Design of silica nanocarriers: Tuning the release to embryonic stem cells by simple strategies.

The design of mesoporous silica nanoparticles (MSNs) for drug delivery is attracting increasing interest. Controlled release of their cargo is usually mediated by diffusion and erosion mechanisms, which might not reach the expected therapeutic effects. Here, we report the development and characterization of MSNs which modulate the cargo release in different cell models: fibroblasts and embryonic stem cells. Based on a double strategy: the presence of multimodal pore channels and a chitosan coating, we demonstrated a modulated release. Our results show that MSNs could be used for controlled drug delivery in different cell types, showing the potential of improving pluripotent stem cells differentiation and reprogramming protocols with promising applications in biomedicine.

SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells.

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-ß and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

The pluripotency transcription factor OCT4 represses heme oxygenase-1 gene expression.

In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal, and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the antioxidant system and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.

The pluripotency transcription factor Nanog represses glutathione reductase gene expression in mouse embryonic stem cells.

OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency.

Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.

Long-term Outcomes of a New Surgical Technique for Corneal Remodeling in Corneal Ectasia.

PURPOSE: To analyze the outcomes of a new technique (corneal remodeling) to treat corneal ectasia. METHODS: Sixty-nine cases that underwent corneal remodeling were analyzed. Anesthetic drops were instilled and a femtosecond laser platform was used to perform an 8-mm diameter keratectomy (180°, 270°, or 360°). Once ablation was completed, the edges of the resection were sutured with 8 to 12 interrupted stitches. RESULTS: Three-year follow-up data are presented. The age of the population was 30.83 ± 12.65 years (range: 16 to 48 years). At 36 months postoperatively, 57.2% presented with uncorrected distance visual acuity (UDVA) of 20/100 or worse and 42.8% achieved between 20/80 and 20/40. After performing photorefractive keratectomy in 3 cases, 14.3% presented with UDVA of 20/100 or worse, 57.2% achieved between 20/80 and 20/40, and 28.5% achieved 20/30 or better in 180° arc length keratectomy. Most parameters compared were moderately to statistically significant (P < .05 to < .0000001). CONCLUSIONS: Corneal remodeling is a safe technique that produces corneal flattening, reduction of anterior chamber depth, and decreased optical aberrations. It also offers a possibility to perform complementary refractive procedures. [J Refract Surg. 2019;35(4):261-267.].

Kat6b Modulates Oct4 and Nanog Binding to Chromatin in Embryonic Stem Cells and Is Required for Efficient Neural Differentiation.

Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b-/-) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b-/- cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b-/- cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.

Superoxide dismutase 1 expression is modulated by the core pluripotency transcription factors Oct4, Sox2 and Nanog in embryonic stem cells.

Redox homeostasis is vital for cellular functions and to prevent the detrimental consequences of oxidative stress. Pluripotent stem cells (PSCs) have an enhanced antioxidant system which supports the preservation of their genome. Besides, reactive oxygen species (ROS) are proposed to be involved in both self-renewal maintenance and in differentiation in embryonic stem cells (ESCs). Increasing evidence shows that cellular systems related to the oxidative stress defense decline along differentiation of PSCs. Although redox homeostasis has been extensively studied for many years, the knowledge about the transcriptional regulation of the genes involved in these systems is still limited. In this work, we studied Sod1 gene modulation by the PSCs fundamental transcription factors Oct4, Sox2 and Nanog. We found that this gene, which is expressed in mouse ESCs (mESCs), was repressed when they were induced to differentiate. Accordingly, these factors induced Sod1 promoter activity in a trans-activation assay. Finally, Sod1 mRNA levels were reduced when Oct4, Sox2 and Nanog were down-regulated by a shRNA approach in mESCs. Taken together, we found that PSCs' key transcription factors are involved in the modulation of Sod1 gene, suggesting a relationship between the pluripotency core and redox homeostasis in these cells.

A Novel Corneal Remodeling Technique for the Management of Keratoconus.

PURPOSE: To describe a novel technique to reshape the ectatic cornea by means of crescent keratectomy performed with an excimer laser using a mask. METHODS: A crescent-shaped perforation at the base of the mask allowed the laser ablation to be directed only to the intended region, shielding the remaining cornea. This technique was performed in 3 eyes of 3 patients with keratoconus grade 2 to 3. Arcs of 180° and 360° had been performed depending on the severity of the keratoconus. The edges of the crescent were closed by 10-0 nylon interrupted sutures. RESULTS: At 1 year postoperatively, all cases showed improvement in visual acuity, keratometry, and corneal topography. The treatment also reduced optical aberrations and shortened the anterior chamber depth. CONCLUSIONS: Although the preliminary results are promising, there is a need to standardize a nomogram of this technique for treating keratoconus. [J Refract Surg. 2017;33(12):854-856.].

Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase.

FNA and CNB in the diagnosis of pulmonary lesions: a single-center experience on 665 patients, comparison between two periods.

PURPOSE: To evaluate the diagnostic yield and complication rate of 2 different biopsy techniques (fine-needle aspiration, FNA, and core-needle biopsy, CNB) in the diagnosis of pulmonary lesions in 2 distinct periods, 2010-2012 and 2013-2015. METHODS: We retrospectively analyzed the results of 691 CT-guided lung biopsies in 665 patients who were divided into 2 groups: cohort 1 (January 2010 to December 2012) was composed of 271 consecutive patients with 284 procedures either by FNA or CNB; cohort 2 (January 2013 to December 2015) was composed of 394 patients with 407 CNBs. Univariate and multivariate logistic regression modeling was used for selected outcomes including diagnostic yield, bleeding and pneumothorax. RESULTS: Cohort 1 comprised 165 men and 106 women (mean age 68.5 years) with 180 FNAs and 104 CNBs; cohort 2 comprised 229 men and 165 women (mean age 66.4 years) with 407 CNBs. The diagnostic yield increased in cohort 2 with respect to cohort 1. There was a slight increase in CT procedure complications (pneumothorax and bleeding) from cohort 1 to cohort 2. The overall risk of complications was greater for lesions ≤20 mm and for lesions at >20 mm distance from the pleura. CONCLUSIONS: CT-guided CNB had a higher diagnostic yield than discretional use of either FNA or CNB; there was a slight but acceptable increase in complication rates.

Protein arginine Methyltransferase 8 gene is expressed in pluripotent stem cells and its expression is modulated by the transcription factor Sox2.

Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (Prmt). Although Prmt1 is essential in development, its paralogue Prmt8 has been poorly studied. This gene was reported to be expressed in nervous system and involved in neurogenesis. In this work, we found that Prmt8 is expressed in mouse embryonic stem cells (ESC) and in induced pluripotent stem cells, and modulated along differentiation to neural precursor cells. We found that Prmt8 promoter activity is induced by the pluripotency transcription factors Oct4, Sox2 and Nanog. Moreover, endogenous Prmt8 mRNA levels were reduced in ESC transfected with Sox2 shRNA vector. As a whole, our results indicate that Prmt8 is expressed in pluripotent stem cells and its transcription is modulated by pluripotency transcription factors. These findings suggest that besides its known function in nervous system, Prmt8 could play a role in pluripotent stem cells.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription.

Two-year study of lipophilic marine toxin profile in mussels of the North-central Adriatic Sea: First report of azaspiracids in Mediterranean seafood.

Since the late 1980s, the North-central Adriatic Sea has frequently experienced blooms of harmful algal species, producing marine lipophilic toxins (MLTs) which accumulate in mussels and pose a serious threat to consumer health. Here, we present a 2-year LC-MS/MS study (2012-2014) of the MLT profile in mussels from the North-central Adriatic Sea in the context of the presence of toxic phytoplankton concentrations in seawater. Okadaic acid increased in mussels from all areas during the summer and autumn-winter periods with a rising trend between 2012 and 2014. In the same periods, Dinophysis sp. increased in abundance in seawater, but the highest densities of algae did not always coincide with the highest levels of toxins in mussels. Yessotoxins (YTXs) content in mussel increased sharply in the autumn-winter periods even exceeding the legal limit; although this accumulation did not always correlated with the YTX-producers in water (such as Lingulodinium polyedrum and Protoceratium reticulatum) a massive bloom of Gonyaulax spinifera was reported in November 2013, suggesting the role of this species in YTXs shellfish contamination. Traces of Azaspiracid 2 (AZA-2) were observed often in mussels during the study period, confirming for the first time the presence of this biotoxin in Mediterranean seafood.

Bladder cancer diagnosis: the role of CT urography.

AIMS AND BACKGROUND: To evaluate the diagnostic performance of computed tomography urography (CTU), we first compared it with cystoscopy and subsequently analyzed which CTU phase of acquisition has the highest diagnostic accuracy in identifying bladder cancer. METHODS: In 2013, 177 patients underwent both cystoscopy and CTU. For all acquisition phases, we calculated sensitivity, specificity, diagnostic accuracy, and positive and negative predictive value (PPV and NPV, respectively). We also evaluated the Cohen κ coefficient. RESULTS: Computed tomography urography sensitivity, specificity, diagnostic accuracy, PPV, and NPV were as follows: 96.3%, 86.4%, 92.8%, 92.9%, and 92.7%; concordance calculated with Cohen κ was good: 0.8413. The arterial acquisition phase showed the highest diagnostic accuracy, identifying 93.4% of all lesions. CONCLUSIONS: Computed tomography urography is an accurate examination for the diagnosis of bladder cancer, and the arterial acquisition phase provides the best diagnostic information.

Defaecography and colonic transit time for the evaluation of female patients with obstructed defaecation.

PURPOSE: Colonic transit time and defaecography are well known, commonly used studies for evaluating patients with chronic constipation. The aim of this study was to compare colonic transit time with radiopaque markers and defaecography in female patients with obstructed defaecation. MATERIALS AND METHODS: In a prospective observational study, between January 2010 and December 2012, a total of 30 female patients, mean age 60 years, with symptoms of obstructed defaecation were subjected to colonic transit time and defaecography, and divided into two groups: normal or abnormal colon transit time. The results were statistically compared using the Chi-square test. RESULTS: The comparison of data between colonic transit time and defaecography showed the following groups: group 1 (6/30 = 20 %) with normal colonic transit time but abnormal defaecography, and group 2 (24/30 = 80 %) with abnormal colonic transit time; the latter was further divided into two subgroups: group 2a (4/24 = 17 %), patients with inertia coli; group 2b (20/24 = 83 %), patients with impaired defaecation demonstrated at defaecography. There was a significant statistical difference between the radiological findings in these groups. CONCLUSIONS: This study confirmed the value of both defaecography and colonic transit time in assessing clinically obstructed women. Obstructed defaecation might not always be associated with abnormal colonic transit time. Likewise, not all constipated patients had signs of obstructed defaecation. The differential diagnosis between colonic slow transit constipation and constipation due to pelvic floor disorders is essential for an adequate strategy of care.

Trans-thoracic biopsy of lung lesions: FNAB or CNB? Our experience and review of the literature.

PURPOSE: This study was performed to determine the type and incidence of complications of fine-needle aspiration biopsy (FNAB) and core biopsy (CNB) performed under computed tomography (CT) guidance to characterise lung lesions, and assess the diagnostic accuracy of the two techniques. MATERIALS AND METHODS: In 2009-2011, we performed 124 lung biopsies (66 CNB and 56 FNAB) on 121 patients with a mean age of 72.4 years. Exclusion criteria were pulmonary resection, pleural lesions and/or effusions, and inadequate blood-coagulation profile. All examinations were acquired after contrast-agent administration in a craniocaudal direction from the lung apex to base during a single inspiratory breath-hold, with standardised parameters. Each lesion was scanned with 13-15 slices that could be repeated whenever necessary to document the needle track and for lesion centring, by positioning a metallic marker perpendicular to the centring light to indicate the point of needle access. Unless otherwise clinically indicated, 4 h after the procedure chest radiography was performed. RESULTS: Age was found to be a factor influencing the complications: pneumothorax in young subjects (31 %) and parenchymal haemorrhage in the elderly (30 %), with CNB but not with FNAB. We had more complications with the right lung: 50 % of pneumothorax cases in the upper lobe with CNB and 40 % of cases of haemorrhage in the lower lobe with FNAB. The anterior approach gave rise to more complications with CNB, while the posterior approach with FNAB. CNB had more complications than FNAB for lesions ≤ 3.5 cm (31 vs. 18 % pneumothorax), and >3.5 cm (34 vs. 9 % haemorrhage). There was no significant correlation with lesion histology, needle calibre or number of passes (probably due to the small number of procedures done with needles other than 18 G in CNB or 22 G in FNAB or involving more than one needle pass). The diagnostic accuracy of FNAB, done with a pathologist's extemporaneous assessment of sample adequacy, was 94.83 % against 81.82. % of CNB. CONCLUSIONS: FNAB under CT guidance is subject to a lower rate of complications and, if performed in the presence of the pathologist, has a greater diagnostic accuracy compared to CNB.

Revaluation of breast cytology with pathologist on-site of lesions with suspicious sonographic features.

OBJECTIVE: Evaluating correlation estimation between diagnostic ultrasound (U.S.) of breast lesions and fine needle aspiration cytology (FNAC), and the correlation between cytology and histology (I) of these lesions undergo surgery. MATERIALS AND METHODS: In 2010 we performed 1589 ultrasound breast. We identified 210 suspicious lesions to be subjected to FNAC, which was performed with pathologist on site, and extemporaneous analysis of the sample to assess their appropriateness. We classified the lesions in 5 ultrasound (U) classes according to the criteria defined by Echographic BIRADS Lexicon. The results of cytology were classified in 5 classes (C) according to the guidelines of F.O.N.Ca.M. Then we evaluated the diagnostic correlation between U.S. and FNAC, and between FNAC and Histology. RESULTS: The distribution of lesions in U classes was: 57U2, 55U3, 36U4 and 62U5. The diagnostic concordance between U and FNAC was 96.7%, with a sensitivity of 98%, specificity 93%, negative and positive predictive value respectively of 94.9% and 97.3%, and diagnostic accuracy of 96.6%. The 98 patients with C4-C5 lesions were subjected to surgery and the histology confirmed high-grade malignancy of lesions with a concordance of 99.7%. CONCLUSIONS: Having achieved high diagnostic concordance between U and FNAC, and then between FNAC and histology, we may say that the FNAC, less invasive and traumatic for the patient than needle biopsy (CB), may be still a valid method when performed with pathologist on-site to assess the adequacy of the sample taken.