Polyethylenimine as a Versatile Simultaneous Reducing and Stabilizing Agent Enabling One-Pot Synthesis of Transition-Metal Nanoparticles: Fundamental Aspects and Practical Implications.

The large surface area of metallic nanoparticles provides them with particular optical, chemical, and biological properties, accordingly enabling their use in a wide array of applications. In this regard, facile and fast synthetic approaches are desirable for ready-to-use functional materials. Following early investigations focused on the direct synthesis of polymer-coated gold nanoparticles, we herein demonstrate that such a strategy can be used to manufacture different types of d-block transition-metal nanoparticles via a one-pot method in aqueous media and mild temperature conditions. Gold (Au3+), palladium (Pd2+), and silver (Ag+) ions could be reduced using only polyethylenimine (PEI) or PEI derivatives acting simultaneously as a reducing and stabilizing agent and without the aid of any other external agent. The process gave rise, for instance, to Pd urchin-like nanostructures with a large surface area which confers to them outstanding catalytic performance compared to AuNPs and AgNPs produced using the same strategy. The polymer-stabilized AgNPs were demonstrated to be biocide against a variety of microorganisms, although AuNPs and PdNPs do not hold such an attribute at least in the probed concentration range. These findings may provide significant advances toward the practical, facile, and ready-to-use manufacturing of transition-metal nanoparticles for a myriad of applications.

The distribution of reproductive risk factors disclosed the heterogeneity of receptor-defined breast cancer subtypes among Tanzanian women.

BACKGROUND: Recent epidemiological studies suggest that reproductive factors are associated with breast cancer (BC) molecular subtypes. However, these associations have not been thoroughly studied in the African populations. The present study aimed to investigate the prevalence of BC molecular subtypes and assess their association with reproductive factors in Tanzanian BC patients. METHODS: This hospital-based case-only cross-sectional study consisted of 263 histologically confirmed BC patients in Tanzania. Clinico-pathological data, socio-demographic characteristics, anthropometric measurements, and reproductive risk factors were examined using the Chi-square test and one-way ANOVA. The association among reproductive factors and BC molecular subtypes was analyzed using multinomial logistic regression. The heterogeneity of the associations was assessed using the Wald test. RESULTS: We found evident subtype heterogeneity for reproductive factors. We observed that post-menopausal status was more prevalent in luminal-A subtype, while compared to luminal-A subtype, luminal-B and HER-2 enriched subtypes were less likely to be found in post-menopausal women (OR: 0.21, 95%CI 0.10-0.41, p = 0.001; OR: 0.39, 95%CI 0.17-0.89, p = 0.026, respectively). Also, the luminal-B subtype was more likely to be diagnosed in patients aged ≤ 40 years than the luminal-A subtype (OR: 2.80, 95%CI 1.46-5.32, p = 0.002). Women who had their first full-term pregnancy at < 30 years were more likely to be of luminal-B (OR: 2.71, 95%CI 1.18-4.17, p = 0.018), and triple-negative (OR: 2.28, 95%CI 1.02-4.07, p = 0.044) subtypes relative to luminal-A subtype. Furthermore, we observed that breastfeeding might have reduced odds of developing luminal-A, luminal-B and triple-negative subtypes. Women who never breastfed were more likely to be diagnosed with luminal-B and triple-negative subtypes when compared to luminal-A subtype (OR: 0.46, 95%CI 0.22-0.95, p = 0.035; OR: 0.41, 95%CI 0.20-0.85, p = 0.017, respectively). . CONCLUSION: Our results are the first data reporting reproductive factors heterogeneity among BC molecular subtypes in Tanzania. Our findings suggest that breast-feeding may reduce the likelihood of developing luminal-A, luminal-B, and triple-negative subtypes. Meanwhile, the first full-term pregnancy after 30 years of age could increase the chance of developing luminal-A subtype, a highly prevalent subtype in Tanzania. More interventions to promote modifiable risk factors across multiple levels may most successfully reduce BC incidence in Africa.

Optical chemosensor for the detection of cyanide in water based on ethyl(hydroxyethyl)cellulose functionalized with Brooker's merocyanine.

Ethyl(hydroxyethyl)cellulose was functionalized with Brooker's merocyanine. The modified polymer was easily transformed in a film, which could be used as a highly selective chromogenic and fluorogenic chemosensor for the detection of cyanide in water, with detection limits of 1.9 × 10(-5) and 1.0 × 10(-7) mol L(-1). The film was successfully applied to the detection of cyanide in cassava (Manihot esculenta Crantz) roots, which are a well-known source of endogenous biological cyanide.

The interplay between XPG-Asp1104His polymorphism and reproductive risk factors elevates risk of breast cancer in Tanzanian women: A multiple interaction analysis.

BACKGROUND: Reproductive history and genetics are well-known risk factors of breast cancer (BC). Little is known about how these factors interact to effect BC. This study investigated the association of ten polymorphisms in DNA repair genes with BC susceptibility in the Tanzanian samples and further analyzed the association between reproductive risk factors and disease risk METHODS: A hospital-based case-control study in 263 histopathological confirmed BC patients and 250 age-matched cancer-free controls was carried out. Allelic, genotypic, and haplotype association analyses were executed. Also, multifactor dimensionality reduction (MDR), and interaction dendrogram approaches were performed. RESULTS: The frequency of genotypic and allelic variants of XRCC1-Arg399Gln (rs25487), XRCC2-Arg188His (rs3218536), XRCC3-Thr241Met (rs861539), XPG-Asp1104His (rs17655), and MSH2-Gly322Asp (rs4987188) were significantly different between the groups (p < 0.05). Moreover, XRCC1-Arg399Gln (rs25487), XRCC3-Thr241Met (rs861539), and XPG-Asp1104His (rs17655) were associated with the increased risk of BC in co-dominant, dominant, recessive, and additive genetic-inheritance models (p < 0.05). XRCC1-Arg/Gln genotype indicated a 3.1-fold increased risk of BC in pre-menopausal patients (p = 0.001) while XPG-His/His genotype showed a 1.2-fold increased risk in younger BC patients (<40 years) (p = 0.028). Asp/His+His/His genotypes indicated a 1.3-fold increased risk of BC in PR+ patients and a 1.1-fold decreased risk of BC in luminal-A patients (p = 0.014, p = 0.020, respectively). MDR analysis revealed a positive interaction between BC and the XPG-Asp1104His (rs17655) together with family history of cancer in the first-degree relatives. Dendrogram analysis indicated that the XPG-Asp1104His (rs17655) and family history of cancer in first-degree relatives were significantly synergistic and might be associated with an elevated risk of BC in Tanzania. CONCLUSIONS: The XPG-Asp1104His (rs17655) might exert both independent and interactive effects on BC development in the Tanzanian women.

Balancing gene transfection and cytotoxicity of nucleic acid carriers with focus on ocular and hepatic disorders: evaluation of hydrophobic and hydrophilic polyethyleneimine derivatives.

Polyethyleneimine (PEI) derivatives substituted by lactose, succinic acid or alkyl domains were evaluated as nonviral gene delivery vectors towards balancing gene transfection and cytotoxicity. The investigations were focused on pDNA transfection into arising retinal pigment epithelia (ARPE-19) and human hepatocellular carcinoma (HepG2) cell lines. The first mentioned cell line was chosen as motivated by the non-negligible number of ocular disorders linked to gene aberrations, whereas the second one is a cell line overexpressing the asialoglycoprotein receptor (ASGP-R), which can bind to galactose residues. The presence of short alkyl domains (C4 and C6), and particularly the succinylation of the PEI chains, improved the biological outputs of the gene vectors. The presence of hydrophobic units possibly enhances lytic activity, whereas the incorporation of succinic acid slightly reduces polymer-DNA interaction strength, thereby enabling more efficient intracellular unpacking and cargo release. Succinylation is also supposed to decrease cytotoxicity and avoid protein adsorption to the polyplexes. The presence of long carbon chains (for instance, C12) nevertheless, results in higher levels of cytotoxicity and respective lower transfection rates. The sugar-decorated polyplexes are overall less cytotoxic, but the presence of lactose moieties also leads to larger polyplexes and notably weak polymer-DNA binding, which compromise the transfection efficiency. Yet, along with the presence of short lytic alkyl domains, the double-substitution of PEI synergistically boosts gene transfection probably due to the uptake of higher DNA and polymer amounts without cell damage. Overall, the experimental data suggest that ocular and hepatic gene therapies may be potentialized by fine-tuning the hydrophobic-to-hydrophilic balance, and succinic acid is a favorable motif for the modification of PEI.

Reduced cytotoxicity of nanomaterials driven by nano-bio interactions: Case study of single protein coronas enveloping polymersomes.

The protein adsorption onto poly(acrylic acid)-block-polystyrene (PAA22-b-PS144) polymersomes has been investigated with regard to structural features, thermodynamic aspects and biological consequences. The light scattering measurements revealed the formation of protein coronas enveloping the polymeric capsules regardless of the chemical nature of the biomacromolecules. The experiments were conducted by using lysozyme, immunoglobulin G - IgG and bovine serum albumin - BSA as model proteins due to their differences concerning size and residual surface charge at physiological pH. The protein adsorption was further confirmed by isothermal titration calorimetry, and the experimental data suggest that the phenomenon is mainly governed by hydrogen bonding and van der Waals interactions. The pre-existing protein layer via the pre-incubation in protein environments notably attenuates the cytotoxicity of the nanomaterial compared to the pristine counterparts. This approach can possibly be extended to different types of assemblies when intermolecular interactions are able to induce protein adsorption and the development of protein coronas around nanoparticles. Such fairly simple method may be convenient to engineer safer nanomaterials towards a variety of biomedical applications when the nanotoxicity is an issue. Additionally, the strategy can possibly be used to tailor the surface properties of nanoparticles by adsorbing specific proteins for targeting purposes.

Ready-to-use room temperature one-pot synthesis of surface-decorated gold nanoparticles with targeting attributes.

Gold nanoparticles (AuNPs) can be used in diagnostic and therapeutic applications. The development of facile and fast synthetic approaches is accordingly desirable towards ready-to-use biomedical materials. We report a practical one-pot method for the synthesis in aqueous media and room temperature of surface-decorated AuNPs with enhanced biological responses. The gold ions could be reduced using only polyethyleneimine (PEI) derivatives containing sugar and-or alkyl chains acting simultaneously as reducing and stabilizing agent, without the aid of any other mediator. The process is possibly potentialized by the presence of the amino groups in the polymer chains which further confer colloidal stability. The kinetics of AuNPs nucleation and growth depends on the chemical nature of the polymer chains. Particularly, the presence of lactose moieties conjugated to the PEI chains conducted to surface-decorated AuNPs with low cytotoxicity that are remarkably faster uptaken by HepG2 cells. These cells overexpress asialoglycoprotein (ASGP-R), a galactose receptor. These findings may kick off significant advances towards the practical and ready-to-use manufacturing of functionalized AuNPs towards cell-targeting since the methodology is applicable for a large variety of other ligands that can be conjugated to the same polymer chains.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

Electrospun blends comprised of poly(methyl methacrylate) and ethyl(hydroxyethyl)cellulose functionalized with perichromic dyes.

Ethyl(hydroxyethyl)cellulose (EHEC) was modified with 4-(2- (pyridin-4-yl)vinyl) phenol (PBM) and 4- [4-(dimethylamino)styryl]pyridine (DMASP), which are precursors of two well-known perichromic/fluorescent dyes, Brooker's merocyanine (BM) and 4- [4-(dimethylamino)styryl]-1-methypyridinium iodide (DSMI). The EHECs were mixed with poly(methyl methacrylate) (PMMA) to allow electrospinning and the nanofibers obtained were characterized by scanning electron microscopy, fluorescence microscopy, ATR-FTIR spectroscopy, thermogravimetry analysis, differential scanning calorimetry and fluorescence spectrophotometry and the absolute quantum yield and contact angle were determined. The PMMA/EHEC blends formed hydrophilic nanofibers, with high water absorption, in contrast to the pure PMMA nanofibers. PMMA/EHEC-PBM nanofibers exhibited cyan emission when excited in the UV region and PMMA/EHEC-DMASP showed intense green emission under excitation with laser in the blue region. Electrospinning of PMMA/modified EHEC in the proportion of 5:2 (wt/wt) provided nanochain-like nanofibers, which were verified by confocal microscopy. The potential for the use of the electrospun PMMA/EHEC-PBM nanofibers in the detection of CN- in water was demonstrated.

Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe.

Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems.

Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression.

The use of sugar-functionalized polyplexes as a nonviral gene delivery vector with lower cytotoxicity than the well-known polymeric carrier branched polyethyleneimine (BPEI) is investigated. The substitution of primary amine groups in the BPEI chains with lactose residues leads to larger polyplexes, presumably due to the higher amount of polymer required to complete DNA condensation. Nevertheless, the sugar functionalization substantially reduces the cytotoxicity of the assemblies. The nanocomplexes are taken up by the cells to a greater extent, whereas the levels of gene expression are maintained compared to those obtained using BPEI, which is known for its excellent transfection efficiency. Accordingly, the preparation of lower-cytotoxicity polyplexes while maintaining gene expression, which is highly relevant to the field, is demonstrated.

Curcumin-loaded dual pH- and thermo-responsive magnetic microcarriers based on pectin maleate for drug delivery.

Magnetic microgels with pH- and thermo-responsive properties were developed from the pectin maleate, N-isopropyl acrylamide, and Fe3O4 nanoparticles. The hybrid materials were characterized by infrared spectroscopy, scanning electron microscope coupled with X-ray energy dispersive spectroscopy, wide angle X-ray scattering, Zeta potential, and magnetization hysteresis measurements. Curcumin (CUR) was loaded into the microgels, and release assays were carried out in simulated environments (SGF and SIF) at different conditions of temperature (25 or 37°C). A slow and sustainability CUR release was achieved under external magnetic field influence. Loaded CUR displayed stability, bioavailability and greater solubility regarding free CUR. Besides, the cytotoxicity assays showed that magnetic microgels without CUR could suppress the Caco-2 cells growth. So, the pectin maleate, N-isopropyl acrylamide, and Fe3O4 could be tailored to elicit hybrid-based materials with satisfactory application in the medical arena.

SOCS3 expression in SF1 cells regulates adrenal differentiation and exercise performance.

Many hormones/cytokines are secreted in response to exercise and cytokine signaling may play a pivotal role in the training adaptations. To investigate the importance of cytokine signaling during vertical ladder climbing, a resistance exercise model, we produced mice lacking SOCS3 protein exclusively in steroidogenic factor-1 (SF1) cells (SF1 Socs3 KO mice). SF1 expression is found in steroidogenic cells of the adrenal cortex and gonads, as well as in neurons of the ventromedial nucleus of the hypothalamus. Histological markers of the fetal adrenal zone (or X-zone in rodents) were still present in adult males and postpartum SF1 Socs3 KO females, suggesting a previously unrecognized effect of SOCS3 on the terminal differentiation of the adrenal gland. This change led to a distinct distribution of lipid droplets along the adrenal cortex. Under basal conditions, adult SF1 Socs3 KO mice exhibited similar adrenal weight, and plasma ACTH and corticosterone concentrations. Nonetheless, SF1 Socs3 KO mice exhibited a blunted ACTH-induced corticosterone secretion. The overall metabolic responses induced by resistance training remained unaffected in SF1 Socs3 KO mice, including changes in body adiposity, glucose tolerance and energy expenditure. However, training performance and glucose control during intense resistance exercise were impaired in SF1 Socs3 KO mice. Furthermore, a reduced counter-regulatory response to 2-deoxy-d-glucose was observed in mutant mice. These findings revealed a novel participation of SOCS3 regulating several endocrine and metabolic aspects. Therefore, cytokine signaling in SF1 cells exerts an important role to sustain training performance possibly by promoting the necessary metabolic adjustments during exercise.

A simplified surveillance case definition of AIDS derived from empirical clinical data. The Clinical AIDS Study Group, and the Working Group on AIDS case definition.

A clinical AIDS case definition is needed for surveillance in countries where the CDC case definition is not practical. To derive such a definition, we compared 110 HIV-seropositive and 135 randomly selected HIV-seronegative adult medical-ward inpatients in Brazil. Multivariate analysis of clinical signs and symptoms and simple diagnoses resulted in a discriminant function with sensitivity of 89% and specificity of 96% in predicting for AIDS. These data were the empirical basis for a clinical definition of AIDS in adults drafted in a Caracas, Venezuela, workshop sponsored by the Pan American Health Organization. The revised "Caracas" definition presented here requires a positive HIV serology, the absence of cancer or other cause of immunosuppression, plus > or = 10 cumulative points, as follows: Kaposi's sarcoma (10 points); extrapulmonary/noncavitary pulmonary tuberculosis (10); oral candidiasis or hairy leukoplakia (5); cavitary pulmonary/unspecified tuberculosis (5); herpes zoster < 60 years of age (5); CNS dysfunction (5); diarrhea > or = 1 month (2); fever > or = 1 month (2); cachexia or > 10% weight loss (2); asthenia > or = 1 month (2); persistent dermatitis (2); anemia, lymphopenia, or thrombocytopenia (2); persistent cough or any pneumonia except TB (2); and lymphadenopathy > or = 1 cm at > or = 2 noninguinal sites for > or = 1 month (2). This definition has a sensitivity of 95% and a specificity of 100% (91% without HIV serology) when applied to the Brazilian patients in this study. The Caracas definition has been adopted by Brazil, Honduras, and Surinam, and is in validation elsewhere. The use of a reasonably sensitive and specific case definition commensurate with available diagnostic resources should facilitate AIDS surveillance in developing countries.

Polyelectrolyte complexes of poly[(2-dimethylamino) ethyl methacrylate]/chondroitin sulfate obtained at different pHs: I. Preparation, characterization, cytotoxicity and controlled release of chondroitin sulfate.

For the first time, polyelectrolyte complex based on poly[(2-dimethylamino) ethyl methacrylate] (PDMAEMA) and chondroitin sulfate (CS) was prepared. The properties of novel material and precursors were investigated by WAXS, FTIR, TGA, SEM and DLS analysis. The PDMAEMA/CS PECs presented hydrophilic-hydrophobic transition at pHs 6.0, 7.0 and 8.0 whereas the non-complexed PDMAEMA showed such a transition at pH 8.0 and not at pHs 6.0 and 7.0. Studies of CS release from PECs at pHs 6 and 8 confirmed that the samples possess the potential to release the CS in alkaline and not in acidic conditions. Since PECs are thermo-responsive due to the reduction of LCST caused by the increase in pH, the release of CS was dependent on temperature and pH factors. Cytotoxicity assays using healthy VERO cells showed that the complexation between CS and PDMAEMA increased the PECs' biocompatibility related to PDMAEMA. However, the biocompatibility depends on the amount of CS present in the PECs.

Properties of aqueous solutions of lentinan in the absence and presence of zwitterionic surfactants.

Morphological and conformational transitions of lentinan (LT), a ß-glucan extracted from Shiitake mushrooms (Lentinula edodes), were investigated at different concentrations of aqueous NaOH, using Small Angle X-ray Scattering (SAXS) technique. At low NaOH(aq) concentration LT chains are self-associated and adopt the triple helix form where as at higher NaOH concentrations the polymer chains undergo a transition to random coil chains. Also, the presence of fractal dimensions was observed through analysis of the exponential decay of the scattering intensity as a function of the scattering angle. In addition, the lateral radius of gyration was determined for LT in different concentrations of NaOH solution, indicating a rigid triple helix present as a small rod-like structure. Interactions of LT with two zwitterionic surfactants were investigated by surface tension, fluorescence, and static light scattering measurements. Experimental data showed that the formation of LT-(surfactant) complexes occurred through a cooperative process.

Properties of aqueous solutions of hydrophobically modified polyethylene imines in the absence and presence of sodium dodecylsulfate.

Four modified hyperbranched polyethylene imines (PEIs) were synthesized by means of the alkylation of PEI. SAXS, viscosity, surface tension, and pyrene fluorescence emission were then used as techniques to examine the conformation and aggregation of the modified PEIs in aqueous solution, in the absence and presence of sodium dodecylsulfate (SDS). Analysis of the SAXS data showed that the radius of gyration decreases with an increase in the alkyl chain length of the polymer, while the viscosity data indicated a decrease in the intrinsic viscosity under the same conditions. The nonmodified PEI was not surface active, while the hydrophobically modified samples showed pronounced surface activity and the presence of hydrophobic domains. On addition of SDS, the onset of the formation of polymer-surfactant complexes was determined, indicating a decrease in the critical aggregate concentration with an increase in the alkyl chain length of the polymer backbone.

Analysis of pluripotent stem cells by using cryosections of embryoid bodies.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm. Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10 µm) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.