s2fieldsofstudy: Biology Medicine

text:
The combined role of allelic variants of IRS-1 and IRS-2 genes in susceptibility to type2 diabetes in the Punjabi Pakistani subjects

Background Diabetes mellitus is a multifactorial disorder characterized by a high level of glucose in the blood. Both genetic and environmental factors interact to cause diabetes. Insulin receptor substrate (IRS) proteins have a significant part in insulin signaling pathways. We aimed to investigate the relationship of type 2 diabetes with a Gly972Arg (G972R) variant of the IRS-1 gene and Gly1057Asp (G1057D) polymorphism of IRS-2 gene in the population of Punjab, Pakistan. Methods We collected 926 samples, 500 healthy controls (fasting blood sugar < 99 mg/dL, random blood sugar < 126 mg/dL) and 426 cases with diabetes (fasting blood sugar > 99 mg/dL, random blood sugar > 126 mg/dL). Several anthropometric measurements were measured. Statistical analysis was performed by using SPSS to determine the allele group/genotype frequency of the selected variants in the study population. Results The genotyping results of G972R by RLFP-PCR showed the allelic frequency of G = 0.68 and R = 0.32 in controls while G = 0.71 and R = 0.29 in the cases. The minor R allele had a slightly higher frequency in the cases than the controls (OR = 0.86, CI 0.706–1.052, p = 0.17). The genotyping results of G1057D showed allelic frequency G = 0.74 and D = 0.26 in the controls while G = 0.961 and D = 0.29 in the cases. The minor D allele appeared to be a risk allele for this SNP although the difference in the allele frequencies was not statistically significant (OR = 1.55, CI 0.961–1.41, p = 0.108). The combined genotype analysis showed that the difference in the allele and genotype frequencies reached statistical difference between the cases and the controls as well as the odds ratio substantially increased when the R allele (G972R) was present together with D allele (G1057D) in any combination. When the association of single variants with the lipid traits was observed, only D allele (G1057D) showed significant association with TG, HDL and LDL, however when the analysis was repeated for combined genotypes using general linear model, many more significant associations between the genotype where D allele and R allele are together, were seen with many lipid traits. Conclusion In conclusion, the single nucleotide polymorphisms with low-modest effect size may not affect the phenotype individually but when in combination, the effect becomes stronger and more visible, therefore, for the SNP association studies, the more the number of SNPs included in the analysis, the more meaningful the results. Electronic supplementary material The online version of this article (10.1186/s13098-019-0459-1) contains supplementary material, which is available to authorized users.

Background Diabetes is characterized by chronic hyperglycemia leading to the imbalance of fat, protein and carbohydrate metabolism due to defective insulin action, secretion or both [1]. The prevalence of diabetes is increasing worldwide due to aging, population growth, urbanization, physical inactivity and obesity [2,3]. The sequelae of diabetes include long term damage and dysfunctioning of various organs leading to their failure [4]. The characteristic symptoms of diabetes mellitus involve polyuria, blurred vision, thirst and weight loss [5,6]. The persons with either type 1 or type 2 diabetes require insulin treatment at some stage of the disease. Type 2 is the most prevalent form of diabetes that is characterized by insulin resistance [7,8].
The insulin receptor substrates (IRSs) are the cytoplasmic receptors, important in insulin signaling [9]. They also play a pivotal role in maintaining basic cellular functions like survival, growth and metabolism. After the activation of these proteins, several signaling cascades are activated. This leads to the activation of many downstream effectors. Finally, they gave the insulin signal to branching series of intracellular pathways. There are four members of the IRS family and are similar in their overall architecture [10].
When insulin binds to its receptor on cell surface, IRS-1, a docking protein is phosphorylated and activates phosphatidyl inositol 3-kinase (PI3-kinase). This initiates a cascade of intracellular signaling resulting in a variety of responses by the cell. One of these responses is the activation of GLUT (glucose transport) receptor resulting in increased uptake of glucose by the cell [11]. The individuals with non-insulin dependent diabetes mellitus (NIDDM) have decreased expression and function of IRS-1 in the fat cells. The Gly972Arg polymorphism is the most commonly studied variant of IRS-1 [12]. Downstream IRS-1, the main docking protein found in the cells is IRS-2 [13]. It acts as the backup protein for the intracellular spread of the insulin signal including the activation of PI3-kinase. However, the hallmark of insulin resistance is that, for the activation of IRS-2, higher concentration of insulin is required. Gly1057Asp (G1057D) is a widely reported variant in IRS-2 gene whereby glycine (G) is replaced by aspartate (D) at 1057 position in the gene [14,15]. These SNPs were selected for analysis in Pakistani subjects because (1) Pakistani population represents a unique ethnic group which allows the study of concentrated risk genetic markers (2) the selected SNPs have been investigated in the Caucasians so their analysis can provide the information on the relationship of these genetic variants with daibetes and (3) these SNPs have not been previously investigated in the Pakistani population and the current study is the first report of their study in our population. The biochemical risk factors included were lipid profile parameters namely total cholesterol (TC), very low density lipoprotein (VLDL), LDL-C, high density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels.

Study subjects
926 subjects (426 diabetic cases and 500 normal controls) were recruited from the local population using a case control design between July 2014 and April 2015. The subject recruitment, inclusion and exclusion criteria have been described previously [3,16]. Inclusion criteria for diabetic subjects were (i) diabetes diagnosed according to etiologic classification of diabetes by the International Diabetes Federation (IDF) and (ii) confirmation that all the grandparents of the subjects are of Pakistani origin. The exclusion criteria were the presence of any infectious disease, conditions where phlebotomy is contraindicated, age below 10 years, body mass index (BMI) ≤ 18.5 kg/m 2 , pregnancy, handicapped/mentally disturbed individuals, obese (obesity defined as: BMI > 23 kg/m 2 as overweight and BMI > 26 kg/m 2 as obese, criteria used for Asian populations as described previously [17], cancer and ethnicity other than Pakistani. The controls were healthy subjects from the general population with normal blood sugar levels ( < 126 mg/dL random or < 99 mg/dL fasting) [16]. All the subjects were genetically unrelated and gave written informed consent. The employed procedures were according to the Helsinki declaration and an ethical approval was obtained from the institutional ethical board.

Blood sampling
Venous blood was collected from the subjects in the fasting state. 5 mL blood was collected from the median cubital vein using aseptic measures. 2.5 mL of blood was dispensed in an EDTA vial and used for DNA isolation and 2.5 mL was poured in a gel clot activator containing vial and used for biochemical analysis.

Genetic analyses
The DNA was isolated by manual method. The isolated DNA was stored at 4 °C or at − 20 °C for further use. The presence of DNA was determined by Agarose gel electrophoresis. After isolation, all DNA samples were quantified before amplification using Epoch Biotek microplate reader. The quantification was performed to equalize the final concentration of DNA samples up to approximately 10 ng/μL. The PCR conditions consisted of initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 45 s for G1057D variant and at 61 °C for 45 s for G972R variant,

Statistical analysis
For conducting statistical analysis, MS Excel and SPSS (Statistical Package of Social Sciences) Package were used. Different variables such as Anthropometric, Biochemical and Genotype were analyzed by calculating mean and standard Deviation. Further the study population was tested for Hardy Weinberg Equilibrium (HWE), Chi-square test was also used to test the significance of difference of Allele and Genotype frequencies among diabetic and non-diabetic. To check the relationship of the SNPs with diabetes, t-test and logistic regression was conducted. Further the association of Polymorphism with Anthropometric and Biochemical parameters was tested through one way ANOVA. An adjusted p-value less than 0.025 was used as a significance cutoff for all such analysis due to inclusion of two SNPs at a time.

Results
The general characteristics of the subjects have been described elsewhere [16] ( Table 1). The study population was tested for Hardy-Weinberg equilibrium by a chisquared test and was found to be in equilibrium (HWE p-value was 0.698 for G972R and was 0.377 for G1057D).

Allele/genotype frequencies of G972R and G1057D polymorphisms
After SNP genotyping, the genotypic and allelic frequencies were calculated.  Table 2). The combined genotype analysis revealed that the coexistence of any combination of the two variants with one or both risk alleles (GG + GD, GG + DD, GR + GD, GR + DD, RR + GD, RR + DD) had significant effect on the outcome as indicated by the odds ratio which increased proportionately when the heterozygous and homozygous genotypes of G972R was analyzes with heterozygous and homozygous genotypes of G1057D (Table 3).

Effect of the G972R and G1057D polymorphisms on biochemical parameters
Biochemical parameters were checked across the three genotypes for both variants individually by one way ANOVA as well as in combination by multivariate general linear model (GLM). For single variant analysis, G972R did not show statistically significant association with any of the lipid traits. For G1057D variant, the presence of risk allele significantly increased TG and LDL and lowered HDL although there was no effect on TC (Table 4). When the genotypes were analyzed in combination, the number of significant associations increased considerably. It was observed that when the common homozygous heterozygous and risk homozygous genotypes of G972R were examined against common homozygous, heterozygous and risk homozygous genotypes of G1057D. The results of this analysis are shown in Table 5. The table shows that the presence of homozygous risk genotype of G972R in any combination with G1057D (GG + GD, GG + DD, GR + GD, GR + DD, RR + GD, RR + DD) had the greatest effect on the change of lipid parameters (significant rise in TC, TG, LDL-C and a decrease in HDL-C).

Discussion
The health care burden of diabetes is increasing at an alarming rate globally. The proper management for diabetes is therefore required to lessen its outcomes and to prevent the further complications [18,19]. The Insulin Receptor Substrates (IRSs) of cytoplasmic adaptor proteins functioning in signal transduction in response to insulin binding at the cell surface consists of IRS-1, IRS-2, IRS-3, and IRS-4, named in order of identification [20,21]. The identification of IRS-1 and IRS-2 gene polymorphisms associated with type2 diabetes was a landmark achievement in the field of diabetes genetics. The Gly972Arg is the most extensively studied polymorphism in IRS-1 gene, present in a highly conserved region of the protein that is involved in the phosphorylation of the substrate [22]. In an in vitro cell culture study of human mesangial cells, the expression of G972R caused a significant reduction in the insulin-stimulated phosphorylation of IRS-1 and Akt kinase [23]. The association studies of G972R SNP report positive as well as negative findings depending upon sample size, genotyping techniques and ethnicity. A previous study reported that carriers of Gly972Arg are at 25% higher risk of havig diabetes as compared to non carriers [24]. Another study reported the association of Gly972Arg with type 2 diabetes in Mexican population [25]. We found contradictory results    compared to the Caucasian Dutch population where Gly972Arg did not appear to play a role in increased risk of type 2 diabetes [26]. However, our results are in accordance with the previous published study to report the association of Gly972Arg with type 2 diabetes [27,28]. The G1057D variant of IRS-2 gene has been reported in different groups but results are controversial as well. Certain studies have indicated that this variant is not linked to type 2 diabetes in Chinese, Finish, Turkish and Tunisian populations [29][30][31]. While some studies have shown that G1057D variant of IRS-2 gene predisposes mice to type 2 diabetes [32]. Another study reported IRS-2 polymorphisms as one of the genetic causes of type 2 diabetes [33]. Studies that included individuals from Kurdish Ethnic Group (in West Iran) and Asian Indians reported association of G1057D polymorphism of IRS-2 gene with type 2 diabetes [34,35]. Similarly, genetic causes that lead to type 2 diabetes in Iranian and Indian population included G1057D polymorphism [36,37].

Table2 Allele and genotypic frequencies of G972R and G1057D polymorphism
In the current study, we determined the association of the variants of IRS-1 Gly972Arg (G972R) and IRS-2 Gly972Arg (G72R) with diabetes. The genotyping results of G972R showed that the minor R allele had a slightly higher frequency in the cases than the controls. The genotyping results of G1057D showed the minor D allele appeared to be a risk allele for this SNP although the difference in the allele frequencies was not statistically significant. The combined genotype analysis showed that the difference in the allele and genotype frequencies reached statistical difference between the cases and the controls as well as the odds ratio substantially increased when the R allele (G972R) was present together with D allele (G1057D) in any combination. When the association of single variants with the lipid traits was observed, only D allele (G1057D) showed significant association with TG, HDL and LDL, however when the analysis was repeated for combined genotypes using general linear model, many more significant associations between the genotype where D allele and R allele are together, were seen with many lipid traits.
There are certain limitations of our study as the sample size was relatively small and the samples were restricted to the Punjab province only. The mean age of controls and cases is significantly different that may cause bias in the study. There are other genes and polymorphisms that affect this association, but could not be included in the current investigation. However, despite these limitations, the results of the current study demonstrate the role of common variants in the progression of complex diseases like diabetes in the Pakistani population and show that if more variants are included in an investigation, more conclusive results can be obtained about the impact of the genetic markers on the phenotype. To our knowledge, the present study is the first attempt to report the association of IRS variants with diabetes in the local population.

Conclusion
In conclusion, there are various environmental factors and genes that work together in predisposition of an individual to complex diseases like diabetes. Moreover the single nucleotide polymorphisms can play an important role in the etiology of the complex diseases, however, is important to consider their combined effect on the phenotype.


extfieldsofstudy: Biology Medicine

text:
The Surface Proteome of Adult Neural Stem Cells in Zebrafish Unveils Long-Range Cell-Cell Connections and Age-Related Changes in Responsiveness to IGF

Summary In adult stem cell populations, recruitment into division is parsimonious and most cells maintain a quiescent state. How individual cells decide to enter the cell cycle and how they coordinate their activity remains an essential problem to be resolved. It is thus important to develop methods to elucidate the mechanisms of cell communication and recruitment into the cell cycle. We made use of the advantageous architecture of the adult zebrafish telencephalon to isolate the surface proteins of an intact neural stem cell (NSC) population. We identified the proteome of NSCs in young and old brains. The data revealed a group of proteins involved in filopodia, which we validated by a morphological analysis of single cells, showing apically located cellular extensions. We further identified an age-related decrease in insulin-like growth factor (IGF) receptors. Expressing IGF2b induced divisions in young brains but resulted in incomplete divisions in old brains, stressing the role of cell-intrinsic processes in stem cell behavior.

INTRODUCTION
In all organisms, developmental processes continue throughout life, overlapping with signs of aging. Many organs maintain stem cell niches after having reached maturation, in which mitotic activity allows for the generation of new differentiated cells. However, the frequency of mitotic activity slowly decreases. Levels of quiescence and cell-cycle entry change following mechanisms that are incompletely understood. This regulation of adult stem cells' quiescence or cell cycle, important both for the unwanted occurrence of cancers and for the desired repair of damaged tissues, remains a fundamental problem in biology.
In vertebrate brains, neural stem cells (NSCs) remain present in defined areas and give rise to new neuronal cells throughout life, albeit in a declining manner. In mammals, well-studied areas are the subependymal/subventricular zone (SVZ) lining the lateral ventricle, giving rise to neuronal cells populating the olfactory bulb as well as the prefrontal cortex in juvenile humans (Lim and Alvarez-Buylla, 2016), the subgranular zone (SGZ) of the hippocampal formation, generating new granule neurons in the dentate gyrus (Goncalves et al., 2016), as well as the hypothalamus (Recabal et al., 2017). The NSCs of these areas display similar morphologies, being tanycytes in the hypothalamus, radial glia in the dentate gyrus of the hippocampus, and radial-shaped astrocytes in the SVZ (Chaker et al., 2016;Fuentealba et al., 2012;Maggi et al., 2014). A special feature of the SVZ is the mixture of radial astrocytes with ependymal cells (Mirzadeh et al., 2008), the latter being recruited only in case of injury (Carlen et al., 2009).
Several parameters influencing the recruitment into the cell cycle of NSCs have been defined: growth factors, celladhesion molecules and the extracellular matrix, contact with blood vessels, cerebrospinal fluid, and metabolism (Goncalves et al., 2016;Lim and Alvarez-Buylla, 2016;Rafalski and Brunet, 2011). As aging progresses, the fraction of mitotically active NSCs declines (Capilla-Gonzalez et al., 2014;Kuhn et al., 1996). Several mechanisms of cell-intrinsic and cell-extrinsic nature, leading to this overall reduction of activity of NSCs, have been revealed. Stem cells have been shown to divide only a limited number of times in the dentate gyrus (Encinas et al., 2011) and reveal signs of depletion in the SVZ too (Calzolari et al., 2015). Progressive lengthening of the G 1 phase of NSCs might be a factor for their less frequent recruitment (Daynac et al., 2014(Daynac et al., , 2016. The milieu has also been shown to play a role in parabiosis experiments, exchanging the lymphatic system of young and old mice (Villeda et al., 2011). The levels of distinct growth factors progressively change with age: insulin-like growth factor 1 (IGF1), fibroblast growth factor (FGF), and vascular endothelial growth factor decline in the dentate gyrus (Kang and Hebert, 2015;Shetty et al., 2005). The immediate surrounding niche, made of the neighboring cells and the extracellular matrix, known to play a role in stem cell activity (Kazanis et al., 2010;Porlan et al., 2014), might also be subjected to agerelated changes influencing mitotic activity (Yamada et al., 2017). A clear picture to explain the individual behaviors within stem cell populations is nevertheless missing.
In the adult zebrafish brain, transcription factors homologous to their mammalian counterparts are at play in the neurogenic process (Diotel et al., 2015), making it a valid comparative vertebrate model organism. Moreover, it presents the advantage of a broader neurogenic activity within all brain regions and a high regenerative potential (Alunni and Bally-Cuif, 2016;Baumgart et al., 2012;Grandel and Brand, 2013). The radial glia in zebrafish display similar features to those of the NCSs of the mammalian neurogenic areas. Their somata are in immediate contact with the ventricles, they express the intermediate filaments glial fibrillary acidic protein (GFAP), Nestin, and Vimentin, and generate new neurons and glial cells (Kroehne et al., 2011;Rothenaigner et al., 2011). Their transitions back and forth between quiescence and cell cycle have been shown by long-term bromodeoxyuridine tracing and by Notch blocking experiments März et al., 2010). Whether every cell or only a subpopulation enter the cell cycle regularly remains uncertain (Dray et al., 2015). The radial glia might give rise to newly born neurons through a direct conversion process without an immediately preceding mitosis (Barbosa et al., 2015). Additionally, progenitors without GFAP expression are also present in close proximity to the somata of the radial glia and are considered intermediate progenitors, even if similar cells organized in pools function as stem cells in other regions of the brain (Galant et al., 2016;Kaslin et al., 2009Kaslin et al., , 2017Than-Trong and Bally-Cuif, 2015).
In addition to the abundance of NSCs, a major advantage of the zebrafish brain is the architecture of its pallium (dorsal telencephalon), where the ventricular surface opens on the outside instead of enclosing a lumen, due to an eversion process during embryonic development (Wullimann and Mueller, 2004), thus becoming accessible to imaging or manipulations (Barbosa et al., 2015;Dray et al., 2015).
We made use of this architecture to isolate the proteins present in an intact population of NSCs. As this cell population reveals an age-related increase of quiescent cells (Edelmann et al., 2013), we identified the surface prote-ome of the young and old ventricular zone to obtain sets of proteins likely implicated in the intercellular communication and in the regulation of cell cycle and quiescence. We focused on two aspects of the dataset. The significant over-representation of filopodial and lamellipodial protein components allowed us to discern new morphological features of NSCs. Furthermore, age-related changes revealed a decline in IGF1-receptor (IGF1R) expression, observed by both mass spectrometry and immunochemistry of the phosphorylated IGF receptor. This decrease was measurable by an altered response to the ligand IGF2b, able to induce complete cell divisions in young, but not in aged animals.

Identifying the Proteome of Neural Stem Cell Surfaces in Intact Brains
The closest contacts between the radial glia are at the level of their cell bodies, adjacent to the brain ventricle. With the aim of defining signaling pathways putatively implicated in their intercellular communication, we designed an approach to isolate all proteins present on the cell bodies in a native configuration of freshly isolated brains. The biotinylation protocol allowed the binding of biotin to the proteins expressed on the surface of the radial glia ( Figures  1A-1C). We tested two different protocols of biotinylation, based on either covalent coupling to glycosyl side chains or covalent coupling to free amino groups of the proteins. We observed a systematic bias toward increased expression levels in old brains with the glycosylation-dependent biotinylation (Figure S1A), which might indicate age-dependent altered total glycosylation levels. While the majority of proteins identified by the glycosylation-dependent biotinylation was also identified by the direct amino acid binding biotinylation ( Figure S1B), we obtained a higher number of proteins with the latter protocol, and concentrated our further analysis on those results. Three replicates of 6-7 brains (2-year-old and 4-month-old, respectively) were prepared in parallel. To verify the localization of biotin anchoring, we fixed two brains and incubated them with streptavidin-AlexaFluor 555. Both young and old brains revealed a specific surface anchorage of biotin with a minor penetration below the row of radial glia , indicating the validity of the method. After biotinylation, brains were dissected into pallium and subpallium ( Figure 1B) in order to separate the apical part of the radial glia located on the dorsal surface, where we expect cell-to-cell communication to take place, from their basal endfeet located on the ventral pial surface. After tissue lysis, biotinylated proteins were bound to streptavidin beads, proteolyzed, and analyzed by mass spectrometry.
The non-biotinylated proteins, representing the remaining material of the telencephalon ( Figure 1C, lysate fraction) were also analyzed by mass spectrometry. We compared the results obtained by this approach, with proteins from sorted radial glia of the telencephalon of gfap:GFP transgenic fish (fluorescence-activated cell sorting [FACS] scatterplots in are shown in Figures S2A-S2F and 1J). These experiments revealed consistent clustering of the GFP-positive and GFP-negative fractions by principal component analysis ( Figure S2G), and an even distribution of proteins respectively up-and downregulated ( Figure S2H). The set of proteins enriched in the GFP-positive fraction contained expected proteins known to be expressed by radial glia (GFP, GFAP, S100b, FABP7a/BLBP, Vimentin) (Ganz et al., 2010;März et al., 2010) while the GFP-negative fraction was enriched in proteins present on differentiated neurons (Elavl3/HuC, Elavl4/HuD) (Adolf et al., 2006;Grandel et al., 2006) ( Figure 1K). A high number of proteins in the biotinylated fraction was not present in the FACS-GFP-positive cells, which might be due to a loss of proteins during the enzymatic dissociation of the cells. Conversely, a large proportion (79.3%) of the proteins isolated from the FACS-GFP-positive cells were also identified in the biotinylated fraction ( Figure 1L). According to our experimental design, this overlap is expected to be incomplete, as FACS-sorted cells contain in addition to plasma membrane the whole cytosolic fraction of radial glia ( Figure 1C). GeneRanker analysis of the FACS-GFP-positive fraction revealed overrepresentation of the insulin pathway, cannabinoid receptor 1 pathway, glial cell line-derived neurotrophic factor (GDNF) and platelet-derived growth factor receptor b (PDGFRb) signaling pathways (Table S1A). While sorting cells by FACS allows for the analysis of the whole content of proteins in a certain cellular type, the advantage of the biotinylation approach was to isolate proteins of intact, non-dissociated cells.
Phobius analysis was used to assess the proportion of proteins with transmembrane domains and signal peptides ( Figure 1M). The surface biotinylation technique led to a protein set enriched in membrane proteins compared with the lysate fraction and the FACS-sorted cells fraction. Of the proteins listed in the Genomatix Pathway System, 645 belong to the plasma membrane, 51 are localized in the cell cortex, 145 are cytoskeletal binding proteins, and 43 are cell-adhesion molecule binding proteins.
Taken together, comparison of the datasets obtained from sorted radial glia, biotinylated surface fraction, and the non-biotinylated lysate of the telencephalon (for original dataset files see Table S6) indicate that the biotinylation approach specifically isolates proteins expressed on the surface of the stem cell compartment.

Identified Proteins Indicate an Epithelial Character and Display a Group Associated with Lamellipodia and Filopodia
The radial glia, as opposed to embryonic neuroepithelial cells, do not form a columnar epithelium, but their somata are cone-shaped and are connected laterally to each other only by a thin apical junctional region. Their highly branched radial processes extend far into the parenchyme. Considering this morphology, it is interesting to define to which extent these cells reveal an epithelial character. In the list of identified surface proteins, members with specific apicobasal localizations appeared (Table 1). For instance, proteins involved in formation of tight junctions (i.e., Discs large 1 and 3, Junctional Adhesion Molecule 3, (B) Cross-section through the telencephalon of a zebrafish. Biotinylated surfaces are depicted as magenta dots. The upper surface of the pallium borders the ventricle, located below the tela choroidea (white arrow) and is composed of the cell somata of the radial glia (depicted in green in the inset). The midline between both hemispheres is also filled by a thin part of the ventricle. After the biotinylation reaction, telencephalons were separated into the dorsal part (pallium) and ventral part (subpallium). (C) Drawing of the fractions isolated by FACS and by biotinylation, depicting in green the radial glia, in dark blue the remaining cells of the telencephalon, in magenta the biotinylated fraction containing the cell surfaces of the radial glia, and in light blue the lysate fraction containing the remainder of these cells as well as the rest of the telencephalon. Metadherin, Striatin, Ankyrin3) and in cell-cell adherens junctions (i.e., Dystroglycan 1, N-Cadherin, Catenin-b1, -a1, and -a2, Desmoplakin) were identified (Table 1). Therefore, even if this adult ventricular surface on the dorsal pallium is classically not considered as a neuroepithelium, these cells that pave the whole ventricular surface express epithelial marker proteins.
We observed a group of proteins involved in filopodia and lamellipodia formation (Table 1), such as CDC42, RAC1 and -2, DOCK 3 and 10, Integrin a 5b2 and b7, RUFY3, BRK1, Formin-like 3, and Fascin 1a and 1b. The high ramification of radial glia in the parenchyme (inset in Figure 2C) might explain this finding. However, as the biotinylation was specifically targeting the apical surface and could not reach the deep processes, we wondered whether filopodia are present apically. Indeed, different expression levels occurred predominantly between the ventral and dorsal domains of the telencephalon (Figure 2A). We performed morphological analyses of single cells by lipofections in vivo, using either a combination of two plasmids (EF1a: Gal4 and UAS: mtdTomato) or a single plasmid (pCS2-Lifeact-RFP) highlighting F-actin, to label the membrane of a few scattered cells. The infusion of the lipofection mix into the brain ventricle led in all cases to an exclusive expression in the radial glia ( Figures 2B-2D, yellow arrows). We took serial confocal pictures to obtain z stacks of whole-mount brains with mtdTomato membrane staining. Lamellipodia were observed in some cells that overlapped with the apical surface of a neighboring cell ( Figures 2E-2G). In the majority of analyzed cells, we identified at least one filopodium-like extension. The extension was either extending apically along borders between two cells ( Figures 2H-2J), reaching the second next cell, or extending from the basolateral cell surface and reaching apical locations of neighboring cells, furthest to the fourth gfap:GFP-positive cell ( Figures 2K-2P). We used a Lifeact construct and analyzed 23 cells from 3 sectioned brains. The cellular extensions contained Lifeact labeling, revealing the presence of F-actin ( Figures 2Q-2S). Co-lipofections of Lyn-GFP and Lifeact-RFP plasmids showed that the cellular extensions could be distinct in their composition, as a few of them were not labeled by Lifeact ( Figures 2T-2V 0 ).
Since the mass spectrometric analysis showed some differences with age in the expression levels of some filopodia-associated proteins, such as the downregulated Neuroligin 1 and FARP1, and the upregulated Flotillin 2, Gelsolin, Talin 2, and Src kinase signaling inhibitor 1 (Figure 2A), we compared morphologies and performed measurements of length and number of filopodia on 16 young (3-monthold) and 26 old (2-year-old) mtdTomato-labeled cells (Figure S3). Neither the mean size of these extensions, nor their numbers, varied significantly between young and old  brains ( Figures S3J-S3K). Nevertheless, possible structural alterations might exist and will need to be examined in future studies. Together, these results reveal cellular extensions between the cell bodies of NSCs, which might promote cell-to-cell communication ranging up to 4 cells apart.
Signaling Pathways Active in the Surface Fraction Besides a possible communication via filopodial extensions, other candidates might relay intercellular signals, such as the gap-junction protein Cx 43, or Cx 28.8 identified in the GFP-positive FACS fraction. We further identified a high number of proteins (557) associated with extracellular exosomes that might convey signals.
We examined pathways significantly overrepresented on the dorsal versus ventral side of the telencephalon, hence likely involved in the communication at the apical location of the radial glia. GeneRanker analysis revealed among others the planar cell polarity, brain-derived growth factor, Semaphorin, and Eph receptor pathways (Table S2).
Cell-surface receptors and their differential expression are listed in Figure S4A. We identified, for instance, Notch3 as well as Dner, another Notch family member, and receptors for GDNF, ciliary neurotrophic factor (CNTF), PDGF, epidermal growth factor (EGF), bone morphogenetic protein (BMP), FGF, and WNT. Many of these receptors and ligands were missing in the proteins identified from cells isolated by FACS, possibly due the enzymatic dissociation. We nonetheless confirmed the expression of these signaling molecules in the radial glia by RNA sequencing (RNA-seq) analysis of FACS-sorted GFP-positive and -negative fractions ( Figure S4B).
Following the intriguing finding of filopodia on the radial glia, we tested whether they would relay signals identified here in the biotinylated fraction, similarly to results obtained in other cells with filopodia (Prols et al., 2016). We investigated the co-localization of two signaling pathways in the cellular protrusions, Wnt and EGF.
The localization of Wnt signals was examined (Stanganello et al., 2015) in NSCs co-lipofected with Wnt8a-mcherry and Lyn-GFP. Lipofected cells did reveal a dotty localization of Wnt8a-mCherry ( Figures S5B, S5E, S5H, and S5K), also at the edges of the cell soma close to neighboring cells. However, no clear co-localization with filopodial extensions could be identified (Figures S5A-S5I). In a second approach, we expressed a GFP-tagged secreted Wnt receptor SFRP1a-GFP in HEK-293 cells and collected the supernatant, then applied it on fixed floating brain sections. As a positive control, lipofected cells with Wnt8a-mCherry did not show a strong specific co-localization of SFRP1a-GFP ( Figures S5J-S5L). Cells lipofected with Fyn-RFP to visualize the filopodia did not reveal co-localization of the SFRP1a-GFP (Figures S5J-S5L). We therefore conclude that with the methods used here, filopodial extensions in adult NSCs do not seem to convey Wnt signaling.
The localization of the EGF receptors was probed with an AlexaFluor 488-EGF complex that binds EGF receptor (EGFR) (Pastrana et al., 2009). Sections with cells lipofected with mtdTomato were incubated with the green fluorescent complex, revealing the localization of EGFR on the radial glia ( Figures S5P-S5W). The AlexaFluor 488-EGF complex did not co-localize specifically with filopodial extensions. Hence, potential signals relayed via filopodia to neighboring radial glia remain to be determined in further studies.
As we were looking for signals involved in the regulation of recruitment to the cell cycle, we next focused on pathways differentially regulated in young and old brains.
The IGF Signaling Pathway Diminishes with Age, and the Mitotic Response to IGF2b Becomes Incomplete The differential protein content in young and old surface fractions revealed an enrichment in proteins involved in G 1 /S transition in the young brain, and an enrichment in proteins involved in the P21 (CDKN1A) pathway in the old brain (Tables S3 and S4), in accordance with previously observed age-related decrease in mitotic activity (Edelmann et al., 2013). Hierarchical clustering and principal component analysis revealed consistent results between replicates ( Figures 3A and 3B). The proteins up-and radial glia are labeled by the gfap:GFP transgene. (C) A small, variable number of cells per brain were labeled by the in vivo lipofection (maximum 12 cells per brain); their somata and branched radial processes into the parenchyme are visible (inset is a higher magnification), revealing the soma at the top (apical side) and the radial process in the parenchyme with numerous branches. All lipofected cells displayed this radial process, but it is not visible on all pictures. downregulated with age were evenly distributed (Figure 3C). Several components of N-Cadherin signaling were upregulated in young surfaces (N-Cadherin signaling components, p value 6.14 3 10 À3 : DAGLA, CDH2, CDC42, CTNNA1, KIF5B), suggesting that cell-cell interactions might be modified with age. However, other components of this signaling pathway remained unchanged (CTNNB1, CNR1, GRIA2, CTTN, GSN, GJA1).
Proteins on the NSCs' surface and in the lysate revealed a decrease in IGF signaling with age ( Figures 4A and 4B), especially for the receptors IGF1R and insulin receptors. In zebrafish, two igf1r genes, igf1ra and igf1rb, are orthologous to the mammalian Igf1R and play complementary roles during development (Schlueter et al., 2006). Both were decreased on the surface fraction of aged brains. The insulin receptor InsR-B also declined with age, while the level of InsR-A expression did not change. IGF ligands were not detected in the proteomic analysis, but were detected by RNA in situ hybridization ( Figure S6), suggesting that they might act as autocrine or paracrine factors, as in mammalian adult neurogenic areas (Bracko et al., 2012;Lehtinen et al., 2011). Phosphorylated (phospho-)IGF1R expression was visible in immunostainings in the layer of radial glia, as well as in a few closely located cells in the pa- IGF2b was cloned into a 53 UAS plasmid for overexpression by in vivo lipofections. Fish were lipofected with a red-labeling plasmid alone or in combination with igf2b. Control lipofections with two differently colored plasmids resulted in cells receiving both plasmids in 90% of the cases (27 of 30 cells co-lipofected with plasmids for a green cytoplasmic staining and for a membrane-bound red staining revealed expression of both colors). After 2-4 days, we observed an increased expression of phospho-IGF1R in the cells lipofected with igf2b ( Figures 5E-5H, arrows in 5G and 5H) as well as in their neighbors, indicating the expected effect of IGF2b expression. The phospho-IGF1R expression in aged brains, upon either control lipofections (Figures 5I-5L) or igf2b lipofections ( Figures 5M-5P), showed a lower basal expression than in young brains, and IGF2b overexpression was able to induce a few dots of phospho-IGF1R expression (arrows in Figures 5O and 5P). However, the expression was not as strongly increased as in young brains, in line with the decreased receptor expression with age observed by mass spectrometry. Quantification of the fluorescence confirmed higher expression levels in igf2b-lipofected cells compared with control lipofections in young brains, but no significantly detectable change in old brains ( Figure 5Q). We tested another downstream target of IGF2b signaling, phosphorylated Akt (Ser473), which revealed only very low levels of expression ( Figure S7).
To test for functional downstream effects of IGF in the adult NSCs, we analyzed the putative proliferative effect 2-5 days after lipofection in 6 independent experiments. Overexpression of IGF2b in young brains (2-4 months old) resulted in a higher proportion of lipofected cells that revealed a recent mitosis, being visible as two neighboring cells immediately abutting each other (Figures 6A-6H and 6X; Table S5). Even if the cell division response to (C-J) Phosphorylated IGF1R (P-IGF1R) immunohistochemistry (Tyr1161) on young (5 months, C-F) and old (2 years, G-J) brains as a closeup view on the ventricular surface of the dorsal pallium depicted as single confocal planes. The radial glia somata, located at the border of the ventricle, express P-IGF1R (yellow arrows). We observe the distinct expression levels of P-IGF1R in young and old brains in a total of 4 young and 4 old immunosamples. Scale bar, 10 mm. Single cells in magenta have been lipofected in vivo 4 days prior to brain fixation with mtdTomato (control, A-D and I-L) or with mtdTomato and igf2b (IGF2b, E-H and M-P), highlighting primarily the cell soma (the radial process is not always in the plane of the optical section, but it is present below the somata of lipofected cells). We observe the distinct expression levels of P-IGF1R in young control (C and D), young + IGF2b (highest, arrows in G and H), old control (K and L), and old + IGF2b (a few dots, arrows in O and P). Insets in (D), (H), (L), and (P) are close-ups of the lipofected cells; nuclei in blue stained by DAPI. Single confocal planes are displayed. Scale bar, 10 mm. (Q) The mean intensity of the phospho-IGF staining was measured on 7 lipofected cells and their immediate neighbors in each condition. Staining intensities differ significantly between control and igf2b-lipofected brains (t test; p = 0.030) in young but not in old brains.
IGF2b was indeed observed in a higher proportion than in control cells, it did not occur in all transfected cells, suggesting that the cells do not all have the same capacity to respond. Note that in Figures 5E-5H, the lipofected cell has divided and encompasses two cell bodies, even if the two cells are incompletely covered by the membrane staining. Overexpression of IGF2b in old brains was able to also induce the formation of two nuclei; however the division was incomplete, lacking cytokinesis, and led to the formation of very large cells ( Figures 6I-6P and 6X). More time might be required for old cells to undergo cytokinesis, so we extended the lapse to 5 days, which revealed, however, the same effect of binucleation in old cells ( Figures 6Q-6S and 6W). We also observed that some cells next to the igf2b-lipofected cells revealed the same phenotype of binucleated cells, as they might respond in a paracrine manner to IGF (Figures 6T-6V). The incomplete divisions might be the result of the lowered levels of IGF receptor, and the exact downstream mechanism will require closer examination. Together, our results show that aged cells are not competent to respond and divide properly to an increase of growth signals encountered, hence indicating significant cell-intrinsic alteration in the course of aging.

DISCUSSION
With the aim of understanding better how NSCs communicate and organize their cell-cycle entries, we identified the proteins expressed on their surface in young and aged brains. The use of the zebrafish brain, offering direct access to NSCs without preceding dissociation, combined with a biotinylation method, permitted us to obtain the surface proteome of an intact NSC population. From there we showed the presence of apically located filopodial structures and a changing response to IGF with age. Hence, two important aspects are revealed herein: a possible path of intercellular communication via cellular extensions, and cell-intrinsic alterations impacting on the proliferative competence of aging cells.
One of the two biotinylation protocols employed in this study, involving a covalent binding reaction between glycosylated residues and biotin, revealed a strong bias for proteins increased in aged samples, suggesting increased glycosylation patterns with aging. Age-related changes in glycosylation patterns in the brain have previously been reported (Miura and Endo, 2016), and a closer investigation of these alterations will be important.
The identified proteins in the surface fraction were in accordance with previous findings in the zebrafish or other adult neurogenic systems. For instance, Connexin 43, identified here on the adult radial glia in zebrafish, plays a role in the adult neuroepithelium in mammals (Lacar et al., 2011). The representation of exosome components in our study is interesting in the context of their newly described roles in adult neurogenesis (Batiz et al., 2015). We also identified Notch3 and Dner, in keeping with the role for the Notch signaling pathway described in previous studies (Alunni et al., 2013;Chapouton et al., 2010;Ehm et al., 2010;Hsieh et al., 2013;Katz et al., 2016).
Our study reveals protein members that allow defining an epithelial identity of the adult NSCs, a question that was also addressed by Grupp et al. (2010), who observed the presence of ZO-1 in those cells. A new feature of the cells described here is the presence of lateral lamellipodial and filopodial extension from the somata of the radial glia. The filopodial extensions are contacting neighboring cells further than the next neighbor (up to the fourth, most frequently the second), an aspect that might be important for cell-to-cell communication over several cell distances. Filopodia can convey signals in developing tissues (Prols et al., 2016), such as Wnt signals (Stanganello et al., 2015) or Notch signals (Cohen et al., 2010). Such signals remain to be identified in these adult cells, and could possibly be involved in coordinating the cell-cycle activity. A feedback signaling between cycling cells and their neighbors has been proposed previously in this system via the Notch signaling pathway .
The activity of the insulin and IGF signaling pathway is important in several developmental and regenerating (Q-W) Brains fixed 5 days after igf2b lipofection also reveal binucleated large cells (arrows in Q, R, and W), visible here in two distinct confocal planes (Q-S and T-V). Some neighboring cells reveal the same phenotype (arrowheads in Q, T, and U). (X) Cells having divided after lipofection were quantified in 6 experiments with a total of 69 young-control, 114 young-igf2b, 64 oldcontrol, and 71 old-igf2b cells. The significance was calculated by a chi-squared test (young, ***p = 0.7 3 10 À3 ; old, *p = 0.01). Scale bars, 10 mm. systems and shows multiple effects at distinct ages. These pathways are generally associated with nutrient sensing, growth, and metabolism, and play important roles in the homeostasis of NSCs (Rafalski and Brunet, 2011;Ziegler et al., 2015). IGF signaling induces proliferation and regulates the length of the cell cycle Mairet-Coello et al., 2009;Nieto-Estevez et al., 2016;Popken et al., 2004;Yuan et al., 2015), in some instances acting in combination with other growth factors, such as the EGFR (Alagappan et al., 2014). With age, systemic and brain levels of IGF ligands are reduced (Rafalski and Brunet, 2011;Shetty et al., 2005). These properties suggest that a maintained IGF pathway would be advantageous for counteracting an agerelated proliferative decline. Indeed, infusion of IGF could restore higher levels of proliferation and neurogenesis in aged rats (Lichtenwalner et al., 2001). In contrast, most of the current literature suggests that inhibition of this pathway is beneficial for animals' longevity (Kappeler et al., 2008;Lopez-Otin et al., 2016), an effect also being observed in dwarf mutant mice (Sun, 2006). For instance, neurogenesis is maintained longer when IGF1R is conditionally deleted in the hypothalamus (Chaker et al., 2016) and SVZ (Chaker et al., 2015). Our results add a new aspect, as they show that NSCs lose their responsiveness to the IGF pathway with age. The reduction takes place both at the level of expression of the receptors and at the level of the proliferative response, remaining incomplete in old brains. Hence, we identify a cell-intrinsic change that might represent a protective response of cells. Collectively these observations lead to the concept that declining levels of neurogenesis with age could be, as opposed to a deficit, an actual benefit linked with healthy aging, whereby cells reduce at the same time their proliferative and general metabolic activity (see Hamilton et al., 2013). Thus, some of the changes observed with age might be the ones that promote longevity.
We have previously shown that aged radial glia are activated in lower percentage by an injury protocol than are young cells (Edelmann et al., 2013). Together with the results presented here, this indicates significant cell-intrinsic changes with age. It will be fundamental in the future to understand the respective contributions of cell-intrinsic changes and signals received from the neighborhood in order to understand under which conditions manipulations to rejuvenate aging tissue can be successful.

EXPERIMENTAL PROCEDURES
See Supplemental Information for details of all experimental procedures.

Animal Maintenance
Zebrafish were bred and maintained in the animal facility of the Helmholtz Zentrum München. Wild-type zebrafish (AB) or the transgenic line gfap:GFP (Bernardos and Raymond, 2006) (mi2001) in the AB background were used. Animal experiments were conducted in accordance with the laws of the government of Oberbayern (animal protocol 55.2-1-54-2531-83-14).
Further information and requests for resources and reagents should be directed to and will be fulfilled by P.C. (chapouton@ helmholtz-muenchen.de).

Supplemental Information includes Supplemental Experimental
Procedures, seven figures, and six tables and can be found with this article online at https://doi.org/10.1016/j.stemcr.2018. 12.005.

AUTHOR CONTRIBUTIONS
J.O. performed the gene ontology analysis, generated all graphs representing the proteomics dataset, and wrote the proteomics methods; F.W. produced the SFRP-EGFP construct; A.K. contributed to the FACS experiments; A.Z. performed the RNA-seq experiments on FACS isolated cells; J.N. analyzed the RNA-seq results; S.M.H. designed the biotinylation protocols and analyzed the mass spectrometry data; P.C. conceived the study, performed the experiments, prepared the figures, and wrote the manuscript. signaling is necessary for epidermal growth factor mediated proliferation of SVZ neural precursors in vitro following neonatal hypoxia-ischemia. Front. Neurol. 5, 79. Alunni, A., and Bally-Cuif, L. (2016). A comparative view of regenerative neurogenesis in vertebrates. Development 143, 741-753.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Glycerylphytate compounds with tunable ion affinity and osteogenic properties

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Results and Discussion
Synthesis and characterization of GPhy derivatives. The main goal of the present work was to obtain phytate derivatives with improved cytocompatibility without sacrificing the interesting biological properties of PA, and at the same time, to enlarge their applications in the biomedical and pharmaceutical fields. Thus, two phytate derivatives have been prepared by introducing glyceryl moieties in the chemical structure of PA, obtaining two compounds that were named as G 1 Phy and G 3 Phy according to their G content, as it will be explained below. This type of strategy has been previously reported for different polyol-phosphate derivatives synthetized as prodrugs with enhancing bioavailability 26 . Figure 1 displays the synthetic procedure applied in the preparation of GPhy. Based on the work of He et al. 27 who proposed a 5-axial/1-equatorial structure of PA at alkaline solutions, we assumed a similar structure for solid PA. As illustrated in the synthetic scheme, there are different possibilities of reaction between the reactants due to the six phosphate groups in the PA structure and the two hydroxylic groups in the G molecule. In addition, other factors such as the PA:G ratio, the conformation of the cyclohexane ring and steric factors will have influence in the final reaction products 28 . In order to have an insight of the stoichiometry of the two condensates, structural characterization was performed. Table 1 shows the results obtained from Elemental analysis (EA) and ICP. The formulas were deduced taking also into consideration the results of loss of water and percentage of residue at 800 °C obtained from (Thermogravimetric Analysis) TGA of PA and both glycerylphytates. Results indicated an average number of 1 glyceryl residue for the compound obtained with a molar ratio PA:G of 5:1 and an average of 3 glyceryl groups for that obtained with a molar ratio PA:G of 1:7 (G 1 Phy and G 3 Phy compounds, respectively). This showed an agreement between the amount of G in the feed and the conjugation of glycerol moieties with phosphate groups. Additionally, the results suggested that the conjugation reaction was also determined by steric hindrance since the number of glyceryl conjugated moieties increased from 1 to 3 when the PA:G molar ratio varied from 5:1 to 1:7. EDX analysis confirmed the presence of expected elements and the absence of impurities (spectra not shown). The quantification of the elements content is shown in Fig. S1 and results mainly reflected an increase of carbon content in the G 3 Phy sample respect to those of PA and G 1 Phy as it was expected due to the higher G conjugation in the former derivative. These results correlated well with those obtained by EA. Figure 2 shows the infrared spectra of PA and GPhy derivatives. PA spectrum showed a band at 3284 cm −1 due to the stretching vibrations of O-H groups, which suffered a broadening in the spectra of GPhy derivatives with the glyceryl content showing the contribution of the additional OH groups. The same observation was found in bands at 2974 and 2892 cm −1 , corresponding to asymmetric and symmetric stretching vibrations of C-H bonds of inositol rings and glyceryl groups. The intensities of these bands increased in both GPhy compounds, but the rise was considerably remarkable in G 3 Phy due to the higher G conjugation. This feature was confirmed by the band at 1460 cm −1 attributed to δ CH 2 groups in glyceryl groups as we can observe in the region of interest (1500-400 cm −1 ) in Fig. 2 29 . PA spectrum also showed a band at 1192 cm −1 corresponding to stretching vibration of P=O groups, and two bands at 1056 cm −1 and 913 cm −1 , which were attributed to P-O and C-O-P stretching vibrations in COPO 3 groups. Similar assignment for other phytates was given by Ishiguro et al. 30 and Guan et al. 31 , who also assigned the two absorption bands at 1192 and 1056 cm −1 to asymmetric and symmetric stretching vibrations corresponding to P-O in HPO 3 − groups in alkaline conditions. In the synthetized glycerylphytates these absorption bands shifted to lower wave numbers respect to PA. In particular, the band due to υ P=O groups shifted from 1192 cm −1 in the PA spectrum to 1185 cm −1 in the GPhy spectra, and the intense peak at 1056 cm −1 attributed to υ C-O alcohols, υ P-O and P-O-C, and υ C-O glycosidic, moved to 1035 cm −1 in the GPhy spectra. Finally, the absorption band at 970 cm −1 assigned to -PO 3 groups, and peaks at 915 and 851 cm −1 assigned to υ P-O and P-O-C bonds shifted respect to PA. All these findings observed in the GPhy spectra indicated changes in the chemical environment of phosphate groups because of the condensation reaction 27,30,32 .
The GPhy compounds were subsequently characterized by NMR spectroscopy. The 13 C NMR spectra of G 1 Phy and G 3 Phy and precursors are shown in Fig. S2a. The spectrum of PA showed four peaks at 74.7, 76.8, 77.5 and 78.9 ppm. The assignment of these peaks was performed according to previous work of Crimella et al. 33 as follows: δ 75.3, C 4 and C 6 ; δ 78, C 2 ; δ 78.5, C 1 and C 3 ; and δ 79.3, C 5 . The 13 C-NMR spectrum of G showed a peak at 63.9 ppm due to CH 2 carbons (C α ) and a peak at 73.5 ppm assigned to the CH carbons (C β ). Spectra of both  derivatives were rather similar to each other and revealed notable changes in their signals respect to precursors, indicating the presence of different C atoms after the condensation reaction. 13 Figure S2b shows the 1 H NMR spectra of both GPhy and precursors. Spectrum of G showed two doublet-of-doublet between 3.53 and 3.66 ppm that were assigned to no chemically equivalent H α protons and a multiplet signal between 3.75 and 3.80 ppm assigned to H β protons 34 . Spectrum of G 3 Phy showed modified signals in the range 3.50-3.82 ppm and two new multiplet signals at lower field; one of them was in the range 3.82-3.98 ppm and it was tentatively attributed to H α and H β protons in the groups CH 2 -O-P and CH-O-P of the glyceryl group. The other multiplet appeared in the range 4.05-4.10 ppm and it was attributed to H β protons of CH-O-P groups in the glyceryl moieties. The spectra of G 1 Phy differed somewhat respect to that of G 3 Phy. The main difference lied in that the new multiplet in the range 3.82-3.98 ppm almost disappeared and the signal in the range 3.98-4.10 ppm attributed to resonance of H β protons in glyceryl CH-O-P groups increased 33,35 . Proton NMR analysis of GPhy derivatives varying in glyceryl content suggested that for low G content in the feed, the reactivity of β hydroxylic groups towards phosphate groups would be favoured whereas an increase in the G content would enhance the reactivity of α hydroxylic.
This hypothesis was further studied through 2D DEPT-HSQC NMR analysis. In this concatenated experiment, the carbon multiplicity information obtained from the DEPT-experiment (CH or CH 2 in our products) was detected in the 2D HSQC spectrum and illustrated in different colours (Fig. 3). In the DEPT spectra of both glycerylphytates, carbons in the range 60-67 ppm appeared in red colour (CH 2 ) and carbons in the range 67-75 ppm appeared in blue colour (CH). Considering this information, in particular, in the 2D DEPT-HSQC spectrum of G 3 Phy the intense signal at 3.8/65.8 ppm was attributed to the glyceryl CH 2 -O groups conjugated with PA through CH 2 -O-P bonds and the signal at 3.8/70.7 ppm of lesser intensity, to conjugated CH-O groups of glyceryl moieties forming CH-O-P bonds. In addition, another two intense signals were present in the 2D spectrum, one of them at 3.5/62.2 ppm that could be due to CH 2 -O groups of reacted G, and the other one at 3.5/72.2 ppm due to reacted CH-O groups in the glyceryl moieties. As far as G 1 Phy is concerned, its 2D DEPT-HSQC spectrum showed that the majority of the signals were due to association of protons with CH carbons (blue colour). Moreover, the 2D plot of G 1 Phy showed less intense signals at 3.8/65.8 and 3.5/62.2 ppm as observed in G 3 Phy and instead, intense signals at 4.1/72.4 and 4.1/78.5 ppm. These findings suggest that conjugation reaction preferably proceeds between the β hydroxylic groups of G when this component is at low concentrations in the feed, whereas for higher G contents, the α hydroxylic groups are more likely to react, confirming the hypothesis previously proposed.
Thermal properties of GPhy derivatives were analysed by Differential Scanning Calorimetry (DSC) and TGA techniques. DSC thermograms revealed a glass transition temperature of 50 °C for both derivatives (thermograms not shown) indicating the amorphous character of these compounds because of the conjugation of glyceryl moieties in their structure. Table S1 summarizes the thermal degradation results of GPhy compounds and their precursors under inert atmosphere. Weight loss of glycerylphytates underwent in three stages in contrast to PA which showed four degradation steps. First stage was due to loss of water, 2 or 3 molecules for G 3 Phy and G 1 Phy, respectively, as explained above in the molecular formula analysis ( Table 1). The second stage involved carbonization of conjugated glyceryl moieties and the dehydration of phytate structure by the decomposition of hydroxylic groups. Finally, the third and last degradation stage of GPhy (T max around 340 °C) corresponded to further decomposition of the phytate moieties (332 °C) 36 . The residues of both derivatives obtained at 800 °C notably decreased in comparison to PA, indicating the content of glyceryl groups after the condensation reaction. Antioxidant properties. PA exhibits a powerful antioxidant activity due to its ion chelating capacity and hence, it is able to prevent Fenton reaction by inhibiting iron-catalysed hydroxyl radical formation 1,14,16,37 . This property has repercussions on the bone mass loss, as it is known that iron-induced oxidative stress is directly linked to the activation of NF-kβ, a key element in osteoclastogenesis 18,21,38,39 . Then, to investigate the antioxidant properties of GPhy compounds, their chelating activity was examined by the ferrozine/FeCl 2 system and compared with that of PA 15 . As it can be observed in Fig. 4a, PA and GPhy antioxidant properties were concentration dependent. Thus, ferrous chelating activity for PA was 33, 60, 73 and 78% at 10, 50, 100 and 300 µg/mL, respectively. Zajdel et al. 16 reported similar results regarding the powerful inhibitory capacity of PA in iron-catalysed oxidative reactions. The GPhy compounds exhibited a significantly lower antioxidant activity than PA at the lowest tested concentration (10 µg/mL). However, their antioxidant activity increased with concentration in both compounds, achieving values of 60% and 54% for G 1 Phy and G 3 Phy respectively, at 100 μg/mL. In particular, the chelating activity of G 3 Phy at all tested concentrations was significantly (p < 0.01) lower compared to PA. G 1 Phy, on the other hand, exhibited similar behaviour to G 3 Phy in the range of 10-100 µg/mL, while antioxidant activity reached to 84% at 300 µg/mL, being no significantly different (p < 0.5) from PA value. These results indicate that antioxidant properties of GPhy compounds measured by ferrozine/FeCl 2 method are not only concentration dependent as occurs with PA, but also composition dependent, showing antioxidant activity in the range of 50-80% for G 3 Phy and G 1 Phy respectively, at the highest studied concentration. This behaviour can be related to the incorporation of glyceryl moieties to PA structure, which reduces the number of ionizable protons and thus chelating properties.
Calcium binding properties. Calcium deficiency plays a crucial role in bone-loss related diseases such as osteoporosis. Although calcium absorption can be affected with age, some dietary factors, and pharmaceutical compounds, can also act as potent chelating agents of calcium ions making them unavailable 40  www.nature.com/scientificreports www.nature.com/scientificreports/ have been focused on the adverse effects of mineral uptake mediated by PA and its strong chelation capacity, concluding that the bioavaibility of these essential minerals can be compromised 40,[42][43][44] . Particularly, magnesium and calcium deficiency has been linked to osteoporosis.
In this work, the Ca 2+ binding capacity of GPhy derivatives was studied and compared with that of PA. After dropping a concentrated solution of the tested compound into a 4 mM solution of CaCl 2 , the precipitation of a solid, white or brown for PA and GPhy compounds respectively, was observed. The pellets were isolated and analysed by ICP spectroscopy. In Fig. 4b, the stoichiometry of the Ca-phytate complexes at pH 7.5 is expressed in moles Ca /moles Phy where Phy can be PA, G 1 Phy or G 3 Phy. The stoichiometry of Ca-G 1 Phy complexes was quite similar to that of Ca-PA complexes. Interestingly, a notable decrease in the chelating capacity of G 3 Phy was obtained. This can be explained by the higher content of glyceryl moieties in this derivative in comparison to G 1 Phy. These results confirm that due to the introduction of glyceryl groups, G 3 Phy derivatives show lower chelating activity than PA and they show how this modification can tune their binding ability.
In vitro biological effect of GPhy derivatives. Lipid peroxidation assay on RAW264.7 cells. Lipid peroxidation inhibition was measured in vitro by MDA-TBA adduct formation after the induction of Fenton reaction with the Fe 2+ /H 2 O 2 system on RAW264.7 cells. Figure 5 shows the results of the inhibitory effect on lipid peroxidation for PA and its derivatives at 100 μg/mL concentration. A higher amount of MDA-TBA adduct is indicative of a higher oxidative stress, therefore a 100% MDA-TBA was the maximum lipid peroxidation induced www.nature.com/scientificreports www.nature.com/scientificreports/ in the cells (positive control). When cells were incubated with PA or its derivatives, iron-loaded related stress was reduced due to their strong ability to chelate iron species. A higher MDA concentration was observed for G 3 Phy as compared to PA, which indicated a decrease in the ability to chelate iron ions as consequence of the higher content of glyceryl groups in its structure. Nevertheless, this compound was still able to prevent lipid peroxidation, as previously described for hydrolysis products of PA which maintain three or more phosphate groups 45,46 . Accordingly, G 3 Phy exhibited reasonable antioxidant iron stress properties in RAW264.7 cells in comparison to positive control. On the other side, G 1 Phy showed lipid peroxidation inhibition results comparable to PA, which correlated well with its antioxidant data obtained in previous analysis (Fig. 5). These results demonstrate that G 1 Phy and G 3 Phy maintain and modulate both iron (II) chelation and antioxidant properties of PA in cell cultures using the RAW264.7 cell line.   49 reported that PA extracted from rice bran was not toxic up to 6 mM for 72 h in untransformed cultures of 3T3 cells, yielding < 10% of dead cells 49 , Similar results were reported by de Lima et al. 50 who did not observe PA cytotoxicity at 4.0 mM for 48 h in lymphocytes isolated from healthy human donors. In this work, cytotoxicity (IC 50 ) of PA and derivatives was tested in MSCs cultures using a standardized Alamar Blue assay. Viability of cells treated with G 1 Phy or G 3 Phy increased respect to PA. IC 50 value of PA was 12.53 ± 2.21 mg/mL (14.5 ± 2.55 mM), while IC 50 values of G 1 Phy and G 3 Phy were 22.81 ± 2.64 (23.41 ± 2.7 mM) and 29.81 ± 2.43 mg/mL (27.45 ± 2.23 mM), respectively. These results suggest that larger amounts of glycerylphytate derivatives than of PA could be safely used in different specific biomedical applications.
Osteogenic properties and biomineralization on MSCs. The osteogenic activity of glycerylphytates and PA was investigated in MSCs cultures using Alamar Blue assay. To that end, firstly MSCs cultures were treated with the tested compound at different concentratios for 1, 7 and 14 d. Figure 6 shows that the metabolic activity of treated cells was affected by each tested compound in a different trend depending on dose and time. Cellular viability progressively decreased with concentration and time at 7 and 14 d and the lowest values were obtained at 150-300 µg/mL. G 3 Phy did not affect cell viability and even enhanced it at 300 µg/mL after long-term incubation time (14 d). The explanation of this enhancement is not clear yet but could be related with the less cytotoxicity of G 3 Phy which has an IC 50 value nearly double than that of PA. The behaviour of G 1 Phy approached that of G 3 Phy for a concentration range between 10 and 150 µg/mL and only a deleterious effect was observed when cells were treated for 14 d with the highest tested dose. These findings indicate that composition of the developed glycerylphytates plays a dominant role in their in vitro cytocompatibility and support previous results on their less cytotoxicity respect to PA.
Since PA has been suggested to be involved in osteogenesis 3,5 , ALP activity, an early marker of osteoblast differentiation, was determined in MSCs treated with the compounds. Figure 7 depicts ALP activity of MSCs treated with 10 μg/mL PA or glycerylphytate compounds for 7 and 14 d in complete or differentiation LG-DMEM media. At the two incubation periods, ALP activity was higher in MSCs cultured in differentiation LG-DMEM than in complete LG-DMEM, either they were untreated or treated with compounds. Interestingly, a different response to the compounds was observed depending on the type of medium. In complete LG-DMEM, ALP activity of cells treated with PA or its derivatives slightly increased respect to control, except for the case of G 1 Phy, in which ALP remained unaffected after 7 d. However, a different behaviour was observed in MSCs cultured in differentiating medium. ALP activity of cells treated with PA or G 1 Phy for 7 d was similar to control, while treatment for 14 d resulted in lower ALP activity. Cells incubated with G 3 Phy showed an interesting behaviour as ALP activity was slightly lower at 7 d respect to control and PA, but at 14 d it reached higher values than those found for PA and G 1 Phy.
The mechanism by which PA participates in osteogenic processes is not well understood, but we can elucidate from these results that bioavailability of phosphate residues may be quite relevant. Furthermore, Addison et al. 3 proposed that the high negative charge of PA molecule also has an impact on cellular interaction and diffusion across the membrane. For this reason, a possible explanation can lie in the enhanced cell-material interactions for G 3 Phy compound due to its major organic content. This hypothesis can be supported by the fact that this kind of modifications has been traditionally used for the protection of active phosphate groups, as those from PA 26 . An improved cytocompatibility would also enhance the metabolic effects that PA can trigger, as it has been demonstrated in this work. Thus, we can conclude that composition of GPhy compounds can tune the differentiation process of MSCs in comparison to PA itself. www.nature.com/scientificreports www.nature.com/scientificreports/ Gene expression of osteogenic markers by RT-PCR. As previously mentioned, treatment of MSCs with PA or GPhy for 14 in complete LG-DMEM d enhanced ALP activity (Fig. 7b). Thus, further studies investigated whether these compounds regulate the expression, at the mRNA levels, of osteogenic differentiation markers in MSCs. Results showed that incubation of MSCs with PA for 14 d in complete LG-DMEM increased COL1A expression (Fig. 8a). Interestingly, COL1A mRNA levels in MSCs treated with G 1 Phy or G 3 Phy were higher respect to PA suggesting that GPhy compounds could potentiate the deposition of a collagen matrix, which nucleates mineral deposition during osteogenesis. On the other side, a previous study reported that treatment of hUC-MSCs with 4 µM PA for 14 d under osteogenic conditions increased ALPL expression 24 . Data obtained in the present work indicated that incubation of MSCs with higher doses (10 μm) of PA in complete LG-DMEM induced an increase in ALPL mRNA levels, suggesting that PA could promote the acquisition of osteoblastic features in MSCs populations even in the absence of osteogenic inductors. Notably, G 1 Phy and G 3 Phy also increased ALPL expression in MSCs and ALPL transcript levels in MSCs treated with G 1 Phy were higher than those obtained in G 3 Phy or PA treated cells (Fig. 8b). It was not surprising that higher ALPL mRNA levels in G 1 Phy treated MSCs were not associated with increased ALP activity levels (Fig. 7b), as gene expression at the transcriptional levels does not necessarily correlate with protein activity, which is regulated by multiple post-transcriptional and post-translational mechanisms. Taken together, our results showed that PA and its derivatives might enhance MSC differentiation toward the osteoblastic lineage by regulating the expression of genes involved in matrix formation and maturation.

conclusions
In this paper, we report two new glycerylphytate derivatives (G 1 Phy, and G 3 Phy) which have different content of glyceryl groups. The reaction conditions allow to modulate chelation properties of the compound through the amount of glyceryl moieties incorporated. The new derivatives have improved cytocompatibility and osteogenic properties in comparison to PA in MSCs. Besides, both of them possess antioxidant and in vitro lipid peroxidation inhibition properties, which are essential features in tissue remodelling processes.
In summary, the chelation activity and biological properties of glycerylphytate compounds can be tuned by the organic content. This approach can offer a library of compounds with different composition to be used as an alternative to PA in biomedical and pharmaceutical applications related with stress oxidative phenomenon and low bone mass density.

Methods
Synthesis of GPhy derivatives. PA sodium salt hydrate (PA) and glycerol (G) were purchased from Sigma-Aldrich and used without further purification. The synthesis of GPhy compounds was carried out at 120 °C in bulk by reacting PA with the corresponding G volume for 12 h. The reaction products were dissolved in water, precipitated twice in 2-propanol, dried under reduced pressure to remove 2-propanol trace and lyophilized. Two ATR-FTIR spectra were obtained on a Perkin-Elmer (Spectrum One) spectrometer equipped with an ATR accessory for all samples. EA was performed with an elemental LECO model CHNS-932 microanalyzer. EDX spectrometry analysis was performed with a Bruker XFlash model with detector 5030. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were carried out in a 4300 DV Perkin-Elmer plasma emission spectrometer using a Gemcone (Perkin-Elmer) nebulizer. DSC experiments were carried out on a DSC model 8500 (PerkinElmer). Three heating-cooling cycles were analysed between 25 °C and 180 °C with a scanning rate of 10 °C/min under nitrogen at 20 mL/min flow rate. From the thermograms, the glass transition temperature (T g ) was determined as the midpoint of the transition. TGA was performed in a thermogravimetric analyzer TGA Q500 (TA instruments) apparatus, under dynamic nitrogen at a heating rate of 10 °C/min in a range of 40-800 °C.
Formation of ions chelate complexes. The iron chelate complexes formation was studied by the method of Dinis et al. 15 PA and GPhy compounds at various concentrations (10-300 µg/ml) in dH 2 O (0.4 mL) were added to a solution of 2 mM FeCl 2 (0.05 mL, Sigma Aldrich). The mixture was shaken vigorously and left at room temperature for 15 min under darkness conditions. Then, 0.2 mL of 5 mM ferrozine (Sigma Aldrich) solution was added, and the total volume was adjusted to 4 mL with ethanol. Absorbance of the solution was measured spectrophotometrically at 562 nm with a NanoDrop ™ spectrophotometer. 10 mM EDTA solution and dH 2 O were used as positive and negative controls, respectively. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± standard deviations (SD). Mean + SD values are relative to untreated MSCs, which were given an arbitrary value of 1. Analysis of variance (ANOVA) of the results for tested samples was performed with respect to untreated cells at significance levels of *p < 0.05, and with respect to PA treated cells at significance levels of # p < 0.05.
The binding capacity of PA and GPhy derivatives to calcium ions was examined with an adapted method from Saw et al. 51 . Particularly, aliquots of 50 µl at a concentration of 396 mg/mL of each compound were added to a 20 mL of a 4 mM solution of CaCl 2 in intervals of 2 min until achieving a final concentration of 5.27 mg/mL of each compound in the solution. The pH was controlled by preparing all the solutions in Tris-HCl 0.1 M buffer pH 7.5. The pellet was isolated by centrifuging at 4 °C and 8000 rpm (Eppendorf centrifuge 5810 R model) for 10 min, and washed with dH 2 O. The final pellet was digested at 65 °C with 65% v/v HNO 3 . Finally, the phosphorus and calcium content of these pellets in 5% v/v HNO 3 solution were measured by ICP. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD. cell culture. Human mesenchymal stem cells from bone marrow (MSCs, Innoprot) were grown and maintained in Mesenchymal Stem Cell Medium Kit (Innoprot) at 37 °C in a humidified atmosphere of 5% CO 2 . Cell culture media was refreshed every 48 h. For subsequent experiments, MSCs were cultured in Low Glucose Dulbecco's Modified Eagle Medium (LG-DMEM), supplemented with 20% fetal bovine serum (FBS), 200 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin (complete LG-DMEM). Differentiation medium consisted of complete LG-DMEM supplemented with dexamethasone (100 nM), ascorbic acid (50 μg/mL), and β-glycerolphosphate (10 mM). All the experiments were performed using MSCs at passages 4 to 8.
Murine RAW 264.7 macrophage cell line (Innoprot) were grown and maintained in DMEM supplemented with 10% FBS, 200 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin, at 37 °C in a humidified atmosphere of 5% CO 2 . Cell culture media was refreshed every 48 h and these cells were used at passages 5 to 6 for all the experiments.
Lipid peroxidation assay. Lipid peroxidation inhibition was tested in RAW264.7 cells, as macrophages are the major sources of oxidative stress and are used to evaluate the antioxidant function of dietary natural compounds 52 . 2 × 10 6 RAW264.7 cells were seeded in 6-well plates and incubated for 24 h with the corresponding solution of PA, G 1 Phy or G 3 Phy at final concentration of 100 μg/mL. For positive (lipid peroxidation induction but without any studied agent) and negative (no lipid peroxidation induction) controls, the same volume of dH 2 O than for the studied samples was added. Lipid peroxidation was induced by incubating the samples with 50 μM Fe 2 SO 4 ·7H 2 O and 200 μM H 2 O 2 solution at 37 °C for 4 h in order to trigger the Fenton reaction 53 . Lipid peroxidation was evaluated using the Lipid Peroxidation Assay Kit (abcam), following the manufacturer instructions. The percentage (%) of lipid peroxidation inhibition was calculated respect to the positive control, considering it as the 100% of MDA producer. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD.
Cytotoxicity. Cytotoxicity of PA and GPhy compounds was evaluated in MSCs cultures using the Alamar Blue assay. A stock solution of the corresponding tested compound in DMEM lacking FBS and containing 2% w/v D-sorbitol was prepared and successive dilutions were tested (5, 15, 20, 30, 50 mg/mL). 9000 of MSCs were seeded in 96-well plates and incubated for 24 h. Then, the medium was replaced and the cells were treated with the tested compounds for 24 h. A 10% v/v Alamar Blue (Invitrogen) solution was prepared in DMEM without phenol red and the plates were incubated at 37 °C for 3 h. Medium was collected and, after laser excitation at 590 nm, emitted fluorescence at 530 nm was quantified using a Biotek Synergy HT plate reader (Biotek Synergy HT spectrophotometer). cell viability. Alamar Blue assay was also used to analyse cell viability after incubation of MSCs with the tested compounds for 3, 7 and 14 d of. For this experiment, 104 of MSCs were seeded in 48-well plates and cultured in Mesenchymal Stem Cell Medium Kit for 24 h. Then, the medium was replaced, and the cells were treated with the tested solutions at various concentrations in complete LG-DMEM (10, 50,100, 150, and 300 µg/mL), and media containing the corresponding compounds were refreshed every 2 d. Medium was collected and, after laser excitation at 590 nm, emitted fluorescence at 530 nm was quantified using a Biotek Synergy HT plate reader (Biotek Synergy HT spectrophotometer). The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD. ALP activity quantification. ALP activity was evaluated after 3, 7 and 14 d of incubation of MSCs with the tested compounds. Total DNA amount was measured using the PicoGreen dSDNA quantitation kit (Molecular Probes). Determination of the ALP/DNA ratio is indicative of the amount of ALP activity per cell. For this experiment, 10 4 of MSCs were seeded in 48-well plates and cultured in Mesenchymal Stem Cell Medium Kit for 24 h. Then, the medium was replaced, and the cells were treated with PA and its derivatives solutions at 10 µg/mL. The media containing the corresponding compounds were refreshed every 2 d. Negative and positive controls correspond to cells cultured in complete or differentiation LG-DMEM media, respectively. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD.
Analysis of differential gene expression by reverse transcription (RT) and real-time quantitative pcR (qpcR). For this experiment, 10 6 of MSCs were seeded in 6-well plates and cultured in complete