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Nanoemulsions (NE) are lipid nanocarriers that can efficiently load hydrophobic active compounds, like palmitoyl-L-carnitine (pC), used here as model molecule. The use of design of experiments (DoE) approach is a useful tool to develop NE with optimized properties, requiring less experiments compared to trial-and-error approach. The optimized formulation was studied for its stability, scalability, pC entrapment, loading capacity, and biodistribution in mice. NE were prepared by the solvent injection technique. DoE was implemented for designing pC-loaded NE, using a two-level fractional factorial design as model. NE were fully characterized by a combination of techniques, and biodistribution was studied ex-vivo after injection of fluorescent NE in healthy mice. We selected the optimal formulation of NE, named pC-NEU, after DoE analysis of four variables. pC-NEU incorporated pC in a very efficient manner, with high entrapment efficiency and loading capacity. pC-NEU did not change its initial colloidal properties stored at 4ºC in water during 120 days, nor in buffers with different pH values (5.3 and 7.4) during 30 days. The scalability process did not affect NE stability properties. Biodistribution study showed that pC-NEU formulation was predominantly concentrated in the liver, with minimal accumulation in spleen, stomach and kidneys.

Additional file 1. Figure S1. Optical appearance of the colloidal suspensions before A) and after B) treatment with the SHDS. Figure S2. Size distribution of the samples directly incubated in SIF or after complete SHDS-treatment of A) LSNPs; B) CNPs, C) FNPs; D) HNPs. Figure S3. HNPs dissolution. Figure S4. Degradation of LSNPs by lipase. Table S1. Mean dH and PDI of NMs in DMEM+10% FBS (100 mg/ml), 24 h incubation. Figure S5. Aspect of the suspension of the (*) untreated and (**) SHDS-treated A) LSNPs, B) CNPs, C) FNPs and D) HNPs in cell medium. Figure S6. SDS-PAGE analysis showing the hard corona of (*) SHDS-treated and (**) untreated A) LSNPs, B) CNPs, C) FNPs and D) HNPs in cell medium and obtained by centrifugation, and E) ctrl without NBMs. Figure S7. LFQ intensity for different proteases inhibitor for FNPs untreated and SHDS-treated. Figure S8. Cells viability of A) Caco-2 cells and B) HCoEpiC cells after 24 h of incubation with SHDS fluids. Figure S9. Genotoxicity of HCT116 cells after 24 h of incubation with untreated and SHDS-treated NMs. Table S2. Tran-Epithelial Electrical Resistance (TEER) values of Caco-2 barrier model after 24 h of incubation. Figure S10. Scheme of representative SDHS treatment.

Described here is the implementation of a lung ultrasound course for physiotherapists focused on the acquisition and retention of knowledge and skills. Initially, we provided online lectures in a virtual learning environment (VLE), in which we taught the semiquantification of edema through a lung ultrasound score (LUS). Afterward, the physiotherapists participated in face-to-face lectures (which resumed the online lectures), followed by hands-on training and simulation with ultrasound. We assessed knowledge acquisition through a multiple-choice test with 30 questions (totaling 10 points). The test was applied before accessing the VLE (pre-VLE), before the face-to-face course and at its end (pre- and post-course). Physiotherapists collected actual patients' ultrasound scans, which were uploaded to the VLE and assessed by three supervisors, who performed a consensus LUS calculation and gave virtual written feedback. Thirteen physiotherapists collected 59 exams. The test results were 3.60 ± 1.58 (pre-VLE), 5.94 ± 1.45 (pre-course) and 8.50 ± 0.71 (post-course), with plt; 0.001 for all. The intraclass correlation coefficient for LUS between physiotherapists and supervisors was 0.814 (plt; 0.001), with moderate-to-weak agreement for LUS of the lung apical, median and basal zones, with κ = 0.455.334, and 0.417 (plt; 0.001 for all). Trainees were found to have increased short-term acquisition and retention of knowledge and skills, with a good intraclass correlation coefficient between them and the consensus of supervisors for the LUS of actual patients.

Polymethine dyes (PMD) have proved to be excellent candidates in the biomedical field for potential applications in both diagnostic and therapeutic. However, PMD application in biomedicine is hindered by their poor solubility and stability in physiological conditions. Therefore, the incorporation of these dyes in nanosystems could be important to prevent the formation of dye aggregates in aqueous environment and to protect their photophysical characteristics. In the present work, two PMD based on the benzoindolenine ring (bromine benzo-cyanine-C4 and bromine benzo-squaraine-C4) were incorporated into Solid Lipid Nanoparticles (SLN) to solubilize and stabilize them in aqueous solutions. Obtained SLN showed a high incorporation efficiency for both PMD (≈90%) and not only preserved their spectroscopic properties in the NIR region even under physiological conditions but also improved them. Viability assays showed good biocompatibility of both empty and loaded nanocarriers while the cellular uptake and intracellular localization showed the effective internalization in MCF-7 cells, with a partial mitochondrial localization for CY-SLN. Moreover, in vitro phototoxicity assay showed that cyanine loaded-SLN (CY-SLN) is more photoactive than the free dye.

Plastic pollution is now considered to be one of the main threats to the marine environment. Plastics are ubiquitous and persistent pollutants that tend to undergo fragmentation and be concentrated in oceanic gyres and coastal fronts by the combined actions of winds and currents. When these garbage patches overlap with key habitats for sea turtles, the consequences for these species are dramatic. Ingestion, bioaccumulation, and entanglement, among others, are frequent impacts of plastic pollution on sea turtles’ health. Since 1999, the local NGO Karumbe has been working tirelessly for the conservation of marine turtles in Uruguay. This chapter discusses the experiences of the Karumbe initiative in the last 20 years.

Objective To investigate the effectiveness and safety of office hysteroscopic metroplasty by diode laser for the treatment of septate uteri. Study design We performed a prospective cohort study including 41 nulliparous women diagnosed with septate uterus and a history of primary infertility or recurrent miscarriage. All patients underwent hysteroscopic metroplasty for the treatment of the uterine septum, which was performed in-office setting using a 4.3 mm continuous-flow hysteroscope and a 15-watt diode laser at 1470 nm. Surgical and reproductive outcomes after 2 years of follow-up are reported. Results The procedure was well tolerated by 93% of patients. No surgical complications were reported. Thirty women out of 38 (78.9%) became pregnant: 17 (56.7%) pregnancies were spontaneously conceived, and 13 (43.3%) were obtained with assisted reproductive techniques. Twenty-four (80%) of the 30 pregnant women had a live birth, with a cumulative live birth of 63.2% among the 38 women (95% CI: 45.9-78.2%). There were no cases of post-partum hemorrhage or uterine rupture among 14 (58.3%) women who had vaginal deliveries. Conclusion Office hysteroscopic metroplasty for septate uterus using diode laser appears to be a feasible and safe alternative to other available techniques and has sufficient efficacy in terms of reproductive outcomes to be considered for further investigations.

We report 8 new records of Lepidochelys olivacea marine turtle in the Uruguayan waters, indicating this area as thesouthernmost limit of distribution for this species in the western Atlantic Ocean. In addition, 1 specimen was subjectedto genetic analysis, revealing its population origin in the western Atlantic nesting colonies (Surinam, French Guiana,and Brazil). This report represents an update of the distribution of L. olivacea in the southwestern Atlantic and providesinsight into the morphological and genetic characterization of the species at temperate waters.

The ingestion of anthropogenic marine debris can lead to injuries in the digestive system of marine turtles through blockages, lacerations and enteritis, as well as sub-lethal effects from bioaccumulation of adhered chemicals and toxic substances leached out into tissues and blood. The early detection of these impacts is central for the treatment and recovery of turtles in a rehabilitation setting. In this study, we provide baseline data on gastrointestinal transit times in healthy green turtles (Chelonia mydas) to enable non-intrusive detection of digestive motility disorders. We conducted two experiments with juvenile green turtles (N = 14) (curved carapace length range 33.7–47.0 cm) using inorganic (inert plastic discs) and organic (corn kernels) markers respectively, in order to estimate gastrointestinal transit times and assess the effectiveness of each marker type in recording them. Gastrointestinal transit times for the inorganic marker trial group (n = 6 turtles) ranged from 14.6 ± SD 3.6 days for the first markers recovered to 22.5 ± SD 4.2 days for the last markers recovered. The corresponding data for the organic marker trial group (n = 8 turtles) ranged from 6.63 ± SD 1.6 days to 17.3 ± SD 3.3 days respectively. We obtained 96% recovery success of markers in the inorganic marker trial versus 72.5% in the organic marker trial. Thus, inorganic markers proved to be more efficient in reporting gastrointestinal transit times because they do not degrade or discolour as they pass along the digestive process, enabling higher recovery success. Opportunistically, veterinarians diagnosed an obstruction caused by plastic fragments, which had been swallowed in the wild prior to the trial, in one of the experimental animals after we failed to recover any markers. This incident is evidence that gastrointestinal transit time assessment is a useful approach for providing early warning of digestive system blockages. Furthermore, this knowledge on transit times could be of interest for toxicology studies regarding exposure to chemicals lixiviated from debris ingested, as an index of the time spent by these substances inside the organism.

Exonic mutations are usually classified as missense, synonymous or nonsense mutations, however, they can affect pre-mRNA splicing either by disrupting splice sites, by creating new ones or by changing splicing regulatory sequences. In this study, we examined 21 mutations of the PKD2 gene, encoding polycystin-2, previously reported as missense or synonymous for their possible effects on pre-mRNA splicing using bioinformatics tools. All these mutations except one have been identified in patients with autosomal dominant polycystic kidney disease, a common genetic disorder characterized by the development and progressive enlargement of cysts in the kidneys leading to end-stage renal disease. We selected 12 missense mutations and 1 synonymous variant for the minigene assay, and found that three, c.1532A>T (p.D511V), c.1716G>A (p.K572K) and c.2657A>G (p.D886G) caused alterations in pre-mRNA splicing. Mutation c.1532A>T resulted in skipping of exon 6 and incorporation of a defective exon lacking the 3' end, while c.1716G>A led to skipping of exon 7. Mutation c.2657A>G resulted in incorporation of an incomplete exon 14, which is in agreement with previous results obtained with the patient's lymphoblast RNA. Our findings should be taken into account with regard to the pathogenicity of these PKD2 exonic mutations. These results together with previous reports highlight the importance to evaluate the effects of exonic single nucleotide substitutions in autosomal dominant polycystic kidney disease.

The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

BACKGROUND The aim of this study was to describe the trends in surgical routes in obese women who underwent hysterectomy for benign disease at our center and compare complications in different groups. METHODS Retrospective study done between 2011 and 2015 in women with a Body Mass Index≥30 who underwent hysterectomy for benign disease at Virgen de las Nieves Universitary Hospital in Granada, Spain. We studied three groups based on the surgical route chosen for hysterectomy: vaginal, abdominal or laparoscopic. The rates of intraoperative and postoperative complications, major complications, reintervention and days of hospital stay were compared. RESULTS Abdominal hysterectomy was associated with the highest risk of postoperative complications and the longest hospital stay. Laparoscopic hysterectomy had a higher risk than vaginal hysterectomy of major complications. There were no significant differences between groups for any of the other variables. CONCLUSIONS In obese women vaginal hysterectomy was associated with the lowest morbidity, and should be the approach of choice whenever feasible.

Dent's disease is an X-linked proximal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. This disorder is frequently caused by mutations in the CLCN5 gene encoding the electrogenic chloride/proton exchanger ClC-5. Occasionally, Dent's disease has been associated to atypical cases of asymptomatic proteinuria with focal glomerulosclerosis. Twelve unrelated patients with Dent's disease, including two who presented with asymptomatic proteinuria and developed glomerulosclerosis, were studied. Mutational analysis of the CLCN5 gene was performed by DNA sequencing. We identified thirteen distinct CLCN5 mutations in the twelve patients. Seven of these mutations, p.P416fsX(*)17, p.[H107P, V108fs(*)27], p.G466D, p.G65R, p.G462S, p.Y164(*) and c.723+1GT, were novel and possibly pathogenic. In one family, the patient's mother was not a carrier of the respective mutation. Our results increased the spectrum of CLCN5 disease causing defects with seven new pathogenic mutations and established a de novo origin in one of them. Remarkably, three new missense mutations, p.G466D, p.G65R and p.G462S, affect highly conserved glycine residues located in transmembrane α-helix GxxxG packing motifs. The two atypical cases further support that the diagnosis of Dent's disease should be considered in children with asymptomatic proteinuria and focal glomerulosclerosis and without evidence of primary glomerular disease.

The correct splicing of precursor-messenger RNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and two synonymous mutations induce significant defects in pre-mRNAsplicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G>A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A>T (p.G109G) and c.11257C>A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level.

The hypoglycemic activity of Psacalium decompositum (Asteraceae) was investigated in fasting healthy mice and alloxan-diabetic mice. The freeze-dried water decoction significantly reduced the blood glucose in normal mice (from 50.9 +/- 4.7 to 32.5 +/- 3.1 mg/dl) and in mild diabetic mice (from 208.5 +/- 13.0 to 52.3 +/- 7.0 mg/dl), 240 min after intraperitoneal administration (P < 0.005). This preparation also diminished fasting glycemia in severe diabetic mice, but the effects were minor (from 394.4 +/- 9.4 to 289.3 +/- 39.5 mg/dl). The main sesquiterpenoid constituents from P. decompositum roots, cacalol, cacalone and maturin, as well as the transformation product cacalol acetate, did not show a hypoglycemic effect on healthy mice. Nevertheless, two polysaccharide fractions (F1 and F3) obtained from the freeze-dried water extract significantly reduced the fasting glycemia in healthy mice. The best results were obtained with the F1 fraction.

The effects of Psacalium decompositum, Psacalium peltatum and Acourtia thurberi (Asteraceae) on blood glucose levels were investigated in fasting mice and temporally hyperglycemic rabbits. The root decoction of P. decompositum reduced the blood glucose of normal mice from 49.1 +/- 3.8 to 35.7 +/- 2.0 mg/dl after intraperitoneal administration (P < or = 0.005) and significantly lowered the hyperglycemic peak (17.1%) in rabbits with temporal hyperglycemia. P. peltatum and A. thurberi decoctions also diminished fasting glycemia in mice and hyperglycemia in rabbits, but the effects were minor. A preliminary phytochemical study using thin layer chromatography showed that water decoctions of the three roots contained alkaloids and sugars. P. decompositum and P. peltatum showed the presence of maturine. However, other furoeremophylanes, such as cacalol and cacalone were only present in P. decompositum. A. thurberi root water decoction showed the presence of the benzoquinone perezone, and its derivative pipitzol.

Tesis (Cirujano Dentista, Especialización en Endodoncia) Hoy en día la terapia endodóntica, conocida por la mayoría de las personas como "tratamiento de conductos", se ha convertido en una posibilidad bastante socorrida que permite optar por mantener una pieza dentaria original con su funcionalidad y estética, situación que hasta unos pocos años atrás, comúnmente terminaba en exodoncia. Lo anterior debido a que se puede conservar gracias a esta técnica, que de otro modo, su principal indicación seria la extracción. El tratamiento de conductos consiste en la extirpación total de la pulpa camera! y de los conductos de una pieza dentaria producto de una serie de diagnósticos, tales como: caries profundas, fracturas dentarias indicaciones protésicas entre otras. Debido a la compleja anatomía que presentan los conductos radiculares, la sola preparación mecánica no basta para la eliminación total de restos pulpares y detritus desde el interior del sistema de conductos y ciertos tratamientos pueden fracasar debido a que quedan zonas, sobre todo a nivel apical, que no son alcanzadas por la instrumentación, por lo que la complejidad anatómica juega un papel relevante en los resultados de dicha preparación. Es por esto que toma un papel relevante el uso de sustancias que ayuden, por medio de acciones físicas y químicas, eliminar bacterias y residuos pulpares del sistema de conductos, por medio de la irrigación, debido a que esta alcanza lugares inaccesibles para los instrumentos endodónticos. Dentro de los irrigantes más utilizados se encuentra el hipoclorito de sodio por su gran capacidad de disolución de tejidos, y gran potencial bactericida, pero por otro lado presenta una alta citotoxicidad (4,5). Sin embargo, otro componente que ha tomado relevancia, es el ácido etilendiamino tetracético (EDTA). Este es un agente quelante inorgánico usado durante la instrumentación de conductos estrechos y como complemento para remover la capa de desecho dentinario. Bystrom et al. En 1985, demostraron una mejor acción antibacteriana al usar una mezcla de hipoclorito de sodio y EDTA, comprobando que si estas dos sustancias se utilizan alternadamente entre cada instrumento, el conducto estará libre de restos desbridados. La combinación de ambas soluciones demostró un efecto muy importante en la remoción de materia orgánica e inorgánica del lumen del conducto, facilitando la limpieza del sistema de conductos. Muchas técnicas de irrigación son conocidas, como las convencionales o pasivas con jeringas monojet, y por otro lado, la irrigación mediante sistemas ultrasónicos que se han introducido debido a sus grandes capacidades en cuanto a la eliminación de restos en los conductos radiculares, debido al fenómeno de cavitación principalmente y oscilación entre otros.

Introduction Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. Materials and Methods Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. Results HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. Conclusions Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.