A 9.3-M LiBr aqueous solution was prepared and used to dissolve SF fibers at 60 °C for 4 h. The obtained solution was placed into dialysis cellulose tubes  and dialyzed against distilled water at room temperature for 72 h. The SF final concentration was 8.16% w/v and was determined by freeze-drying known SF volumes  at −50 °C, 8 × 10−1 mbar for 72 h.SF was extracted by ocedures ,28. Briew/v and then added dropwise to the acetone, where curcumin was previously solubilized at a concentration of 0.08 mg/mL. SFN suspension was dialyzed against distilled water at room temperature for 72 h using dialysis cellulose tubes (12 kDa MWCO), freeze-dried at 8 × 10−1 mbar and −50 °C and stored at room temperature until use (maximum 2 months).SF nanoparticles (SFNs) were prepared by a desolvation method according to the procedures previously reported by our group ,9. SF aqBefore functionalizing SFNs with RDG, we performed bioconjugation with a fluorescent compound named Azide-fluor 545 (Molecular Weight 631.70 g/mol) . Azide-fg for 10 min, and supernatants were analyzed. A calibration curve (correlation coefficient R2 > 0.9899) was obtained to measure the Azide-fluor concentration; we consider the Azide-fluor concentration range of 0–10 µg/mL (deionized water was considered as control solution). Each measurement was performed in triplicate.The quantification of Azide-fluor conjugated on SFNs was evaluated with spectrophotometric analysis  at 554 nm. First, 2 mg of SFNs was suspended in 300 µL of distilled water, maintaining magnetic stirring overnight. After incubation, nanoparticles were centrifuged at 3250× 3 group. The key step, i.e., the Huisgen cycloaddition between FNPs and the azide-functionalized cRGD, was carried out in phosphate buffer in the presence of CuSO4 and sodium l-ascorbate. The final dialysis (cut-off 3–5 kDa MWCO) of the reaction mixture allowed the obtainment of pure cRGD-SFNs.The synthesis of cRGD-functionalized SFNs (cRGD-SFNs) was carried out by exploiting a “click reaction” between the azide group of the RGD-based cyclopentapeptide and a triple bond on the surface of SFNs . SFN funBoth nude and functionalized SFNs were characterized as follows.SFN production yield (Y%) was calculated as follows:v/v ethanol (0.1 mg/mL), maintaining mild magnetic stirring in the dark, and the absorbance was measured at 254 nm by a spectrometer . Ethanol was considered as blank. To extrapolate the drug content, a calibration curve was prepared using curcumin in the concentration range 0.25–10.00 μg/mL; R2 was 0.9957. The drug loading (% w/w) was calculated, dividing the total drug content  and the concentration of analyzed SFNs. All experiments were performed in triplicate.The amount of curcumin loaded into SFNs was quantified by spectrophotometric analysis. Briefly, SFNs were dissolved in 96% The encapsulation efficiency (EE%) was calculated as the percentage ratio between the drug entrapped in SFNs and the drug dissolved in the acetone solution.Particle size and size distribution of both naked and functionalized SFNs were measured by Nanoparticle Tracking Analysis . The instrument was fitted with a flow-cell top plate and a 405 nm laser. The temperature was set at 25 °C and the detection angle at 90°. Before carrying out NTA analyses, SFNs were dispersed in water, vortexed and sonicated for 15 min. Measurements were repeated 3 times for each sample, and results were analyzed with the NTA software 3.0.The polydispersity index (PDI) and zeta potential of both naked and functionalized SFNs were measured using ZetasizerNano ZS . Aqueous suspensions (1 mg/mL) of SFNs were prepared and allowed to equilibrate overnight at room temperature with gentle stirring. Then, the nanoparticle dispersions were filtered with a 0.45 μm filter and analyzed. The measurements were performed in triplicate for each sample. To define the Z-potential of the nanoparticles, 1 mM KCl was added to the aqueous suspension of 1 mg/mL.Morphology of both naked and functionalized SFNs was evaluated using SEM . Before the analysis, freeze-dried samples were gold-sputter-coated under argon.g and 4 °C using Millipore’s Amicon® Ultra Filters  with a Nominal Molecular Weight Limit (NMWL) of 10 kDa and load capacity of 500 µL. Deionized water was used as a washing solution. The collected samples were injected into the HPLC system. The instrument was an Agilent HPLC series 1100 system , equipped with a quaternary pump, an autosampler, a mobile phase online degasser, a thermostated column compartment and a diode array detector. The analytical column was a TSKgel SuperSW3000 (4.6 × 300 mm). SEC analysis was performed according to a previously reported method [2SO4 and 0.05% (w/v) NaN3 in 0.1 M phosphate buffer, pH 6.7. The elution was performed in isocratic conditions at the constant flow of 350 μL/min. The column temperature was maintained at 25 °C, and the injection volume was set at 5 μL. UV absorption was monitored at 280 nm. Calibration was performed using protein standards  at 60 °C for 4 h. The solution obtained was ultrafiltered 2 times (10 min each) at 10,000× d method . The mobards see . Analysev/v) FBS, 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericin B. MSCs fulfilled adherence to the International Society for Cellular Therapy criteria [Mesenchymal stem cells (AD-MSCs) were isolated from adipose tissue samples collected from patients undergoing abdominoplasty surgery after informed consent was obtained  and according to previously reported procedures ,31,32. Acriteria .Human fibroblasts (HF) were obtained from informed donors subjected to skin biopsy. Skin samples were digested as previously reported . Obtaine2 in DMEM-HG supplemented with 10% fetal bovine serum, non-essential amino acids (NEAA) 100×, penicillin 100 U/mL, streptomycin 100 μg/mL, amphotericin 0.25 μg/mL, glutamine 4 mM, sodium pyruvate 1 mM.Caco-2 cells were cultured at 37 °C and 5% COv/v) FBS, 1 mM glutamax, 1% v/v antibiotics (penicillin-streptomycin) and 1 mM pyruvate at 37 °C, under a humidified atmosphere containing 5% CO2.HeLa were cultured in DMEM with a low content of glucose (1 g/L), supplemented with 10%  and cultured with their culture media as described in sample is the mean value of the measured absorbance of the tested samples, and Absctr is the mean value of the measured absorbance of cells not incubated with SFNs. All experiments were performed in triplicate.The cytocompatibility of both naked and functionalized SFNs was evaluated by the MTT test on all the cell lines reported in 2. After 24 h of incubation with culture medium  glutaraldehyde solution in PBS for 3 h at 4 °C and washed four times with PBS. Naked and functionalized nanoparticles taken up by fibroblasts ECV and Caco-2 cells were visualized under a Zeiss Axioskop 40 microscope  equipped with a 40 × 0.75 objective lens and an RT Slider camera .The experiments were repeated on Caco-2 and ECV cells at 60, 180 and 360 min. These two cell lines display opposite integrin subunit expression patterns: Caco-2 cells express very low integrin subunit mRNA levels, particularly β3, while in ECV, integrin mRNA levels appear to be higher, and β3 is very abundant see . TherefoThe microscopy fluorescence images acquired were firstly pre-processed using ImageJ  and thenp < 0.05. Unless otherwise specified, data are expressed as mean ± standard deviation.Raw data were processed by STATGRAPHICS XVII . For data with a normal distribution, a linear generalized analysis of variance model (ANOVA) was used and combined with Fisher’s least significant difference (LSD) procedure to evaluate the differences between the groups. For all the analyses, the statistical significance was set at w/w), with an encapsulation efficiency of 83.58%. These results agree with our previous works [w/w. The alkyne linker, consequently the Azide-fluor, selectively reacted and attached the Lysine residues on SFNs’ surfaces with a 1:1 stoichiometric ratio reaction. Starting from this assumption, we considered the molecular weight of Azide-fluor (631.70 g/mol) to obtain the bioconjugation data expressed as 3.64 nmol of reacted Lysine for mg of SFNs.SFNs were prepared by a desolvation method. The addition of acetone as a desolvating agent reduces fibroin chains’ hydration until fibroin protein precipitates in the form of nanoparticles . After fus works ,38, thusp < 0.05). Naked SFNs showed a mean diameter of 128.9 ± 2.4 nm, and the d10, d50 and d90 were 84.9 ± 0.8, 109.8 ± 0.9 and 201.5 ± 12.6 nm, respectively . For cRGD-functionalized SFNs, the mean diameter was 142.7 ± 2.7 and the d10, d50 and d90 were 96.7 ± 1.8, 129.1 ± 2.8 and 205.0 ± 3.3 nm, respectively   A,B. Part, n = 3) C. LargerAnalytical size exclusion chromatography (SEC) was selected to compare the elution profiles of naked and functionalized SFNs due to its ability to elute analytes in order of their molecular size. The SEC profile of solubilized naked SFNs A revealsp > 0.05, see Drug release studies have been performed on both naked and functionalized SFNs, as reported in the Before starting treatments with naked and functionalized nanoparticles, the cells used in this study were tested for RGD-binding integrin mRNA expression by real-time qRT-PCR, as described in the p < 0.05), and especially on the Caco-2 cell line, where the cell metabolic activity was reduced to 27.9 ± 6.594 already at the lowest concentration considered (0.01 mg/mL). No dose-dependent effect was observed (p = 0.06). It is not clear what caused the apparent decrease in the viability for functionalized nanoparticles at concentrations above 0.01 mg/mL, but since there is no dose-dependent continuous decrease, it is hard to say that the nanoparticles caused severe cytotoxicity [toxicity .p < 0.001); at the high concentrations tested , no significant differences were observed among the uptake of functionalized or naked SFNs by all the cell lines considered. However, at the lowest concentration tested (0.01 mg/mL), functionalized SFNs were more effectively taken up by ECV cells, which overexpress integrin receptors  was further confirmed by microscopy (exploiting the fluorescence of fibroin and curcumin) using Caco-2 (which do not overexpress integrin receptors) and ECV (which overexpress integrin receptors). For Caco-2 cells, no different behavior was revealed among naked and functionalized SFNs at 1 and 3 h. Only after 6 h of incubation, a slight internalization of functionalized SFNs is detectable as an increase in fluorescence in the vicinity of the cell membrane (p < 0.001). For Caco-2 cells, fluorescence intensity was not significantly influenced by the functionalization (p > 0.05); for ECV cells, the treatment time was significant (p = 0.0495), and an increasing trend was detectable. These results confirm what was observed visually and support the supposed uptake mechanism.To further confirm what was observed, the images acquired were processed to extract the average intensity values of fluorescence. ECV cells fluoresce significantly more than Caco-2 cells (lization A. For EClization B. For CaCurcumin-loaded SFNs were prepared by a desolvation method and then functionalized with cRGD to provide active targeting towards tumor cells that overexpress integrin receptors. A “click reaction” was exploited between the RGD-based cyclopentapeptide carrying an azide group and triple-bond-functionalized nanoparticles. SFNs showed round-shaped morphology, and the mean diameter was 128.9 ± 2.4 and 142.7 ± 2.7 for naked and functionalized SFNs, respectively. After functionalization, a higher-MW protein population was observed by comparing the SEC elution profiles, suggesting proper functionalization. The functionalization of SFNs’ surfaces with cRGD provided active internalization by cells overexpressing integrin receptors, such as ECV. In detail, a marked uptake for functionalized SFNs was observed for ECV cells already after 1 h. In contrast, for Caco-2 cells, which do not overexpress integrin receptors, no differences were revealed between naked and functionalized SFN uptake at 1 and 3 h. Only after 6 h of incubation, a slight internalization of functionalized SFNs is detectable as an increase in fluorescence in the vicinity of the cell membrane. Overall, SFN functionalization provided in vitro active targeting for site-specific delivery of anticancer drugs.