Prox1 Regulates the Notch1-Mediated Inhibition of Neurogenesis

During development of the spinal cord, Prox1 controls the balance between proliferation and differentiation of neural progenitor cells via suppression of Notch1 gene expression

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

Distributed Nonlinear Model Predictive Control by Sequential Linearization and Accelerated Gradient Method

A suboptimal approach to distributed NMPC for nonlinear interconnected systems subject to constraints is proposed. The objective is to develop a computationally efficient approach. The suggested method is based on a sequential linearization of the nonlinear system dynamics and finding a suboptimal solution of the resulting Quadratic Programming problem by using distributed iterations of the dual accelerated gradient method. The benefits of the approach are reduced complexity of the on-line computations, and simple software implementation, which makes it appropriate for embedded distributed convex NMPC. The proposed method is illustrated with simulations on the model of a quadruple-tank system

Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study

Horse serum potentiates cellular viability and improves indomethacin-induced adipogenesis in equine subcutaneous adipose-derived stem cells (ASCs)

ABSTRACTSubcutaneous fat tissue is an accessible and abundant source of multipotent stem cells for cell therapy in regenerative medicine. Successful trilineage differentiation is required to define the stemness features of the obtained mesenchymal cells, and adipogenesis is a part of it. Since indomethacin is bound to serum albumin, replacing foetal bovine serum (FBS) with horse serum (HS) in adipogenic induction protocols would suppress its cytotoxic effect and reveal a better adipogenic potential in equine MSCs. The equine subcutaneous adipose-derived stem cells (ASCs) were separately induced in adipogenesis by three different concentrations of 3-isobutyl-1-methylxanthine, IBMX (0.5 mM; 0.25 mM and 0.1 mM) and indomethacin (0.1 mM; 0.05 mM and 0.02 mM) for 48 h. In contrast to the IBMX, indomethacin in all concentrations caused dramatic cellular detachment. Further, the same induction concentrations were used in FBS and HS conditions for adipogenic induction. The MTT assay revealed that the culture media supplemented with HS raised cellular vitality by about 35% compared to those cultured in FBS. Based on those results, an adipogenic cocktail containing indomethacin (0.05 mM) and IBMX (0.5 mM), supplemented with HS and FBS, respectively, was applied for 18 days. The adiponectin gene expression was significantly up-regulated in HS-supplemented media since established changes in PPAR-gamma were insignificant. The tri-lineage differentiation was successful, and a cross-sectional area of adipocytes was performed. The albumin concentration was higher in HS than in FBS. In conclusion, our study revealed that HS is an appropriate supplement in induced adipogenesis since it probably suppresses the indomethacin-related cytotoxic effect and increases adipogenic ability in equine subcutaneous ASCs

Evaluation of the Anticancer and Probiotic Potential of Autochthonous (Wild) <i>Lacticaseibacillus paracasei</i> Strains from New Ecological Niches as a Possible Additive for Functional Dairy Foods

Probiotics such as Lactobacillus spp. could modulate the intestinal microbiota composition, supporting gastrointestinal tract barrier function and benefiting human health. To evaluate the anticancer and probiotic properties of potentially active autochthonous Lacticaseibacillus paracasei strains on proliferating and differentiated enterocytes, human colon adenocarcinoma cell line HT29 was used as a model. The lactic acid bacteria (LAB) were isolated from new ecological niches—mountain anthills populated by redwood ants (Formica rufa L.). Human colorectal adenocarcinoma cells (HT29, ATCC, HTB-38™) were treated for twenty-four hours with supernatants (SNs) derived from four strains of Lacticaseibacillus paracasei: P4, C8, C15 and M2.1. An MTT assay, alkaline phosphatase activity, IAP, Bax and Bcl-2 gene expression analysis (RT-qPCR) and the Bax/Bcl-2 ratio were evaluated. The MTT assay revealed that the observed effects varied among groups. However, 10% neutralized supernatants from P4, C8, C15 and M2.1 strains did not show cytotoxic effects. In contrast to non-differentiated cells, a significant (p p p p p p L. paracasei strains possess anticancer and probiotic properties and could be used as additives for functional dairy foods and thus benefit human health

Effects of beta-blockers in an 18-year old thalassemia patient with severe cardiac siderosis

Introduction: Patients with transfusion dependent Thalassemia have cardiac complications as a re­sult of chronic high output state and the iron overload of the myocardium. Gradually, a high output state leads to high output heart failure. This is the leading cause of mortality among thalassemic pa­tients.Materials and methods: 18-year old Thalassemia major patient who was regularly receiving he­motransfusions since early childhood was admitted to the hospital. By using T2* cardiovascular mag­netic resonance, severe myocardial iron overload 7, 02 was noted. Since January 2017, the patient has been receiving regular check-ups due to the complaints of weakness and dyspnea. Electrocardi­ography performed after the patient received hemotransfusion showed sinus rhythm with the heart rate (HR) of 95 beats per minute (b.p.m.). Echocardiography showed a reduced ejection fraction (EF) 45, 6% and in tissue Doppler imaging reduced myocardial velocities were examined. Patient started treatment with a beta-blocker, bisoprolol 2,5mg oral per day (o.d.).Results: After bisoprolol 2,5mg o.d. treatment for a month and a half, a follow-up echocardiography was performed. On echocardiography examination, a significant improvement in contractile func­tion, EF 58, 5%, HR 73b.p.m. was observed. Echocardiography performed seven months later showed EF of 60% and an increase in the myocardial systolic velocity (Sm) compared with the first echocar­diographic examination. The complaints were decreased to a minimum.Conclusion: Beta-blockers are used in the standard treatment for the patients with heart failure who have reduced ejection fraction. However, beta-blockers should be used with caution for patients with thalassemia since these patients tend to have low blood pressure in nature. Significant improvement on patients` severe myocardial siderosis and low myocardial contractile function of the left ventricle was achieved after starting the treatment with beta-blockers. The symptoms of heart failure due to the severe myocardial iron deposition due to hemotransfusions was successfully managed with beta-blockers

Multidrug-Resistant Proteus mirabilis Strain with Cointegrate Plasmid

Proteus mirabilis is a component of the normal intestinal microflora of humans and animals, but can cause urinary tract infections and even sepsis in hospital settings. In recent years, the number of multidrug-resistant P. mirabilis isolates, including the ones producing extended-spectrum &beta;-lactamases (ESBLs), is increasing worldwide. However, the number of investigations dedicated to this species, especially, whole-genome sequencing, is much lower in comparison to the members of the ESKAPE pathogens group. This study presents a detailed analysis of clinical multidrug-resistant ESBL-producing P. mirabilis isolate using short- and long-read whole-genome sequencing, which allowed us to reveal possible horizontal gene transfer between Klebsiella pneumoniae and P. mirabilis plasmids and to locate the CRISPR-Cas system in the genome together with its probable phage targets, as well as multiple virulence genes. We believe that the data presented will contribute to the understanding of antibiotic resistance acquisition and virulence mechanisms for this important pathogen

Genomic and Phenotypic Analysis of Multidrug-Resistant <i>Acinetobacter baumannii</i> Clinical Isolates Carrying Different Types of CRISPR/Cas Systems

Acinetobacter baumannii is an opportunistic pathogen being one of the most important causative agents of a wide range of nosocomial infections associated with multidrug resistance and high mortality rate. This study presents a multiparametric and correlation analyses of clinical multidrug-resistant A. baumannii isolates using short- and long-read whole-genome sequencing, which allowed us to reveal specific characteristics of the isolates with different CRISPR/Cas systems. We also compared antibiotic resistance and virulence gene acquisition for the groups of the isolates having functional CRISPR/Cas systems, just CRISPR arrays without cas genes, and without detectable CRISPR spacers. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as phylogenetic analysis of full-length cas gene sequences, predicted prophage sequences and CRISPR array type determination. For the first time the differences between the isolates carrying Type I-F1 and Type I-F2 CRISPR/Cas systems were investigated. A. baumannii isolates with Type I-F1 system were shown to have smaller number of reliably detected CRISPR arrays, and thus they could more easily adapt to environmental conditions through acquisition of antibiotic resistance genes, while Type I-F2 A. baumannii might have stronger “immunity” and use CRISPR/Cas system to block the dissemination of these genes. In addition, virulence factors abaI, abaR, bap and bauA were overrepresented in A. baumannii isolates lacking CRISPR/Cas system. This indicates the role of CRISPR/Cas in fighting against phage infections and preventing horizontal gene transfer. We believe that the data presented will contribute to further investigations in the field of antimicrobial resistance and CRISPR/Cas studies