Cardiac adipose tissue may have local paracrine effects on epicardial arteries and the underlying myocardium, promoting calcification and affecting myocardial microcirculation. We explored whether the total amount of cardiac adipose tissue was associated with coronary artery calcium score (CAC) and myocardial flow reserve in persons with type 1 or type 2 diabetes and healthy controls.82Rb positron emission tomography/computed tomography (PET/CT) scanning. Cardiac adipose tissue volume , CAC, and myocardial flow reserve  were evaluated using semiautomatic software. We applied linear regression to assess the association between cardiac adipose tissue, CAC and myocardial flow reserve.We studied three groups: (1) 30 controls, (2) 60 persons with type 1 diabetes and (3) 60 persons with type 2 diabetes. The three groups were matched for sex and age. The three groups derived from retrospective analysis of two clinical studies. All underwent cardiac Mean (SD) cardiac adipose tissue volume was 99 (61) mL in the control group, 106 (78) mL in the type 1 diabetes group and 228 (97) mL in the type 2 diabetes group. Cardiac adipose tissue was positively associated with body mass index in all three groups (p ≤ 0.02). In the controls, cardiac adipose tissue was positively associated with CAC score (p = 0.008) and negatively associated with myocardial flow reserve (p = 0.005). However, cardiac adipose tissue was not associated with CAC or myocardial flow reserve in the groups including persons with type 1 or type 2 diabetes (p ≥ 0.50).In contrast to what was found in healthy controls, we could not establish a relation between cardiac adipose tissue and coronary calcification or myocardial microvascular function in person with type 1 or type 2 diabetes. The role of cardiac adipose tissue in cardiovascular disease in diabetes remains unclear. Cardiac adipose tissue is a highly metabolic active fat depot surrounding the heart and coronary arteries. It includes epi- and pericardial adipose tissue. There has been considerable interest in the cardiac adipose tissue as a risk marker . High leLittle is known about the possible mechanisms that could link cardiac adipose tissue to an increased risk of cardiovascular disease. It has been demonstrated that adipose tissue is an active endocrine tissue, implied in the production of numerous pro-inflammatory mediators . Thus, aEpicardial adipose tissue embeds the coronary arteries and autonomic nerve fibers, and shares microcirculation with the underlying myocardium. We were interested in whether the association between cardiac adipose tissue and cardiovascular disease might be mediated by micro- or macrovascular changes or autonomic nerve damage. Due to the proximity of the structures, cardiac adipose tissue could through paracrine signalling promote calcification or impair the myocardial microcirculatory function , 10. KnoThe aim of this retrospective analysis was to explore whether the total amount of cardiac adipose tissue  was associated with coronary artery calcium score (CAC), myocardial flow reserve and/or cardiac sympathetic nerve integrity in persons with type 1 or type 2 diabetes and healthy controls.The present study is a retrospective analysis of two clinical studies performed at a single center: A study from 2013 in 60 persons with type 2 diabetes and 30 controls ; and a sMain exclusion criteria were renal failure and contraindications for cardiac positron emission tomography (PET)/computed tomography (CT). Cardiovascular disease [history of coronary artery disease or other cardiovascular disease (including stroke) or heart symptoms] was an exclusion criterion for the type 2 diabetes group and the controls, but not for the type 1 diabetes group. None of the participants had symptoms of angina at time of inclusion. History of cardiovascular disease was assessed from patient electronic health records and information on symptoms from the heart was obtained from thorough interviews with all participants using the WHO Rose questionnaire.Cardiac adipose tissue was measured using the cardiac software, Syngo.via Frontier—Cardiac risk assessment , which uses non-contrast CT data to automatically quantify the adipose tissue volume located on the outside of the myocardium by tracing myocardial and adipose tissue borders based on the specific density of fat (− 150 to − 50 Hounsfield units). The software does not distinguish epi- and pericardial fat thus total cardiac adipose tissue above the entire heart was measured. Two separate investigators examined and verified the accuracy of the automatically generated adipose tissue tracings.82Rb . Cardiac PET/CT was performed at rest and during stress . Myocardial blood flow was calculated as the ratio between maximal induced and resting myocardial blood flow, as previously described in detail [All participants underwent a dynamic, gated cardiac PET/CT on a hybrid PET/CT scanner in 3D mode  after administration of 1100 MBq n detail . The myon detail . Myocard123I to allow imaging. Cardiac 123I-MIBG uptake was semiquantified using images taken 240 min after intravenously tracer injection (200 MBq 123I-MIBG). We calculated the heart to mediastinum ratio, by drawing regions of interest following the epicardial contour and the upper mediastinum (avoiding the thyroid gland) in the planar anterior view [We performed cardiac radionuclide imaging using the nonmetabolized norepinephrine analogue metaiodobenzylguanidine (MIBG). Sympathetic nerve terminals actively take up MIBG, and by quantifying the cardiac uptake, it is possible to directly assess cardiac sympathetic nerve integrity. MIBG is radioactively labeled with 123I-MIBG scintigraphy was performed in all 60 participants with type 1 diabetes, in 29 of the participants with type 2 diabetes, and in 14 of the controls [As previously described, cardiac controls , 21.1c and lipids were measured by standard methods. The CKD-EPI equation was used to calculate eGFR. In the type 1 diabetes group, urine samples were taken three consecutive mornings and urinary albumin creatinine rate (UACR) was measured by an enzyme immunoassay. For the persons with type 2 diabetes and the controls, two 24-h urine collections were taken and urinary albumin excretion rate (UAER) was measured by an enzyme immunoassay. The 24-h blood pressure was recorded using a cuff-device  in the type 1 diabetes group and a tonometric wrist-device  in the type 2 diabetes group and in the controls.HbANormal distributed continuous variables are provided as mean and standard deviation (SD), non-normal distributed  as median with interquartile range (IQR) and categorical variables as total numbers and percent. UACR, UAER and CAC were log2 transformed in all analyses.2 test or Fisher’s exact test, as appropriate, when comparing categorical variables.Independent samples t-test was applied to compare continuous variables between participants with cardiac adipose tissue below or above the median, and the χLinear regression analysis was used to determine whether any association existed between total cardiac adipose tissue and clinical characteristics or measures of cardiac function. The three groups were analyzed separately.A two-tailed p < 0.05 was interpreted as significant. Missing data was not replaced.The characteristics of the controls, and the participants with type 1 and 2 diabetes are shown for all within the groups and according to median cardiac adipose tissue level in the three groups in Table Reversible ischemia was observed on the cardiac PET/CT in 1 control person, 10 participants with type 1 diabetes (7 of whom were known with coronary artery disease) and 11 participants with type 2 diabetes. Five participants with type 1 diabetes and three with type 2 diabetes had irreversible ischemia (fixed perfusions defects) which was not found in any control persons.Mean (SD) cardiac adipose tissue level was 99 (61) mL in the control group; 106 (78) mL in the persons with type 1 diabetes and 228 (97) mL in the persons with type 2 diabetes. The cardiac adipose tissue level was comparable in controls and persons with type 1 diabetes, however significantly higher in persons with type 2 diabetes compared to controls (p < 0.0001) and type 1 diabetes (p < 0.0001). These differences remained significant after adjustment for age, sex, BMI and UACR/UAER (p ≤ 0.02).We evaluated the associations between cardiac adipose tissue and clinical characteristics separately in the three groups Table . Cardiac123I-MIBG uptake, separately in the three groups (Table 123I-MIBG uptake in the three groups (p ≥ 0.08).Furthermore, we evaluated the associations between cardiac adipose tissue and myocardial flow reserve, left ventricular ejection fraction, CAC score and The main finding of our study was that total cardiac adipose tissue was not associated with advanced measures of cardiac micro- and macrovascular function including CAC score, myocardial flow reserve, and cardiac sympathetic integrity in persons with type 1 or type 2 diabetes. In contrast we found that cardiac adipose tissue was positively associated with CAC score and negatively associated with myocardial flow reserve in the controls.The hypothesis that cardiac adipose tissue influences the development of coronary atherosclerosis through endocrine and paracrine activities has provoked considerable interest in the association between cardiac adipose tissue and coronary atherosclerosis. Large studies including the Framingham Heart Study have reported distinct associations between perivascular fat and CAC score in the general population . We exte82Rb cardiac PET/CT it is possible to measure myocardial flow reserve, reflecting both the function of the large epicardial arteries and the microcirculation. We report an association between cardiac adipose tissue and myocardial flow reserve in non-diabetic controls, but not in persons with type 1 or type 2 diabetes. The few published reports on myocardial flow reserve and cardiac adipose tissue are in line with our results with divergent findings in non-diabetes and diabetes cohorts. A retrospective study, including 46 persons with and 153 persons without diabetes all evaluated for coronary artery disease, observed an association between myocardial flow reserve and epicardial adipose tissue only in the group of persons without diabetes [82Rb PET/CT imaging for clinical indications, retrospective analysis revealed an association between higher epicardial adipose tissue and presence of impaired myocardial flow reserve  in the total cohort [Due to the proximity of cardiac adipose tissue and the epicardial arteries and underlying myocardium, it is possible that cardiac adipose tissue has local effects on myocardial microcirculation and thereby influences the development of myocardial ischemia. Major advances in non-invasive imaging enable the investigation of new aspects of the microcirculation and with diabetes . In a mil cohort .Why do we find associations between cardiac adipose tissue and CAC and myocardial flow reserve in the controls and not in the persons with diabetes? We speculate that (1) pathophysiological processes promoting coronary atherosclerosis and microvascular dysfunction other than paracrine effects of cardiac adipose tissue are dominant in diabetes; or (2) medical treatment may counteract the adverse effects of the cardiac adipose tissue. Differences in medication between controls and persons with diabetes could then explain the divergent findings.We report no relationship between cardiac adipose tissue and cardiac sympathetic nerve integrity. To the best of our knowledge, this has not been investigated in persons with diabetes before.How might our findings impact on clinical practice? Our findings indicate that total cardiac adipose tissue is not a parameter for use in the clinical setting as a marker of microvascular  or macrovascular (CAC) cardiovascular disease in persons with diabetes.As the epicardial adipose tissue is a transducer of the harmful effects of systemic inflammation and metabolic disorders on the heart it may represent an important treatment target , 23. A r123I-MIBG scintigraphy was used for direct assessment of cardiac sympathetic integrity. However, we acknowledge that our study has limitations. The limited size of our study population was only powered to detect stronger correlations and accordingly we may have overlooked smaller correlations. Nevertheless, as the control population was smaller than the diabetic populations an even lower power was present for the controls where we did find the anticipated associations. Also, our sample size is large compared to clinical studies applying similar advanced imaging modalities in persons with diabetes [We applied robust methodology: CT acquisition and assessment followed standard methods and cardiac adipose tissue was automatically quantified using the cardiac software, Syngo.via Frontier , 27. Thediabetes , 12. Estdiabetes . FinallyIn contrast to what was found in healthy controls, we could not establish a relation between cardiac adipose tissue and coronary calcification or myocardial microvascular function in person with type 1 or type 2 diabetes. The role of cardiac adipose tissue in cardiovascular disease in diabetes remains unclear.


Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. Streptomycete bacteria are prolific producers of specialized metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalization or sub-functionalization. To test whether expanded gene families in  RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialized metabolite gene clusters  was also influenced by carbon source.Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters. Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial  Streptomyces. As a result, homologous functions often combined into a single flux pathway that may not reflect the physiological nature of each gene product. This is especially stark when attempting to understand the supply of precursor molecules from central metabolism to the production of specialised metabolites.Streptomycete bacteria are major source of clinically useful bioactive natural products including antibiotics, immunosuppressive and anti-cancer agents. A remarkable feature of their genomes is that there are often several genes that appear to encode the same biochemical function . This isStreptomyces and their impact on central metabolism and specialised metabolism, an RNA-Seq approach was taken using S. coelicolor grown either on glucose or tween as sole carbon source. Cultures were compared at a single point during growth (mid-log phase) to understand how transcription of central metabolic genes varies when the cultures are growing primarily via glycolysis (glucose as the sole carbon source) or gluconeogenically (with the mono-oleate tween). It was found that expanded gene families exhibit different responses to growth on different carbon sources. These data will enable prioritization of targets for metabolic engineering and will be informative for the construction of metabolic models.It was hypothesized that gene families that have undergone gene-expansion events would exhibit different expression profiles if they have diverged functionally. To understand the role of these gene expansions in S. coelicolor M145 [an A3(2) strain] was grown respectively on glucose and Tween40 as sole carbon source. Growth of liquid cultures was monitored by cell dry weight and samples were removed for total RNA extraction at mid-exponential phase, which for glucose was 19 h and for tween was 36 h . None of the pigmented antibiotics (actinorhodin or undecylprodigiosin) could be detected at time points RNA was harvested. The specific growth rate of S. coelicolor M145 was 0.18 h−1 when grown on glucose and 0.1 h−1 for growth on tween .To investigate the differences between glycolytic and gluconeogenic growth conditions, RNA samples from these cultures showed global effects on transcription, when analysed by RNA-Seq, when the strain was grown on different carbon sources. Growth on tween resulted in 644 genes being differentially expressed when compared to growth on glucose. Of the 644 differentially expressed genes, 37 % were predicted to encode hypothetical proteins, 9 % encoded regulatory genes, 12 % encoded central carbon metabolic enzymes, 6 % were transporters, 4 % were associated with specialized metabolism, 2 % encoded genes associated with stress metabolism and 1 % of genes were associated with DNA replication, nitrogen metabolism and genes associated with metal metabolism (see Tables S1–6). Growth on Tween40 resulted in an upregulation of genes associated with gluconeogenesis and fatty acid degradation when compared to cultures grown on glucose , pyk1 (SCO2014) and pyk2 (SCO5423), one of the three GAPDH (SCO7511) and a single copy of the pyruvate dehydrogenase E1 complex (SCO2183), it was noted that all genes belonged in the oxidoreductase gene-ontology grouping.Gene-ontology (GO) enrichment analysis (using PANTER logy.org ), was usGO enrichment analysis for genes upregulated during growth on Tween were found to be those associated with fatty acid catabolism, gluconeogenesis  and another of the three GAPDH homologues (SCO7040).To examine transcription of expanded gene families, two categories of expression profiles were considered to reflect changes across the gene family that are predicted to encode the same enzymatic function: type 1, where all genes within an expanded gene family behaved similarly under the growth conditions, and type II where members of an expanded gene family exhibit differential gene expression, i.e. one or more member of the family increased, while expression of other members of the gene family, increased to a different degree, decreased or remained the same (glk (SCO2126) remained unchanged under both conditions. GAPDH also showed type II expression profiles, with expression of SCO7040 increased on tween, whereas the other two copies of the genes encoding GAPDH had reduced expression (SCO1947 and SCO7511). A previous proteomic-based study also identified an increase in abundance of the SCO7040 protein when grown on a non-glucose carbon source , whereas expression of the primary glk SCO126 remaippdk1 (SCO0208) upregulated 11-fold up and ppdk2 (SCO2494) upregulated 30-fold (ppdk2). This suggests that, under these conditions, S. coelicolor may be using unconventional gluconeogenic routes for anaplerotic reactions rather than via the glyoxylate shunt, as expression of ICL and both MS enzymes remain unchanged during growth on either carbon source.Overall, genes involved in gluconeogenesis were upregulated when cultures were grown on tween compared to glucose – as would be expected. The only expanded gene family encoding gluconeogenic function is that encoding the two PPDKs, which both displayed substantially increased expression (type I expression), with zwf, with expression of SCO6661 reduced when cells were grown on tween, whereas expression of SCO1937 was unchanged between the two conditions. This was consistent with the work of Gubbens et al. [S. coelicolor [Expression of the pentose phosphate pathway showed little differential expression under the two conditions studied. Type II expression profiles were observed for elicolor  these daS. coelicolor of genes encoding the PHDCE1 subunit exhibited a range of expression changes – expression profiles for SCO1269, SCO7124, SCO2371 and SCO3816/3817 were the same for both carbon sources, whereas SCO2183 had a 3.5-fold decrease and SCO1270 a sixfold increase on tween.In general, expression of genes encoding enzymes of the tricarboxylic acid (TCA) cycle remained stable under both growth conditions as would be expected, given this core part of metabolism is required for biosynthesis under all physiological conditions. The exception was expanded gene families, which exhibited differential expression (type II): expression of the fumarase (SCO5042) was reduced twofold on tween whilst SCO5044 expression remained unchanged; one copy of the malic enzyme (SCO2951) showed a twofold decrease in expression, compared to SCO5261, a second malic enzyme, which remained unchanged under both conditions; the extensive expansion in S. coelicolor was grown on tween, especially cholesterol esterase (SCO5420), which had an increase of almost 37-fold. The enzyme catalyses the hydrolysis of the head group of tween from the fatty acid palmitate, which are then utilized as carbon source [As expected fatty acid utilization genes were expressed more when n source . The othpyk1 (SCO2014) and pyk2 (SCO5423) from glycolysis, ppdk1 and ppdk2 from gluconeogenesis with primary sigma factor hrdB (SCO5820) as control. These two pairs of genes were chosen as representative of expanded families, identified in a previous study [hrdB  showed no difference under the two conditions tested. The fold-change difference in the qPCR data was similar to that observed in RNA-Seq experiments, corroborating the wider results .To verify the RNA-Seq data, we performed qPCR on the RNA samples used for the RNA-Seq experiment using primer pairs for five different genes – pyk1 SCO014 and p– 2.2-fold, SCO5250 – 4.4-fold) when grown on glucose (Table S2). Given the only difference between the cultures was the carbon source, we hypothesize that cultures experienced oxidative stress. This is in part supported by the GO enrichment analysis where 51 genes with GO terms matching ‘oxidoreductases’ exhibiting increased expression on Tween as the sole carbon source, with a sevenfold increase in expression of a superoxide mutase (SCO0999) and an eightfold increase of expression of a putative bacterioferritin co-migratory protein . Caution should be exercised however as many genes in primary metabolism also share GOs with ‘oxidoreductases’.Genes involved in isorenieratene biosynthesis (SCO0185-0191), showed an increase in transcription in cultures grown on tween, with a three to eightfold increase in expression across the entire operon. Isorenieratene is associated with blue-light exposure, with the pathway present in green photosynthetic bacteria and a few actinobacteria . IsoreniA non-ribosomal peptide synthetase (NRPS) pathway (SCO6429-6438), the siderophore coelichelin biosynthetic gene cluster (SCO0491-0498) and the actinorhodin cluster (SCO5071-5092) all had decreased expression when grown on tween compared to glucose by two to fivefold, two to threefold and three to 11-fold, respectively  increasing expression on tween when the other two copies show decreased expression under the same conditions. This knowledge will help inform choices and enable prioritization of metabolic engineering targets for industrial streptomycetes and the supporting dataset will also inform researchers on the role of hypothetical proteins and regulators, during growth on two different carbon sources that drive metabolism along two different pathways.Studies such as this can inform metabolic modelling approaches for strain improvement enabling expression data and metabolic flux analysis to be taken into account with respect to isoenzymes within expanded gene families rather than combining the activities of all copies of a gene family into a single flux . The ideS. coelicolor A3(2) M145 [°C and 250 r.p.m. until emerging germ tubes were visible under a microscope [(2) M145  was usedcroscope  in 2 l fS. coelicolor biomass (15 ml sample) from liquid cultures was harvested by centrifugation . Cell pellets were resuspended in an equal volume of RNAprotect (Qiagen) for 5 min at room temperature. Following centrifugation  biomass was then resuspended in 1 ml 1× TE buffer containing 15 mg ml−1 lysozyme. Tubes were vortexed for 10 s and incubated at room temperature for 60 min whilst shaking. Then, 1 ml RLT buffer (Qiagen RNA Isolation Kit)+10 µl β-mercaptoethanol was added and vortexed and a phenol chloroform extraction followed by an ethanol precipitation was carried out. The sample was then purified using a commercial RNA isolation Kit (Qiagen). The isolated RNA was treated with RNAse free DNase  as specified by the manufacturer.hrdB (SCO5820).Quantification of RNA was carried out using Qubit (Life Technologies). The quality and integrity of the RNA was assessed using a Bioanalyzer (Agilent). Furthermore, the RNA samples were also used as templates for a generic PCR in order to check for DNA contamination using primers for To enrich the samples for mRNA, rRNA depletion was performed  according to the manufacturer’s instructions. The rRNA-depleted samples were then precipitated with ethanol and resuspended according to the manufacturer’s instructions. The quality and integrity of the samples were then analysed on the BioAnalizer (Agilent) and the concentration was determined using Qubit (Life Technologies).cDNA synthesis and library preparation was carried out using the Ion Total RNA-Seq Kit v2 Revision E from Ion Torrent, Life Technologies. The manufacturer’s protocol for less than 100 ng rRNA depleted samples was followed. The yield and size distribution of the amplified cDNA was assessed using BioAnalizer (Agilent). The three samples harvested from glucose cultures and three samples from tween grown cultures were barcoded and pooled to a concentration of 20 pM as specified by the manufacturer. The Ion OneTouch 2 system using the Ion PGM Template OT200 Kit (Life Technologies) was used for template preparation which includes the steps of emulsification, amplification and enrichment of the library. The libraries were checked using the quality control assay for the Qubit (Life Technologies).https://www.ncbi.nlm.nih.gov/sra/PRJNA566372).Sequencing of the samples was carried out using a Ion Torrent Personal Genome Machine System  on a 316v2 chip following the procedures in the manual, Ion PGM Sequencing 200 Kit v2 User Guide Revision 3.0. All sequence data were deposited on the Sequence Read Archive (SRA) under the BioProject: PRJNA566372 . The colour code was expressed relative to the highest and lowest expression change in the data shown ranging from green to red, respectively.Sequencing data were downloaded from the Ion Torrent server version Torrent Suite 4.0.2 in Fastq format. Reads were trimmed and low-quality data removed. Reads were mapped to the reference genome of licolor ; GenBankus value . The difus value . The rawhttps://www.ncbi.nlm.nih.gov/pubmed/23868073) and summarized in Table S7.Gene-ontology enrichment analysis was performed using Panther classification from gene ontology at NCBI (qPCR was performed on the same samples as the RNA-Seq in order to provide independent confirmation of the data obtained. Each primer pair was tested using genomic DNA as the template. cDNA was synthesized from the RNA samples using qPCRBIO cDNA synthesis Kit (PCR Biosystems) following the manufacturer’s instructions. Quantification of the cDNA was carried out using the QuantiFluor ssDNA system (Promega) with the Qubit Fluorometer (Life Technologies).−1 and each reaction contained 10 ng of cDNA. Samples were mixed with qPCRBio MasterMix and the respective primers . Using a Corbett Research 6000 (Qiagen) machine, PCR reactions were subjected to a three-stage thermocycling reaction  using purified PCR product from a genomic DNA PCR. This template was diluted to create seven different standards ranging from 101 to 107 molecules/per reaction  and was used to calculate the concentrations of the unknown samples obtained in the RNA sequencing.All cDNA samples were diluted to a concentration of 10 ng µl


Group 1 and Transbond™ Plus self-etching-primer adhesive for Group 2 were considered. The brackets were tested under cycling loading force of 10-N and a crosshead speed of 300 mm/min and 40 cycle/min. The frequency of strokes that the brackets failed were determined and these data were analyzed by statistical analysis using an independent sample t-test and one-way analysis of variance (ANOVA). The level of significance was set at p < 0.05. Generally, differences between the frequency of shearing strokes of the bracket failures were found to be statistically significant depending on the type of adhesives and brackets (p < 0.05). The bonding technique for Group 1 was found to have a significantly higher shear bonding strength than Group 2. It is also seen that different types of bracket belonging to the same or different brands had different shear bonding strength. It may be concluded that: (i) all bracket types used in this study can be applied with both bonding techniques, (ii) in order to minimize the risk of hard tissue damage, ceramic brackets should be carefully bonded using the self-etching primary adhesive technique.Based on the development of many adhesive systems and bonding techniques, bonding strength of orthodontic brackets has become even more important in modern clinical orthodontics. The aim of this study was to determine mean shearing stroke frequency of different orthodontic bracket types and bonding agents under cycling loading. Therefore, 10 different types of orthodontic bracket from 4 different brands were divided into 2 groups. Two different adhesives, namely Transbond™ XT etch-and-rinse for  Modern orthodontic developments have focused on lessening orthodontic treatment time and achieving the perfect and stable treatment outcomes ,2. BrackThe bonding of orthodontic attachments is one of the most important phases of the entire treatment process. The bonding technique can be applied incorrectly not only by an inexperienced clinician, but also by an experienced orthodontist who does not follow the procedures carefully . SuccessAdvances in orthodontic adhesive and bracket base manufacturing have created many options for clinicians, and some combinations have been reported to perform best ,10. It hBonding failure of orthodontic brackets is an undesired situation for orthodontists and patients, causing increased appointments, prolonged chair time and relapse in tooth movements . During Shear bonding strength tests were first applied in the late 1970s and are widely used in measuring the bonding strength of orthodontic materials . Shear fThe aim of this study was to determine mean shearing stroke frequency of different orthodontic bracket types and bonding agents under cycling loading.Ethics committee approval for the study was given by the Clinical Research Ethics Committee at Zonguldak Bulent Ecevit University (2016/05: 2016-21-27/01). Three hundred upper premolar human teeth were divided into two subgroups with 15 samples. The sample size was determined using the data from Dall’Igna et al. . A samplThe teeth were kept in distilled water with 0.1% thymol crystals to prevent dehydration and deterioration of the enamel structure after removing blood, organic residue, etc. . Teeth wThe following 10 different types of upper premolar brackets were considered in this study see :a) APC (Adhesive Pre-coated) Victory metal ,(b) Victory metal ,(c) Gemini metal ,(d) Gemini Clear ceramic ,(e) Gemini SL self-ligating metal ,(f) Clarity Advanced ceramic ,((g) Clarity SL self-ligating ceramic , h) Equilibrium® metal , (j) Mini Sprint® metal , (k) Mini Master metal .(Group 1 (etch-and-rinse), Transbond™ XT Light Cure Adhesive Primer  and Transbond™ XT Light Cure Adhesive Paste  were used to bond the brackets. In Group 2 (self-etching-primer), Transbond™ Plus Self Etching Primer  and Transbond™ XT Light Cure Adhesive Paste  were used to bond the brackets.Vestibular surfaces of the teeth were cleaned by fluorine-free pumice-water mixture using polishing rubber, rinsed and dried. According to the information provided by the manufacturer , the compositions of the adhesive agents used in the study are summarized in Group 1 (etch-and-rinse), acid-etching was applied with 37% phosphoric acid for 30 s and then rinsed with water for 30 s and air dried for 10 s. After seeing the chalky white surface, Transbond™ XT Light Cure Adhesive Primer  was applied as a thin layer on the tooth surface.For Group 2 (self-etching-primer), routine care control was completed so that the buccal surfaces of the teeth were clean. Transbond™ Plus Self Etching Primer  was applied to the tooth surface for five seconds. Consequently, air was feebly applied to the surface with an air-water syringe. Then, APC brackets were removed from their protective boxes and placed in their correct position on the tooth surface. Transbond™ XT Light Cure Adhesive Paste  was placed on the base of the remaining brackets. All brackets were placed in the precise position on the tooth surface and the flashes were removed. All brackets were cured from mesial and distal for 20 s using 3M Espe Elipar S10  which has the intensity of 430–480 nm at about 1200 mW/cm2, respectively.For Fatigue tests were carried out with a closed-loop controlled, low-cycle fatigue machine with 10 N capacity and a crosshead speed of 300 mm/min and 40 cycle/min. During the test, attention was paid to the force coming perpendicular to the bracket groove. After fixing the acrylic blocks, the test was conducted to obtain frequency of strokes according to the bracket failure see .t-test and one-way analysis of variance, post-hoc Tukey test and Tamhane’s T2 were chosen. p value less than 0.05 was considered statistically significant.Statistical analysis was performed using SPSS for Windows . For comparisons between groups, an independent sample According to the descriptive statistical analysis of the groups, the homogeneity test results of the groups are shown in The number of brackets used in the study, the mean frequency of strokes, the standard deviation, the minimum and maximum frequency of strokes are given in Group 1 and Group 2, no statistically significantly difference was found between the mean shearing stroke frequency of APC metal brackets and Victory metal brackets (p > 0.05). The shearing stroke frequency was found to be statistically significantly higher in APC metal brackets compared to Mini Sprint® and Mini Master metal brackets (p < 0.05).For both Group 2, a statistically significantly lower shearing stroke frequency was found in APC metal brackets compared to Equilibrium® 2 metal brackets (p < 0.05).For Group 1 and Group 2, even though the mean shearing strokes frequency of Clarity Advanced ceramic brackets were higher than APC metal, Victory metal, Equilibrium® 2 metal brackets, no statistically significant differences was found (p > 0.05). In spite of the mean frequency of shearing strokes of Clarity Advanced ceramic brackets were higher compared to Clarity SL self-ligating ceramic brackets, no statistically significant difference was found. The mean shearing stroke frequency of Victory series metal brackets was found to be statistically significantly higher than that of Mini Sprint® metal and Mini Master metal brackets (p < 0.05).For both Group 1, there was no statistically significant difference between Equilibrium 2 metal brackets and Victory series metal brackets in mean shearing stroke frequency (p > 0.05). The mean frequency of shearing strokes of Clarity Advanced ceramic brackets was found to be statistically significantly higher except for APC Victory, Victory and Equilibrium® 2 metal braces (p < 0.05).For Group 2, a statistically significantly higher mean frequency of shearing strokes was found in Equilibrium 2 metal brackets compared to Victory series metal brackets. Mean shearing strokes frequency was found to be significantly higher than that of Mini Master and Mini Sprint® metal brackets (p < 0.05).For Group 1 and Group 2, mean shearing stroke frequency of Gemini SL self-ligating metal brackets were found to be statistically significantly higher than Mini Master and Mini Sprint® metal brackets. Mean shearing stroke frequency of Equilibrium® 2 metal brackets was found to be statistically significantly higher than Gemini SL self-ligating metal brackets (p < 0.05). Additionally, there was no statistically significant difference between the mean frequency of shearing strokes of Clarity SL self-ligating ceramic brackets and Clarity Advanced ceramic brackets (p > 0.05).For both Group 1, mean shearing stroke frequency of Clarity SL self-ligating ceramic brackets was found to be statistically higher than Gemini SL self-ligating metal brackets (p < 0.05). However, for Group 2, there was no statistically significant difference between each other (p > 0.05).For Group 1 and Group 2, mean shearing stroke frequency of 3M Unitek and Dentaurum brand metal brackets were found to be statistically significantly higher than Forestadent and American Orthodontics brand metal brackets (p < 0.05). Moreover, there was no statistically significant difference between the shearing strokes frequency of 3M ceramic brackets (p > 0.05).For both Group 1, there were mostly no statistically significant differences between the mean shearing strokes frequency of 3M brand and Dentaurum brand metal brackets. On the other hand, for Group 2, mean shearing strokes frequency of Dentaurum brand brackets were found to be significantly higher than 3M brand metal brackets (p < 0.05).On the one hand, for Group 1, there were no statistically significant differences between mean shearing strokes frequency of Forestadent brand and American Orthodontics brand metal brackets (p > 0.05). On the other hand, for Group 2, the mean shearing strokes frequency of American Orthodontics brand metal brackets were found to be statistically higher than Forestadent brand metal brackets (p < 0.05). In both groups, Mini Sprint® metal and Mini Master metal brackets showed statistically significantly lower mean shearing stroke frequency, the standard deviation, and the lower minimum and maximum number of strokes (p < 0.05).In a similar way, on one hand, for Group 1 brackets were statistically significantly higher than that of Group 2 brackets.For Clarity Advanced ceramic brackets, samples were renewed due to breakage in the bracket wings in four samples and severe hard tissue damage (enamel + dentin) in two samples see . Most ofShearing forces are applied to orthodontic brackets mostly in shear bonding strength tests . AlthougThe factors affecting in vitro orthodontic bonding strength testing were reported as: test conditions, storage conditions of the samples which were embedded to blocks, polymerization time, and crosshead speed . HoweverIn orthodontic bracket bonding strength tests, shearing forces are applied on the bracket base, ligature groove or bracket wings. These three different locations where shearing forces are applied display significant differences between studies. Klocke at al. reported a 49.3% decrease in SBS values and 25% increase in bonding failure when the shearing force was transferred from the bracket–resin interface to the ligature groove . Thomas Group 1 and Group 2, mean shearing strokes frequency of APC series metal brackets and Victory series metal brackets were similar and no statistically significantly difference was found. On one hand, APC Victory metal brackets in Group 1 and Group 2 had a statistically significantly higher shearing stroke frequency when compared to Mini Sprint® metal and Mini Master metal brackets. On the other hand, APC metal brackets in Group 2 possessed a statistically significantly lower shearing stroke frequency than that of Equilibrium® 2 metal brackets.Bonetti et al. reported that there was no statistically significant difference in their study comparing the bonding failure mode of APC II Victory (3M Unitek) series metal brackets and Victory metal (3M Unitek) brackets . Abd andGroup 1 and Group 2 were evaluated as higher than metal brackets. In Group 1, Clarity Advanced ceramic brackets had statistically significantly higher shearing stroke frequency compared to Mini Master metal, Mini Sprint® metal, Gemini metal and Gemini SL self-ligating metal brackets. In Group 2, a statistically significantly higher shearing stroke frequency was found in Clarity Advanced ceramic brackets compared to Mini Master metal and Mini Sprint® metal brackets. Although the frequency of shearing strokes was noted to be higher than the remaining metal brackets in both Group 1 and Group 2, no statistically significantly difference was found.Mirzakouchaki et al. reported in their study that the bonding strength of metal brackets  was statistically significantly higher than ceramic brackets  . Liu et ®, 3M Unitek) was statistically significantly higher than conventional metal brackets  [Group 1 and Group 2, a statistically significantly higher shearing stroke frequency was observed in Gemini SL self-ligating metal brackets compared to Mini Master metal and Mini Sprint® metal brackets. Additionally, a statistically significantly higher shearing stroke frequency was calculated in Equilibrium® 2 metal brackets compared to Gemini SL self-ligating metal brackets.Sfondrini et al. reported in their study that the shear bonding strength of self-ligating metal brackets  . Northru; Leone) . For botGroup 1 and Group 2 was similar to that of the Clarity Advanced ceramic brackets, and no statistically significant difference was found.Theodorakopoulou et al., in their in vitro study comparing the shear bonding strength of polycrystalline  and monocrystalline  ceramic brackets, reported that the bonding strength was similar in both ceramic bracket types and no statistically significant differences were found . In thisZielinski et al. reported that there were 8 bracket breakages in different types of ceramic and plastic bracket groups investigating the shear bonding strength . Stein eJeiroudi reported enamel damage in the upper right central and lateral incisors which was broken due to an accident in the patient and treated using a polycrystalline sapphire ceramic bracket . Viazis Sheibaninia et al. reported that Equilibrium 2 metal brackets showed the statistically significantly highest bonding strength in distilled water compared to self-ligating metal brackets, while a statistically significantly decrease in bonding strength in acetic acid medium . SimilarGroup 1 and Group 2 that:(i)® metal and Mini Master metal brackets,statistically significantly higher shearing strokes frequency was found in Victory series metal brackets when compared with Mini Sprint(ii)equilibrium 2 metal brackets showed statistically significantly higher shearing stroke frequency than that of Gemini SL self-ligating metal brackets.In this study, it was clearly seen for both Group 1 were computed to be statistically significantly higher than that of self-etching brackets for Group 2.Bishara et al., in their in vitro study investigating the bonding strength using metal brackets , reported statistically significantly higher bonding strength in the acid-etching group compared to the self-etching group . Türk etFarahani et al., reported that bonding strength was statistically significantly higher in 3M brand brackets  and significantly lower in American Orthodontics brand brackets  in the control group without aluminum oxide sandblasting. There was no statistically significant difference between American Orthodontics and Dentaurum brand brackets  . Dalaie Group 2. In both Group 1 and Group 2, breakage was observed at statistically significantly lower frequency of strokes in Forestadent and American Orthodontics brand metal brackets among all brands. In addition, difference between the shearing stroke frequency of Forestadent and American Orthodontics brand metal brackets were not found to be statistically significant in Group 1, while in Group 2, frequency of shearing strokes of American Orthodontics brand metal brackets were statistically significantly higher than Forestadent brand metal brackets. There was no statistically significant difference between 3M branded ceramic brackets. In both Group 1 and Group 2, there was no statistically significant difference found between Dentaurum branded metal brackets and 3M branded ceramic brackets.In this study, the shearing strokes frequency of 3M Unitek and Dentaurum brand metal brackets were found to be statistically significantly higher than those of Forestadent and American Orthodontics brand metal brackets. Dentaurum brand brackets were found to be statistically significantly higher shearing strokes frequency than that of 3M brand metal brackets in Although it is actually obvious that this study does not totally reflect the complexity of the in vivo conditions, it presents a novel approach on evaluation of shear bonding strength of brackets with a different method in which this is tested under cyclic loading. However, another important limitation of the study is only one adhesive agent brand (3M Unitek) is used . The reaFor the sake of planning according to time span, frequency of shearing strokes plays an important role for understanding orthodontic brackets performance.As per the primary results of the study, it is suggested that all types of brackets can be bonded with both adhesive agents. However, it was underlined that in order to reduce the risk of hard tissue damage, ceramic brackets could be bonded with the self-etching primary technique carefully. In spite of same type of bonding agent being used between different brands of metal bracket, there were differences in mean shearing stroke frequency. The reason for this difference was thought to be due to different techniques and designs used in the production of brackets.® metal and Mini Master metal brackets showed lower mean shearing stroke frequency, standard deviation, and lower minimum and maximum frequency of strokes. In addition, at least one of the bracket wings of all Gemini Clear ceramic brackets was broken during the fatigue tests. However, Equilibrium® 2 metal and Victory metal brackets showed higher mean shearing stroke frequency with both bonding agents.As per the secondary results of the study, independently of the adhesive agent (etch-and-rinse or self-etching primer), Mini SprintConsidering the limitations arising from this in vitro study, the effectiveness of fatigue tests should be supported by advanced in vitro studies.


Transplantation of the neural crest cells into both acute and chronic rat cervical spinal cord injury models promoted remodeling of descending raphespinal projections and contributed to the partial recovery of forelimb motor function. The results achieved in this proof-of-concept study demonstrates that human embryonic stem cell–derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of spinal cord injury.Spinal cord injury results in irreversible tissue damage and permanent sensorimotor impairment. The development of novel therapeutic strategies that improve the life quality of affected individuals is therefore of paramount importance. Cell transplantation is a promising approach for spinal cord injury treatment and the present study assesses the efficacy of human embryonic stem cell–derived neural crest cells as preclinical cell-based therapy candidates. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated  Primary physical impact leading to neuroglial and vascular damage, blood-spinal cord barrier disruption, hemorrhage, and ischemia during the initial acute phase of SCI (ASCI) gradually develops into a secondary injury cascade, which can last for several weeks and is characterized by the expansion of tissue damage, neuroglial degeneration, demyelination, and macrophage-mediated axonal dieback46.The majority of spinal cord injuries (SCI) occur at the cervical level, and affected individuals are left with severe physical and mental health challenges due to sensorimotor and autonomic impairment in addition to neuropathic pain7. Other characteristic milestones of CSCI are persistent neuroinflammation with suppressed systemic immunity in addition to delayed secondary degeneration of neurons in descending and ascending long spinal tracts8. The development of effective therapeutic strategies that attenuate the immune response and foster functionally significant de novo axonal regeneration and sprouting following SCI is therefore important to improve the life quality of affected individuals10.Several months after patients are discharged from hospital they are considered to be in the chronic phase of SCI (CSCI). At this stage the lesion site often contains cysts and cavities and is surrounded by a thick scar with fibrotic and glial components1013. Several possible mechanisms that underlie the therapeutic effects of cell transplants have been proposed and encompass neuroprotection, the stimulation of axonal regeneration and sprouting in addition to the modulation of the neuroinflammation and remyelination responses2. Human embryonic stem cells (hESCs) possess the greatest conceptual potential for the development of novel cell-based SCI therapies since they are infinitely renewable and amenable to molecular manipulation14. To this end, hESC-derived neural progenitors have been shown to support functional recovery in animal SCI models in addition to showing promise in human Phase I/II clinical trials10. However, studies have also demonstrated that neural stem cells can induce deleterious side effects such as allodynia-like hypersensitivity and the formation of ectopic colonies following focal transplantation16. The identification of hESC-derived transplantation candidates that circumvent these side effects is therefore of significant importance for the development of the future treatment strategies for SCI.Cell transplantation therapies have become a promising preclinical strategy for the treatment of SCI, and various human derived cell types including neural stem cells, Schwann cells, olfactory-ensheathing cells, and mesenchymal stromal cells have been tested both experimentally and clinically as suitable candidates1719. Moreover, our previous study demonstrated that neural crest cells could be rapidly differentiated from hESCs and their transplantation into a peripheral nerve injury model promoted robust sciatic nerve regeneration without tumor formation20. The work presented in this study therefore extends on our previous investigation and assesses the efficacy of hESC-derived neural crest cells for the treatment of acute and chronic cervical SCI. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated the in vitro outgrowth of rat primary dorsal root ganglia (DRG) neuronal cultures. Transplantation of the neural crest cells into a rat cervical SCI model promoted remodeling of descending motor tracts and partial motor recovery of forelimb function in both ASCI and CSCI scenarios. The results achieved in our proof-of-concept study therefore demonstrates that hESC-derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of SCI.hESC-derived neural crest cells are uniquely qualified as cell-based SCI therapy candidates due to their multipotent capability and ontological similarity to spinal cord stem cells21 in Dulbecco’s modified Eagle’s medium/F12 supplemented with 20% (v/v) KnockOut Serum Replacement, 1% (v/v) nonessential amino acids, 100 mM l-glutamine, 0.1 mM β-mercaptoethanol, 1% (v/v) penicillin–streptomycin (PeSt), and 4 ng/ml basic fibroblast growth factor (bFGF). hESC colonies were enzymatically passaged by collagenase IV treatment as described previously22. All hESC culture reagents were purchased from Thermo Fisher Scientific, Stockholm, Sweden.hESCs  were cultured on irradiated embryonic fibroblasts isolated from CF-1 mice23 and the NGFR+ neural crest population was subsequently isolated by magnetic activated cell sorting (MACS) using a CD271 human microbead kit . The NGFR+ neural crest cell population was then seeded on Matrigel-coated culture vessels in N2 media23 supplemented with 10 ng/ml bFGF and 20 ng/ml epidermal growth factor (EGF). Neural crest cell cultures were enzymatically passaged onto Matrigel-coated culture vessels by accutase treatment for a maximum of five passages prior to transplantation. A total of three independent differentiation experiments were performed during this study.Neural progenitor cells were differentiated from hESCs by small molecule inhibition of SMAD and WNT-signaling pathwaysn = 17), (ii) neural crest cell transplantation into chronic SCI , and (iii) time-matched SCI controls with medium injections (n = 17). Normal uninjured rats (n = 15) served as baseline controls . The animals selected for the transplantation into ASCI and matching SCI control animals were mounted into a stereotaxic instrument (see below) and cell injection (ASCI) or injection of a cell medium (SCI control) was begun within 30 min after initial surgery. Transplantation into CSCI was performed 7 weeks postoperatively. In experimental rat models of SCI, the chronic injury period is defined as one that begins at least 4 weeks post-trauma4. The animals were anesthetized as described above, the C3–C4 cervical vertebrae were re-exposed and the widow in the vertebrae created during previous laminectomy was carefully extended both rostral and caudal. The position of the injury site was identified and parts of the connective tissue covering the spinal cord above and below the injury site were carefully dissected. Special care was taken not to damage spinal cord tissue and connective tissue directly above the injury site. To simplify the initial penetration of the thin glass micropipette into the chronically injured spinal cord, a small opening in the meninges was made using 27G needle. For transplantation, neural crest cells were detached by accutase treatment and suspended at a concentration of 100,000 cells per µl in N2 medium. The cells were then transferred into a siliconized glass micropipette (100 µm O.D.) attached to a 5 µl Hamilton syringe. The cell suspension was slowly pressure injected into the spinal cord (1.5 µl over 10 to 12 min) at a depth of 1 mm and positions approximately 1 mm rostral and caudal to the lesion site using a Stoelting’s Lab Standard Stereotaxic Instrument . The micropipette was left in place for additional 3 min prior to being gently removed. The injury and transplantation sites were subsequently covered with fibrin glue  and a thin layer of Spongostan  soaked in normal saline. The muscles and skin were then closed in successive layers and the transplanted animals were administered fluixin , normal saline , and benzylpenicillin . All experimental animals with SCI were treated with cyclosporine A  for 3 weeks after cell transplantation or medium injection. The duration of CsA treatment was based on our previous studies demonstrating markedly reduced expression of neurotrophic factors by the transplanted human stem cells at 3 weeks postoperatively24 and unexpected significant additional neuroprotective effects of CsA following long-term administration25.Transplantation experiments were performed on adult (10- to 12-week-old) female Sprague–Dawley rats under isoflurane anesthesia in combination with buprenorphine . Following dorsal cervical C3–C4 laminectomy on the left side, a small 3 × 2 mm window was created in the bone using Friedman-Pearson Rongeurs with a 0.5 mm cup size. The dura mater was cut longitudinally and the border between the C3 and C4 dorsal root entry zone and the dorsal funiculus was identified prior to being vertically penetrated with a 23G needle. After introducing one blade of a microserrated Vannas spring scissors into the stab wound and the other blade between the lateral surface of the spinal cord and the dura mater, the lateral funiculus and adjacent gray matter were transected from the lateral side. Care was taken to avoid additional damage to the dura mater. This injury interrupts descending bulbospinal motor tracts from the brainstem in addition to ascending sensory and propriospinal pathways. The corticospinal tract in the dorsal column was spared. Immediately after the injury, the animals were randomly divided into three experimental groups: (i) neural crest cell transplantation into acute SCI  and enzyme linked immunosorbent assay (ELISA), C3 and C4 spinal cord segments  were removed and divided sagittally prior to freezing in liquid nitrogen. For immunohistochemical analysis, animals were transcardially perfused with Tyrode’s solution followed by 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Spinal cord segments  were then removed and transferred into 4% (w/v) PFA in 0.1 M phosphate buffer (pH 7.4) at 4°C for 4 h. The tissue blocks were then cryoprotected in 10% (w/v) and 20% (w/v) sucrose in 0.1 M phosphate buffer (pH 7.4) for up to 5 days prior to being frozen in liquid isopentane and cryosectioned. Serial horizontal or transverse cryosections (16 µm) were dried overnight at room temperature prior to being stored at −85°C.Total RNA was isolated from hESCs, neural crest cells, and spinal cord segments using the RNeasy Mini Kit  and reverse transcribed using the iScript cDNA Synthesis Kit . The resulting templates were analyzed by qPCR using a CFX96 Real Time System  and the SsoAdvanced Universal SYBR-Green Supermix . Species-specific oligonucleotides were employed in this study. These were designed by BLAST alignment of the requisite human and rat gene sequences to identify nonhomologous regions and species-specific primer pairs were designed against domains that contained the greatest base pair mismatches. The specificity of each primer pair was verified by RT-PCR using total RNA isolated from neural crest cells and rat C4 spinal cord segments using a T100 Thermal Cycler  and the OneStep RT-PCR kit . Details of all oligonucleotide sequences used in this study are given in Supplemental Table S2.26. The cells were washed three times for 5 min with PBST [PBS and 0.1% (v/v) Triton X-100], blocked for 1 h at room temperature in 10% (v/v) fetal calf serum (FCS) in PBST and then incubated overnight at 4°C with the required primary antibodies diluted in 10% (v/v) FCS in PBST. Cells were subsequently washed three times for 5 min with PBST and then incubated for 1 h at room temperature with 5 ng/ml 4’,6-diamidino-2-phenylindole (DAPI)  and the required secondary antibodies  diluted in 10% (v/v) FCS in PBST. Cells were finally washed three times for 5 min in PBST and mounted using Vectashield Mounting Medium . Spinal cord cryosections were rehydrated two times for 30 min in PBSAB [PBS containing 0.1% (w/v) sodium azide and 0.1% (w/v) bovine serum albumin]. The slides were then incubated for 1 h in blocking solution  in PBSAB prior to being incubated for 2 h with the required primary antibodies diluted in PBSAB. The slides were then washed with PBSAB and subsequently incubated for 1 h with the required secondary antibodies  diluted in blocking solution. The slides were finally washed three times for 5 min in PBS and mounted using ProLong antifade mounting medium with DAPI . All incubations were performed at room temperature. Antibody specificity was verified by omission of primary antibodies. Details of all primary antibodies used in this study are provided in Supplemental Table S3.Neural crest cells were fixed in ice-cold 4% (w/v) PFA in phosphate buffered saline (PBS) for 10 min and immunostaining as described previously2) and cultured in N2 medium supplemented with 10 ng/ml bFGF and 20 ng/ml EGF. Dispersed hESCs were seeded as monolayers on Matrigel-coated culture vessels 23 and grown in hESC medium containing 10 ng/ml bFGF and 20 ng/ml EGF. Conditioned medium was harvested from all cultures after 48 h incubation and filtered using a Steriflip Filtration System .Neural crest cells were plated onto Matrigel-coated flasks . ELISAs were performed on C3 spinal cord protein extracts and conditioned media using RayBio Sandwich ELISA kits .Spinal cord segments were homogenized in ice-cold RIPA Buffer  containing Protease Inhibitor Cocktail  using a TissueRuptor . The homogenate was agitated on an orbital shaker at 4°C for 2 h prior to the insoluble material being pelleted by centrifugation at 13,000 × n = 6) as described previously24. The isolated neurons were seeded on 20 µg/ml laminin coated culture vessels and grown for 24 h in Neurobasal-A supplemented with B27, 0.5 mM l-glutamine, and 1% (v/v) PeSt. The primary cultures were then briefly washed in PBS prior to the addition of conditioned media (0.5 ml). The cultures were then incubated for a further 72 h. Total neurite outgrowth analyses were performed as described previously20.Primary sensory neurons were dissociated from the DRG of adult female Sprague-Dawley rats , and Image-Pro Plus . Density of the descending motor 5-hydroxytryptamine (5-HT+) raphespinal axons was quantified in the trauma zone and in the Rexed lamina IX of the C2 and C6 ventral horns (+) sensory axons into Rexed lamina III was studied in the C2 dorsal horn  that were randomly placed on each image prior to analysis using custom macros written in Adobe Photoshop.Images were captured using a Zeiss LSM 710 confocal or Nikon Eclipse E80i microscope coupled to a Nikon DS-U2 digital camera. All images were analyzed and compiled using Fijial horns , B. Abersal horn , and the28. All experimental animals were recorded for 5 min upon placement within the cylinder and the extent of motor recovery of the forelimb was assessed from these video recordings. The following parameters were scored: (i) independent or simultaneous use of the injured and uninjured forelimb for initial wall contact during a full rear, (ii) independent or simultaneous use of the injured and uninjured forelimb for wall exploration following a full rear, and (iii) independent or simultaneous use of the injured and uninjured forelimb for landing following exploration. These counts were then employed to calculate the percentage of independent injured, independent uninjured, or simultaneous forelimb use for rearing, exploration, and landing, respectively. Testing was performed at 8-, 12-, and 16-week post-transplantation.Spontaneous forelimb use was assessed using vertical exploration of a clear plastic cylindert-tests, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test or a repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test were used to determine all statistical significances. Data on all graphs are presented as the mean and the standard error of the mean. P-values are indicated as follows: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.Statistical analyses were performed using GraphPad Prism 7 . Unpaired two-tailed Student’s 23 and the NGFR+ (nerve growth factor receptor) cell population was enriched by MACS at 14 days in vitro (DIV14). The isolated cells were then expanded for a maximum of five passages in the presence of bFGF/EGF (DIV14 to DIV30) (+ cell populations (DIV14 or DIV30) 29 30 (+ cell population (DIV14) compared to the hESC cultures (DIV0). Furthermore, subsets of the NGFR+ population (DIV30)  (n = 87/333), POU4F1  , and TFAP2A  17  exhibited multipotent characteristics as demonstrated by the robust expression of the intermediate neurofilaments PRPH (peripherin)  and NEFH (neurofilament heavy polypeptide)  (n = 107/117) and S100B    . We firsr DIV30) , C. A doOCT-4)29  and paraSNAI2)30  was obse 67/308)  expresse2/369)17 . Taken t130/137) , I in ad143/159) , K31,32.+ population (DIV30) with that of the parental hESCs (DIV0 and DIV2) by qRT-PCR and ELISA  but reduced levels of angiogenic factor expression (ANGPT1 and VEGFA) was observed in the NGFR+ cells (DIV30) relative to the hESCs (DIV0) (+ cells (DIV30) but not in the hESC population (DIV2) (In vitro neurite outgrowth assays were therefore performed to assess the bioactivity of these secreted trophic factors. Exposure of rat primary DRG neurons to NGFR+ conditioned medium (DIV30) induced the elaboration of TUBB3+ (tubulin beta class 3) neurites that were consistently longer than the corresponding neurite outgrowth of primary neuronal cultures exposed to hESC-conditioned medium (DIV2) (We next compared the trophic factor expression profile of the enriched NGFRnd ELISA . An incrs (DIV0) . We alson (DIV2) . In vitrm (DIV2) .in vitro experiments demonstrated that multipotent neural crest cells could be rapidly differentiated from hESCs and that the enriched cells secreted biologically active trophic factors that stimulated rat primary DRG neuron outgrowth. These features suggested that the neural crest cells could be viable cell-based therapy candidates. We therefore assessed their in vivo regenerative potential in a rat cervical SCI model (+ (human nuclear antigen) neural crest cells in both ASCI and CSCI 3 weeks after transplantation , mesenchymal cells/fibroblasts (human-specific Thy-1/CD90), adipocytes (Glut4), and alpha-smooth muscle actin. We did not find any cells stained for these additional neural crest lineage markers. Interestingly, qRT-PCR analysis using a panel of human-specific primers demonstrated that the neural crest cells continued to express elevated levels of BDNF transcripts in C4 spinal cord segments in both ASCI and CSCI animals for up to 3 weeks following transplantation  and ELISAs for trophic molecules  that were detected during the in vitro characterization of the neural crest cells were undetectable in C4 spinal cord segments. Furthermore, qRT-PCR analyses demonstrated that cell transplantation did not affect the expression of Ptprz1, Bcan, Ncan, and Vcan chondroitin sulphate proteoglycan genes when compared to SCI alone  and aberrant sprouting of sensory (CGRP+) axons, respectively. Sixteen weeks after SCI we found single 5-HT+ raphespinal axons present within the injury site of control animals  axon sprouting in lamina III of the C2 cervical dorsal horn  microglial cells both rostral and caudal to the lesion and injection sites . However, the neural crest cells had no effect on astrocytic activation in the scar region but reduced astrocytic reactivity rostral and caudal to the injury site when transplanted in CSCI animals  and parallel induction of molecular markers  in the NGFR+ population that was consistent with those of bona fide multipotent neural crest cells40. But despite the reproducibility of our experimental approach, it should be noted that one drawback of our methodology is the reliance on single marker cell sorting as previous studies have also noted42. We cannot therefore completely exclude the possibility that the isolated NGFR+ populations are composed of predominantly neural crest cells  but also smaller populations of alternative dorsal neural tube derived cells. Eliminating this single marker enrichment step is therefore one of the critical obstacles that must be overcome if the routine preclinical use of these cells is to be realized43. This could be accomplished by transcriptome analyses of hESC-derived neural crest cell lines to identify novel signaling pathways that could subsequently be modulated during critical differentiation periods to promote the rapid and guided derivation of homogenous cell populations without the use of cell sorting31.This study corroborates our previous publication and establishes that the use of small molecule inhibitors is a robust approach for deriving neural crest cells from hESCs44. Consistent with previous studies45 we observed that the differentiated cells expressed several trophic factors in vitro  with subsets of these transcripts being actively translated into proteins  that were detectable in conditioned medium. Interestingly, negligible levels of NT-3 and NGF were once again observed that corroborated with our previous study20 and suggests that our differentiation approach yields a population of neural crest cells with a reproducible trophic factor profile. Moreover, these secreted trophic factors were biologically active and were able to induce robust neurite outgrowth of rat primary DRG neurons. Taken together, these results demonstrate that the neural crest cells generated in this study exhibited functional trophic factor profiles that are one of prerequisite traits for promoting neural regeneration and angiogenesis following SCI4648.One important requirement for cell-based SCI therapy is that the transplanted cells must provide trophic support that fosters host tissue survival and regeneration49. In this study, the cavity was found in one animal with SCI alone and in one animal with cell transplantation. The observation that the S100B+ and NGFR+ transplanted cells were present within the injection sites and trauma zone 3 weeks after surgery is consistent with previous studies using xenogeneic stem cell transplantation with cyclosporine treatment45. Although cyclosporine has been found to be neuroprotective in an experimental traumatic brain and peripheral nerve injury models50, it seems to have differential effects upon transplanted stem cells51. Moreover, in compression SCI models the use of cyclosporine induces the upregulation of PDGF-A without any effect on other trophic factors45. Contrastingly, the neural crest cells derived in our study expressed elevated levels of BDNF in both ASCI and CSCI groups. Moreover, no teratoma formation was observed indicating the effective removal of pluripotent cells using our differentiation approach that concurs with our previous use of hESC-derived neural crest cells in a rat peripheral nerve injury model20.The efficacy of the neural crest cells as preclinical therapy candidates was assessed in a rat cervical hemisection model of SCI. In contrast to contusion type of SCI with significant tissue necrosis and formation of cavities in the injury site, laceration cause by sharp bone fragments typically shows very limited cavitationBDNF levels observed in our study indicates the possibility that the regenerative effects of the transplanted cells in both ASCI and CSCI groups were mediated via neurotrophic support to different long spinal tracts. Numerous studies have demonstrated that BDNF has significant growth promoting and neuroprotective effects on bulbospinal tracts including 5-HT+ raphespinal axons and on sensory CGRP+ axons53. BDNF receptor TrkB is downregulated at the epicenter of SCI but remains largely unaffected in the segments rostral and caudal to the lesion site54. That these regions were utilized for cell transplantation in this study might account for the regenerative response observed in reply to the elevated levels of BDNF following transplantation. Although we did not find any signs of aberrant CGRP+ sprouting in the dorsal horn, the transplanted cells promoted ingrowth of 5-HT+ raphespinal axons deep into the trauma zone and induced their sprouting rostral to injury site. In addition, we found that the transplanted cells had no effects on the density of the reactive astrocytes in the scar but reduced their density both rostral and caudal to the trauma zone following CSCI which is in line with a recent report that human neural stem cells can attenuate reaction of astrocytes55. Although the mechanism is not completely understood, the authors demonstrate that the effect of stem cells on reactive astrocytes could be mediated via inhibition of NF-κB activity and associated immune response55. Unexpectedly, we did not find any effect of neural crest cells on host astrocytes following implantation into acutely injured spinal cord. This could possibly reflect the differences in tissue environment in the acutely and chronically injured spinal cord. Moreover, that the neural crest cells in the present study had no effects on neuroinflammation is in contrast to that recently observed for rat neural stem cells56.The elevated 34. It has been shown that this test is not influenced by repeated analysis and does not depend on aversive motivation34. Several reports suggest that injury to the lateral funiculus of the cervical spinal cord results in severe limb use asymmetries during rearing and landing without significant recovery in CSCI58. In contrast to single food pellet reaching test, which is often used to study fine motor control following cervical SCI, the vertical cylinder test does not require animal training before the injury. It has been also found that the outcome of the reaching test can be significantly affected by the differences in the rate of learning a motor task and different compensatory reaching strategies used by experimental animals with SCI59. We observed that neural crest cell transplantation progressively improved forelimb use for landing in both ASCI and CSCI groups. It is unlikely however that our observed improvement of locomotor function was due to axon regeneration across the injury site. Transplantation-induced sprouting of 5-HT+ raphespinal axons rostral to the injury site could indicate some form of neuronal relay formation between descending motor pathways in the lateral funiculus and propriospinal relay neurons9. Such a circuit could therefore transmit the signal to the neurons caudal to the injury site. It has been shown that human glial restricted progenitors could be very efficient in promoting axon regeneration of reticulospinal and raphespinal axons60 and the recovery mechanism of novel relay formation with propriospinal neurons has been reported previously for the corticospinal and reticulospinal tracts after incomplete cervical SCI63. Similar mechanisms could therefore account for the partial recovery of forelimb motor function observed in our study.Regaining upper limb function is of paramount importance to quadriplegic individuals following cervical SCI. Accordingly, axon regeneration for up to two spinal segments below the level of high C3–C4 cervical SCI would allow patients to regain control of both arm and shoulder muscles. To assess forelimb function, we used the vertical cylinder test that is known to be sensitive to the assessment of chronic limb use asymmetriesClick here for additional data file.Supplemental Material, sj-pdf-1-cll-10.1177_0963689720988245 for Human Embryonic Stem Cell–derived Neural Crest Cells Promote Sprouting and Motor Recovery Following Spinal Cord Injury in Adult Rats by Iwan Jones, Liudmila N. Novikova, Mikael Wiberg, Leif Carlsson and Lev N. Novikov in Cell Transplantation


Monocytes originate from bone marrow hematopoietic stem cells and circulate in the bloodstream. Monocyte extravasation and differentiation serve multiple immune functions. The differentiation of monocytes into tissue macrophages at steady state can serve homeostatic functions. Monocytes can also fuel acute inflammatory reactions and anti-microbial immunity by differentiating into inflammatory macrophages. Finally, monocytes also actively contribute to the resolution of inflammation and tissue regeneration.The subset classification of monocytes is a rapidly emerging field. Recent progress in single-cell genomics and high dimensional approaches in phenotyping have highlighted additional subsets of monocytes. Monocytes might also adopt new dynamic transcriptional states associated with inflammation and reflecting their subset heterogeneity. This brings the research community to face a significant challenge of assigning monocyte heterogeneity to specific functions. The goal of this Research Topic is to gather contributions bringing new insights into:Monocyte subsets and their ontogenyMonocyte heterogeneity and inflammatory diseasesMonocyte heterogeneity and the regulation of inflammationMonocyte heterogeneity and cancer+ and CD16+ monocytes in humans and Ly6Chigh and Ly6Clow monocytes in mice.Kapellos et al. provide an updated classification of human monocytes by compiling multiple profiling studies 1. Monocytes are composed of two main subsets: CD14high and CD14+ monocytes are now termed “classical” monocytes. Although classical monocytes constitutively enter tissues during homeostasis, upon inflammation, they rapidly extravasate and, depending on the needs of the environment, can differentiate into multiple cell types such as monocyte-derived macrophages.While originally referred to as “inflammatory” monocytes, Ly6C+ and Ly6Clow monocytes are termed as “non-classical” . Non-classical monocytes display an intra-vascular function, interacting dynamically with endothelial cells . Expression of 6-sulfo LacNac sugar antigen linked to the cell surface protein PSGL-1 is termed as SLAN. SLAN expression defines non-classical CD16+ in humans and provides a practical way to distinguish them from the “intermediate” CD14+CD16+ monocytes .CD16The developmental pathways by which monocytes arise from hematopoietic stem cells have received a lot of attention. Since the identification and characterization of Granulocyte-Monocyte Progenitors by Akashi et al. in 2000 .Wolf et al.). GMP-dependent monocytes would co-exist with MDP-derived monocytes. The later encompass monocytes progenitors endowed with a potential for the generation of monocyte-derived DCs (moDCs). Fate mapping studies with Ms4a3cre/Ms4a3CreERT2 driver lines identifies the GMP-dependent pathway for the generation of monocytes and assesses precisely its contribution to the tissue resident macrophages (TRMs) pool .The differentiation of monocytes into cellular products endowed with DC-like features has been historically evidenced in vitro . Since tDuroux-Richard et al. summarize the current of knowledge on the regulation of monocytes subsets by microRNAs. For instance, mir146a regulate the pool of classical monocytes with a regulatory role in osteoclastogenesis .Kapellos et al.; Patel and Yona). In this topic, Patel et al. and Kapellos et al. provide a fascinating inventory of factors impacting on monocyte populations thereby emphasizing that “steady state is not a single physiological condition” (Patel and Yona) and providing some insights on how changes in lifestyle impact on monocyte subsets .Assessing developmental pathways underlying the production of monocyte subsets during homeostasis should not obliterate the fact that monocytes are highly dynamic and regulated by both environmental and genetic factors .Deciphering the aegis of specific signals is often complicated by the fact that they act not only on monocytes but also on numerous other myeloid lineages. The inflammatory factor GM-CSF provides a good example of such pleiotropy. Kapellos et al.)   . For insDick et al. provide an integrative picture of the remodeling of cardiac macrophages populations imposed by ischemic injury. Some striking features emerging from multiple scRNAseq studies might apply to various cases of acute injury: i) TIMD4+LYVE1+MHCIIlow TRMs tissue resident macrophages rapidly disappear from the inflamed zone but slowly repopulate the organ by local proliferation; ii) monocyte-derived macrophages acquire multiple transcriptional phenotypes including one resembling tissue resident macrophages (despite the absence of TIMD4 and LYVE1 expression) and other transcriptional phenotypes possibly associated to tissue repair functions; iii) TIMD4+LYVE1+MHCIIlow and CCR2+ TRMs pools present before the onset of acute injury orchestrate monocyte infiltration by controlling the diversity of inflammatory macrophages phenotypes with important consequences for the onset of tissue repair . Further research is needed to establish a map of developmental trajectories linking monocyte subsets to macrophages recruited after myocardial injury, possibly involving a heterogenous spatial distribution . Coillard and Segura detail and discuss the various markers and methodological options available, in human immunology settings, to probe the monocytic origin of tissue phagocytes . However, the analysis of macrophage dynamics in the injured myocardium underlines the difficulties to move beyond the transcriptional definition of cellular populations to an integrated understanding of their functional contributions to both injury and tissue repair .Monocyte extravasation supports their function of precursors for macrophages. In particular, Ma et al. provide a broad panorama of the role of monocytes in multiple autoimmune conditions ranging from Systemic Lupus Erythematosus (SLE) to type 1 diabetes (T1D). Understanding monocyte biology has important therapeutic application. In this context, Hamilton delivers a synthesis of the current knowledge and controversies regarding the action of GM-CSF on monocytes and macrophages. A key feature of GM-CSF signaling lies in the activation of the CCL17 axis in monocyte-derived macrophages by a process involving the JMJD3 histone demethylase. Therapeutic blockade of GM-CSF in rheumatoid arthritis reduce CCL17 levels and reduces osteoarthritic pain (Hamilton).Monocytes and macrophages play a major role in autoimmunity. In this topic, Postat and Bousso identify quorum sensing as new mechanism to maintain tissue integrity during the onset of anti-microbial innate immune reactions. In this case, nitric oxide release by differentiated anti-microbial monocytes plays a major role in activating the resolution of inflammation by a mechanism involving the suppression of mitochondrial respiration, thereby limiting cytokine and chemokine secretion (Postat and Bousso). Furthermore, IL-10 is also an important immuno-regulatory factor that can promote the acquisition of an immunoregulatory phenotype in differentiated moDCs associated to CD14 re-expression when combined with TLR stimulation . Undoubtedly, that understanding the mechanisms generating monocyte-derived cells endowed with immunosuppressive properties has a strong therapeutic relevance. For instance, Iglesias-Escudero et al. show in this topic that the presence of regulatory monocytes descendants is regulated by rapamycin in kidney transplant recipients.The role of monocyte subsets in the resolution of inflammation is increasingly considered. The differentiation of classical monocytes into microbicidal macrophages is a central feature of anti-infectious innate immunity . HoweverButenko et al. evidence that post-phagocytic “satiated” monocyte-derived macrophages express type I IFN and exhibit a transcriptomic profile limiting their pro-fibrogenic activity.The resolution of inflammation is a dynamic process relying on the efferocytic capacity of monocytes and their progeny, which support the development of anti-inflammatory responses and subsequently will contribute to the reestablishment of tissue resident macrophage pool. In this regard, Wu et al. discuss the relevance and limitations of the M1/M2 paradigm to understand the diversity of transcriptional states observed in TAMs. Wu et al. propose to re-assess TAM heterogeneity by integrating their ontogeny, activation and localization. Laviron and Boissonnas review the current knowledge on the selective contributions and functional contributions of acutely recruited monocytes in cancer versus pre-existing homeostatic tissue resident macrophage would they be derived from embryonic precursors or adult monocytes.Monocytes play a major role in cancer where CCR2-dependent classical inflammatory monocytes largely contribute to the pool of tumor-associated macrophages (TAMs), often with immunosuppressive and pro-tumoral properties . By contIn summary, the very diverse and multidisciplinary contributions in this Frontiers TOPIC highlight the dynamism of monocyte research in finding new developments related to multiple biological processes and pathologies.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.