Tropical tree foliage supplementation in ruminants improves rumen fermentation and the bacterial profile and decreases methane production.

The main of this study was to evaluate the effect of supplementation of tropical tree foliage in ruminant diets on the in vitro fermentation, bacterial population, volatile fatty acids (VFAs), and enteric CH4 production. Seven experimental diets were evaluated: a control treatment of Pennisetum purpureum (T7) and six treatments of P. purpureum supplemented (30%) with the foliage of Neomillspaughia emargiata (T1), Tabernaemontana amygdalifolia (T2), Caesalpinia gaumeri (T3), Piscidia piscipula (T4), Leucaena leucocephala (T5), and Havardia albicans (T6). The T2, T7, and T5 treatments had the highest (p < 0.05) digestibility of dry matter. Overall, supplementation increased (p < 0.05) the concentrations of propionic and butyric acid and decreased acetic acid. Methanogenic bacteria decreased (p < 0.05) in T1, T2, T5, and T6. Ruminococcus albus decreased in T1, T2, T3, and T5 and Selenomonas ruminiantum increased in T3. Fibrobacter succinogenes increased, except in T5. Methane production decreased (p < 0.05) in T1, T4, T5, and T6. The supplementation with Leucaena leucocephala, Tabernaemontana amygdalifolia, Neomillspaughia emargiata, Piscidia piscipula, Havardia albicans, and Caesalpinia gaumeri is a potential alternative nutritional strategy for ruminants that results in positive changes in VFAs profile, a decrease on CH4 production and methanogenic bacteria, and changes on fibrolytic and non-fibrolytic bacteria composition.HIGHLIGHTSTropical tree foliage supplementation increased propionic and butyric acid and decreased acetic acid concentrations.Fibrolytic, non-fibrolytic, and Methanogenic bacteria were selectively modulated with the supplementation of tropical tree foliage.The enteric methane (CH4) production decreased with the supplementation of tree foliage.The supplementation of Tabernaemontana amygdalifolia and Leucaena leucocephala had the highest digestibility and is a potential alternative nutritional strategy for ruminants.

Electrical stimulation: Effective cue to direct osteogenic differentiation of mesenchymal stem cells?

Mesenchymal stem cells (MSCs) play a major role in bone tissue engineering (BTE) thanks to their capacity for osteogenic differentiation and being easily available. In vivo, MSCs are exposed to an electroactive microenvironment in the bone niche, which has piezoelectric properties. The correlation between the electrically active milieu and bone's ability to adapt to mechanical stress and self-regenerate has led to using electrical stimulation (ES) as physical cue to direct MSCs differentiation towards the osteogenic lineage in BTE. This review summarizes the different techniques to electrically stimulate MSCs to induce their osteoblastogenesis in vitro, including general electrical stimulation and substrate mediated stimulation by means of conductive or piezoelectric cell culture supports. Several aspects are covered, including stimulation parameters, treatment times and cell culture media to summarize the best conditions for inducing MSCs osteogenic commitment by electrical stimulation, from a critical point of view. Electrical stimulation activates different signaling pathways, including bone morphogenetic protein (BMP) Smad-dependent or independent, regulated by mitogen activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) and p38. The roles of voltage gate calcium channels (VGCC) and integrins are also highlighted according to their application technique and parameters, mainly converging in the expression of RUNX2, the master regulator of the osteogenic differentiation pathway. Despite the evident lack of homogeneity in the approaches used, the ever-increasing scientific evidence confirms ES potential as an osteoinductive cue, mimicking aspects of the in vivo microenvironment and moving one step forward to the translation of this approach into clinic.

Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture.

The use of piezoelectric materials in tissue engineering has grown considerably since inherent bone piezoelectricity was discovered. Combinations of piezoelectric polymers with magnetostrictive nanoparticles (MNP) can be used to magnetoelectrically stimulate cells by applying an external magnetic field which deforms the magnetostrictive nanoparticles in the polymer matrix, deforming the polymer itself, which varies the surface charge due to the piezoelectric effect. Poly(vinylidene) fluoride (PVDF) is the piezoelectric polymer with the largest piezoelectric coefficients, being a perfect candidate for osteogenic differentiation. As a first approach, in this paper, we propose PVDF membranes containing magnetostrictive nanoparticles and a biomimetic heparin/collagen layer-by-layer (LbL) coating for mesenchymal stem cell culture. PVDF membranes 20% (w/v) with and without cobalt ferrite oxide (PVDF-CFO) 10% (w/w) were produced by non-solvent induced phase separation (NIPS). These membranes were found to be asymmetric, with a smooth surface, crystallinity ranging from 65% to 61%, and an electroactive ß-phase content of 51.8% and 55.6% for PVDF and PVDF-CFO, respectively. Amine groups were grafted onto the membrane surface by an alkali treatment, confirmed by ninhydrin test and X-ray photoelectron spectroscopy (XPS), providing positive charges for the assembly of heparin/collagen layers by the LbL technique. Five layers of each polyelectrolyte were deposited, ending with collagen. Human mesenchymal stem cells (hMSC) were used to test cell response in a short-term culture (1, 3 and 7 days). Nucleus cell counting showed that LbL favored cell proliferation in PVDF-CFO over non-coated membranes.

Modulation of CYP2E1 metabolic activity in a cohort of confirmed caffeine ingesting pregnant women with preterm offspring.

BACKGROUND: To ascertain interactions of caffeine ingestion, food, medications, and environmental exposures during preterm human gestation, under informed consent, we studied a cohort of Mexican women with further preterm offspring born at ≤ 34 completed weeks. At birth, blood samples were taken from mothers and umbilical cords to determine caffeine and metabolites concentrations and CYP1A2 (rs762551) and CYP2E1 (rs2031920, rs3813867) polymorphisms involved in caffeine metabolism. RESULTS: In 90 pregnant women who gave birth to 98 preterm neonates, self-informed caffeine ingestion rate was 97%, laboratory confirmed rate was 93 %. Theobromine was the predominant metabolite found. Consumption of acetaminophen correlated significantly with changes in caffeine metabolism (acetaminophen R2 = 0.637, p = 0.01) due to activation of CYP2E1 alternate pathways. The main caffeine source was cola soft drinks. CONCLUSION: Environmental exposures, especially acetaminophen ingestion during human preterm pregnancy, can modulate CYP2E1 metabolic activity.

Capacitively coupled electrical stimulation of rat chondroepiphysis explants: A histomorphometric analysis.

The growth plate is a cartilaginous layer present from the gestation period until the end of puberty where it ossifies joining diaphysis and epiphysis. During this period several endocrine, autocrine, and paracrine processes within the growth plate are carried out by chondrocytes; therefore, a disruption in cellular functions may lead to pathologies affecting bone development. It is known that electric fields impact the growth plate; however, parameters such as stimulation time and electric field intensity are not well documented. Accordingly, this study presents a histomorphometrical framework to assess the effect of electric fields on chondroepiphysis explants. Bones were stimulated with 3.5 and 7 mV/cm, and for each electric field two exposure times were tested for 30 days (30 min and 1 h). Results evidenced that electric fields increased the hypertrophic zones compared with controls. In addition, a stimulation of 3.5 mV/cm applied for 1 h preserved the columnar cell density and its orientation. Moreover, a pre-hypertrophy differentiation in the center of the chondroepiphysis was observed when explants were stimulated during 1 h with both electric fields. These findings allow the understanding of the effect of electrical stimulation over growth plate organization and how the stimulation modifies chondrocytes morphophysiology.

Effect of tree foliage supplementation of tropical grass diet on in vitro digestibility and fermentation, microbial biomass synthesis and enteric methane production in ruminants.

The objective of this study was to evaluate the influence of tree foliage species supplemented in ruminant diets based on Pennisetum purpureum on the in vitro digestibility and fermentation, microbial biomass synthesis and enteric methane production. Seven experimental diets were evaluated, including a control treatment based on P. purpureum (PT) grass, and six additional treatments supplemented with 30.0% foliage from Neomillspaughia emargiata (NE), Tabernaemontana amygdalifolia (TA), Caesalpinia gaumeri (CG), Piscidia piscipula (PP), Leucaena leucocephala (LL) and Havardia albicans (HA). A randomised complete block design repeated in two periods (block) was used. The highest gas production (P < 0.05) was recorded in treatments TA and PT (237 and 228 mL g-1, respectively). The highest in vitro digestibility of dry matter (IVDMD) and organic matter (IVOMD) (P < 0.05) was recorded in the control treatment PT (57.9% and 66.1%, respectively). Treatments LL, NE, TA and PP promoted greater microbial biomass synthesis (290, 223, 220 and 213 mg g-1, respectively) (P < 0.05). The proportion of propionic acid also increased in these latter treatments and in treatments CG and HA (P < 0.05). Additionally, treatments LL, PP, NE and TA decreased methane production (25.8, 29.5, 30.6 and 31.8 L kg-1 of digested dry matter, respectively). In conclusion, supplementation with L. leucocephala, P. piscipula, N. emargiata and T. amygdalifolia in ruminant diets based on P. purpureum is one feed alternative that can promote greater efficiency and synthesis of microbial biomass, increase the proportions of propionic and butyric acid and decrease the production of enteric methane by 15.6 to 31.6%.

Effects of quebracho tannin extract on intake, digestibility, rumen fermentation, and methane production in crossbred heifers fed low-quality tropical grass.

The aim of this work was to evaluate the effect of quebracho tannins extract (QTE) on feed intake, dry matter (DM) digestibility, and methane (CH4) emissions in cattle fed low-quality Pennisetum purpureum grass. Five heifers (Bos taurus × Bos indicus) with an average live weight (LW) of 295 ± 19 kg were allotted to five treatments (0, 1, 2, 3, and 4% QTE/kg DM) in a 5 × 5 Latin square design. Intake, digestibility, and total methane emissions (L/day) were recorded for periods of 23 h when cattle were housed in open-circuit respiration chambers. Dry matter intake (DMI), organic matter intake (OMI), dry matter digestibility (DMD), and organic matter digestibility (OMD) were different between treatments with 0 and 4% of QTE/kg DM (P < 0.05). Total volatile fatty acid and the molar proportion of acetate in the rumen was not affected (P < 0.05); however, the molar proportion of propionate increased linearly (P < 0.01) for treatments with 3 and 4% QTE. Total CH4 production decreased linearly (P < 0.01) as QTE increased in the diet, particularly with 3 and 4% concentration. When expressed as DMI and OMI by CH4, production (L/kg) was different between treatments with 0 vs 3 and 4% QTE (P < 0.05). It is concluded that the addition of QTE at 2 or 3% of dry matter ration can decrease methane production up to 29 and 41%, respectively, without significantly compromising feed intake and nutrients digestibility.

miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia.

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Prediction of the "in vivo" mechanical behavior of biointegrable acrylic macroporous scaffolds.

This study examines a biocompatible scaffold series of random copolymer networks P(EA-HEA) made of Ethyl Acrylate, EA, and 2-Hydroxyl Ethyl Acrylate, HEA. The P(EA-HEA) scaffolds have been synthesized with varying crosslinking density and filled with a Poly(Vinyl Alcohol), PVA, to mimic the growing cartilaginous tissue during tissue repair. In cartilage regeneration the scaffold needs to have sufficient mechanical properties to sustain the compression in the joint and, at the same time, transmit mechanical signals to the cells for chondrogenic differentiation. Mechanical tests show that the elastic modulus increases with increasing crosslinking density of P(EA-HEA) scaffolds. The water plays an important role in the mechanical behavior of the scaffold, but highly depends on the crosslinking density of the proper polymer. Furthermore, when the scaffold with hydrogel is tested it can be seen that the modulus increases with increasing hydrogel density. Even so, the mechanical properties are inferior than those of the scaffolds with water filling the pores. The hydrogel inside the pores of the scaffolds facilitates the expulsion of water during compression and lowers the mechanical modulus of the scaffold. The P(EA-HEA) with PVA shows to be a good artificial cartilage model with mechanical properties close to native articular cartilage.

Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior.

An experimental fatigue study of a porous scaffold for the regeneration of articular cartilage.

The aim of this experimental study is to predict the long-term mechanical behavior of a porous scaffold implanted in a cartilage defect for tissue engineering purpose. Fatigue studies were performed by up to 100,000 unconfined compression cycles in a polycaprolactone (PCL) scaffold with highly interconnected pores architecture. The scaffold compliance, stress-strain response and hysteresis energy have been measured after different number of fatigue cycles, while the morphology has been observed by scanning electron microscopy at the same fatigue times. To simulate the growing tissue in the scaffold/tissue construct, the scaffold was filled with an aqueous solution of polyvinyl alcohol (PVA) and subjected to repeating cycles of freezing and thawing that increase the hydrogel stiffness. Fatigue studies show that the mechanical loading provokes failure of the dry scaffold at a smaller number of deformation cycles than when it is immersed in water, and also that 100,000 compressive dynamic cycles do not affect the scaffold/gel construct. This shows the stability of the scaffold implanted in a chondral defect and gives a realistic simulation of the mechanical performance from implantation of the empty scaffold to regeneration of the new tissue inside the scaffold's pores.

Fármacocinética del genérico zidovudina en pacientes cubanos infectados por el virus de la inmunodeficiencia humana / Pharmacokinetic of generic formulations of zidovudine in human immunodeficiency virus infected cuban patients

Objetivo: El objetivo del presente trabajo es caracterizar los parámetros farmacocinéticos de la zidovudina (AZT) en pacientes cubanos seropositivos al virus de la inmunodeficiencia humana (VIH). Materiales y métodos: Para ello se realizó un estudio de dosis única (300mg) a 13 pacientes "naives" seropositivos al VIH-1 donde se midieron, según la cinética establecida, las concentraciones de AZT en plasma y orina. Estas concentraciones se determinaron por cromatografía líquida de alta resolución en fase reversa (RP-HPLC) con detección UV (λ=267nm). Resultados: Los parámetros farmacocinéticos fueron calculados usando técnicas estándares no compartimentales. Entre las variables determinadas están: Concentración máxima (Cmax= 3,35±1,41 μg/mL), Recobrado urinario (RU= 25,36±9,25%), Aclaramiento renal (ClR= 17,56±7,78L/h), Aclaramiento plasmático (CLp= 47,09±29,45 L/h), Tiempo de vida media de eliminación (t½= 1,19±0,30h) y Biodisponibilidad relativa (F= 63,12±16,59%). Conclusiones: Los valores de los parámetros calculados posibilitan la caracterización del perfil farmacocinético del genérico cubano AZT. Esto es útil para posteriores correcciones de los regímenes de dosificación según las especificidades de cada paciente

Aims: The purpose of this study was to characterize pharmacokinetic parameters of zidovudine generic (AZT) in human inmunodeficiency virus (HIV) infected cuban patients. Materials and methods: A single-dose study (300mg AZT) was made in 13 "naive" patientes for determination of the AZT concentrations in plasma and urine, fallowed an established kinetic. These concentration values were measured by reversed-phase liquid chromatography (RP-HPLC) with UV detection (λ=267nm). Results: Pharmacokinetic parameters for zidovudine were estimated from data of concentration in plasma and urine versus time by using noncompartmental methods. The most important parameters obtained were: maximum concentration (Cmax= 3,35±1,41 μg/mL), urinary recovery (UR= 25,36±9,25 %), renal clearance (ClR= 17,56±7,78L/h), plasmatic clearance (CLp= 47,09±29,45 L/h), terminal elimination half-life (t½= 1,19±0,30h), and apparent bioavailability (F= 63,12±16,59%). Conclusions: The values of pharmacokinetic parameters made possible to the establishment of the pharmacokinetic profile for the Cuban generic zidovudine. This is very important for future dose adjustment of patient

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

An "in vitro" experimental model to predict the mechanical behavior of macroporous scaffolds implanted in articular cartilage.

A model is proposed to assess mechanical behavior of tissue engineering scaffolds and predict their performance "in vivo" during tissue regeneration. To simulate the growth of tissue inside the pores of the scaffold, the scaffold is swollen with a Poly (Vinyl alcohol) solution and subjected to repeated freezing and thawing cycles. In this way the Poly (Vinyl alcohol) becomes a gel whose stiffness increases with the number of freezing and thawing cycles. Mechanical properties of the construct immersed in water are shown to be determined, in large extent, by the water mobility constraints imposed by the gel filling the pores. This is similar to the way that water mobility determines mechanical properties of highly hydrated tissues, such as articular cartilage. As a consequence, the apparent elastic modulus of the scaffold in compression tests is much higher than those of the empty scaffold or the gel. Thus this experimental model allows assessing fatigue behavior of the scaffolds under long-term dynamic loading in a realistic way, without recourse to animal experimentation.

[Bicycle accidents treated in emergency departments. A multicentre study]. / Accidentes de bicicleta atendidos en los Servicios de Urgencias. Estudio multicéntrico.

OBJECTIVES: To describe epidemiological characteristics, types of injury, prognosis and medical management of bicycle-related Paediatric Emergency Department (ED) visits and to identify potential preventive measures. PATIENTS AND METHODS: This multicentred, observational prospective study included all children between 3 and 16 years of age treated for bicycle-related injuries in the Emergency Departments of 15 Spanish Hospitals belonging to the «Unintentional Paediatric Injury Workshop¼ of the Spanish Paediatric Emergency Society between the 1(st) of June 2011 and the 31(st) of May 2012. Characteristics of all ED visits, as well as epidemiological data and accident-related information, were collected. RESULTS: A total of 846 patients were included in the study, with a male predominance (72.9%) and a median age of 9.6 ± 3.6 years. Head injury was the third most common injury (22.3%) and the main cause of admission to the Pediatric Intensive Care Unit (PICU) (68.4%). More than three-quarters (77.9%) of the patients did not wear a helmet, which was significantly associated to a higher incidence of head injury and admission to PICU. Older children (OR 1.063) and bicycle injuries involving motor vehicles (OR 2.431) were identified as independent risk factors for worse outcomes. CONCLUSIONS: Since helmet use reduces up to 88% of central nervous system lesions secondary to head injury, promotion of its use should be the main preventive measure, followed by restriction of bike-riding to cycling areas.

Computational methodology to determine fluid related parameters of non regular three-dimensional scaffolds.

The application of three-dimensional (3D) biomaterials to facilitate the adhesion, proliferation, and differentiation of cells has been widely studied for tissue engineering purposes. The fabrication methods used to improve the mechanical response of the scaffold produce complex and non regular structures. Apart from the mechanical aspect, the fluid behavior in the inner part of the scaffold should also be considered. Parameters such as permeability (k) or wall shear stress (WSS) are important aspects in the provision of nutrients, the removal of metabolic waste products or the mechanically-induced differentiation of cells attached in the trabecular network of the scaffolds. Experimental measurements of these parameters are not available in all labs. However, fluid parameters should be known prior to other types of experiments. The present work compares an experimental study with a computational fluid dynamics (CFD) methodology to determine the related fluid parameters (k and WSS) of complex non regular poly(L-lactic acid) scaffolds based only on the treatment of microphotographic images obtained with a microCT (µCT). The CFD analysis shows similar tendencies and results with low relative difference compared to those of the experimental study, for high flow rates. For low flow rates the accuracy of this prediction reduces. The correlation between the computational and experimental results validates the robustness of the proposed methodology.

Magnesium monitoring practice in monoclonal anti-epidermal growth factor receptor antibodies therapy.

WHAT IS KNOWN AND OBJECTIVE: It is now estimated that about 5% of cetuximab-treated patients and about 3% of panitumumab-treated patients will develop grade 3-4 hypomagnesemia. The aim of this study was to assess the extent of magnesium monitoring in patients treated with epidermal growth factor receptor (EGFR)-targeting antibodies and to estimate the incidence of hypomagnesemia in these patients at our institution. METHODS: A 2-year retrospective study was carried out. At least four doses of weekly cetuximab or two doses of bi-weekly panitumumab were required for inclusion. Serum magnesium profiles were reviewed from 1 month before treatment until 3 months after treatment discontinuation, and patients with <2 determinations were excluded. RESULTS AND DISCUSSION: Two hundred and one patients received at least one dose of EGFR-targeting antibodies, but only 68 met the inclusion criteria. Seventy patients had <2 magnesium determinations. The overall hypomagnesemia was 58·82% (40 of 68 patients), with a 4·41% grade 3 hypomagnesemia (three of 68 patients). No grade 4 hypomagnesemia was detected. WHAT IS NEW AND CONCLUSION: There is a lack of magnesium monitoring in these patients. Serum magnesium determinations should be done every 4-8 weeks in patients treated with EGFR-targeting antibodies, as it is a useful surrogate marker for both toxicity and efficacy.

Acute myeloid leukemia with translocation (8;16)(p11;p13) and MYST3-CREBBP rearrangement harbors a distinctive microRNA signature targeting RET proto-oncogene.

Acute myeloid leukemia (AML) with t(8;16)(p11;p13) (t(8;16) AML) has unique clinico-biological characteristics, but its microRNA pattern is unknown. We analyzed 670 microRNAs in seven patients with t(8;16) AML and 113 with other AML subtypes. Hierarchical cluster analysis showed that all t(8;16) AML patients grouped in an independent cluster. Supervised analysis revealed a distinctive signature of 94-microRNAs, most of which were downregulated, including miR-21 and cluster miR-17-92. The mRNA expression analysis of two known transcription factors of these microRNAs (STAT3 and c-Myc, respectively) showed significant downregulation of STAT3 (P=0.04). A bioinformatic analysis showed that 29 of the downregulated microRNAs might be regulated by methylation; we treated a t(8;16) AML sample with 5-aza-2'-deoxycytidine (5-AZA-dC) and trichostatin A and found that 27 microRNAs were re-expressed after treatment. However, there was no difference in methylation status between t(8;16) and other AML subtypes, either overall or in the microRNA promoter. Cross-correlation of mRNA and microRNA expression identified RET as a potential target of several microRNAs. A Renilla-luciferase assay and flow cytometry after transfection with pre-microRNAs confirmed that RET is regulated by miR-218, miR-128, miR-27b, miR-15a and miR-195. In conclusion, t(8;16) AML harbors a specific microRNA signature that is partially epigenetically regulated and targets RET proto-oncogene.

Biomimetic hydroxyapatite coating on pore walls improves osteointegration of poly(L-lactic acid) scaffolds.

Polymer-ceramic composites obtained as the result of a mineralization process hold great promise for the future of tissue engineering. Simulated body fluids (SBFs) are widely used for the mineralization of polymer scaffolds. In this work an exhaustive study with the aim of optimizing the mineralization process on a poly(L-lactic acid) (PLLA) macroporous scaffold has been performed. We observed that when an air plasma treatment is applied to the PLLA scaffold its hydroxyapatite nucleation ability is considerably improved. However, plasma treatment only allows apatite deposition on the surface of the scaffold but not in its interior. When a 5 wt % of synthetic hydroxyapatite (HAp) nanoparticles is mixed with PLLA a more abundant biomimetic hydroxyapatite layer grows inside the scaffold in SBF. The morphology, amount, and composition of the generated biomimetic hydroxyapatite layer on the pores' surface have been analyzed. Large mineralization times are harmful to pure PLLA as it rapidly degrades and its elastic compression modulus significantly decreases. Degradation is retarded in the composite scaffolds because of the faster and extensive biomimetic apatite deposition and the role of HAp to control the pH. Mineralized scaffolds, covered by an apatite layer in SBF, were implanted in osteochondral lesions performed in the medial femoral condyle of healthy sheep. We observed that the presence of biomimetic hydroxyapatite on the pore's surface of the composite scaffold produces a better integration in the subchondral bone, in comparison to bare PLLA scaffolds.

Fibrin-chitosan composite substrate for in vitro culture of chondrocytes.

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.