Daniel Ferreira

The activation of the ERK pathway contributes to the spinal c-fos expression observed after noxious bladder stimulation.

C-fos is an immediate-early gene whose expression in the spinal cord has been extensively used as a marker of peripheral noxious stimulation. The Fos protein accumulates in the nuclei of spinal neurons, reaching detectable levels 2u2009h after stimulation. The ERK pathway is an important signalling pathway in spinal cord neurons. ERK is activated upon phosphorylation on specific amino acid residues. Its activation in the spinal cord, following noxious stimulation, has been shown to contribute to the establishment and maintenance of long-term neuronal alterations associated with chronic pain. Phosphorylated ERK can target several cellular elements, including transcription factors, which indicates that ERK participates in the regulation of gene expression. The relation between ERK and c-fos is at present still unclear. Some in vitro studies have reached the conclusion that ERK contributes to c-fos regulation whereas others have provided evidence of ERK-independent c-fos expression. In fact, in the spinal cord the occurrence of c-fos expression in the absence of ERK phosphorylation has been reported. In this study we investigated in vivo the contribution of ERK to c-fos expression in the spinal cord. By inhibiting spinal ERK activation with intrathecal administration of PD98059, we verified that ERK phosphorylation does contribute to regulate c-fos expression upon noxious bladder stimulation.

Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesis

Disruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced gene IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a-specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data enclose potential for designing targeted therapies to rescue E-cadherin function.

Estimativa da erodibilidade a partir de atributos de solos com horizonte B textural no Brasil

The soil erodibility (K-factor) determination by indirect methods has not been adequate for application in Brazilian soils. This paper aimed the development of K-factor evaluation methods depending on easily-assessed chemical, physical and morphological soil properties. Samples from A and B horizons of 22 Brazilian soils bearing argillic horizon were submitted to several chemical and physical analyses and the morphological description of the soil profile was considered. A stepwise multiple linear regression analysis was run, relating the results of those analyses with the K-factor values directly measured in the field. Several equations with increasing degrees of complexity and precision were obtained. From the selected variables from the A and B horizons, as well as the particle-size distribution with and without chemical dispersant, it was possible to satisfactorily estimate the erodibility of such soils. The informations obtained from the soil profile description, specially about soil structure, as well as organic carbon amounts, are of major importance for the indirect determination of the erodibility (K-factor).

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/E-cadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin- deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset.

Development of low-cost customised hand prostheses by additive manufacturing

ABSTRACT The absence of hands makes human life very difficult. The development of prostheses becomes fundamental to improve living conditions. There are many types of prostheses ranging from the simplest ones, with only aesthetic function, to bionic, which have functionality closer to the human hand. Despite all actual technological progress, prices are still very high, making them inaccessible for a wide range of population. Therefore, the development of a low-cost prosthesis will allow simple actions like holding a cup, promoting a better quality of life of people without economic capacity to buy an expensive one. The e-NABLE project that aims to design and manufacture prosthetic hands in polymeric materials through additive manufacturing, tailored to meet user’s specific needs. These prostheses work by flexion and extension of the wrist that produces finger movements. Currently, the prostheses provided have some limitations and conceptual problems. This work contributes to overcome these problems.

Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles

Graphical abstract Figure. No caption available. &NA; Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic‐co‐glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly‐used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme‐linked immunosorbent assay. Most importantly, Fab‐decorated NP binding to the cell surface receptor was monitored by fluorescence‐activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween‐80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween‐80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab‐decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand‐decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed.

Fab-conjugated PLGA nanoparticles effectively target cancer cells expressing human CD44v6

Targeting of CD44 isoforms containing exon v6 (CD44v6) represents a viable strategy for the therapy and/or early diagnosis of metastatic cancers of the epithelium (e.g. gastric and colorectal cancer). We developed and characterized poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) modified with polyethylene glycol (PEG) and engrafted, by site-directed conjugation, with an engineered human Fab that specifically target human CD44v6 (v6 Fab-PLGA NPs). The v6 Fab-PLGA NPs displayed spherical morphology around 300u202fnm and were negatively charged. They strongly bound to a CD44v6-derived peptide and, more importantly, to cells that endogenously and exogenously express CD44v6, but not to non-expressing cells and cells expressing the standard isoform of CD44. The v6 Fab-PLGA NPs also recognized CD44v6 in tumor sections from cells grown subcutaneously within mice. The NPs had nominal cytotoxicity at 50u202fµg/mL and withstood simulated intestinal fluid exposure. Interestingly, v6 Fab-PLGA NPs cryopreserved in 10% trehalose and stored maintained specific cell binding. In conclusion, we envision NPs targeting CD44v6 as potential in vivo diagnostic agents and/or as anti-cancer agents in patients previously stratified with CD44v6+ carcinomas. STATEMENT OF SIGNIFICANCE: The v6 Fab-PLGA NPs displayed many favorable qualities as a potential CD44v6-targeted drug and/or diagnostic delivery agent. The NPs were designed for optimal ligand orientation and for immediate administration into humans. v6 Fab-PLGA NPs strongly bound to cells that endogenously and exogenously express CD44v6, but not to non-expressing cells and cells expressing the standard isoform of CD44. Binding ability was retained after freeze-drying and long-term storage, providing evidences on the stability of Fab-functionalized NPs. These NPs can potentially be used as an in vivo diagnostic from parenteral or oral/rectal administration.

Cerebral autoregulation is preserved in multiple sclerosis patients

Multiple sclerosis (MS) is an inflammatory disease that may also be associated with vascular dysfunction. One master component of vascular regulation is cerebral autoregulation (CA). We aimed to investigate the integrity of CA in MS patients and study its relationship with autonomic dysfunction (AD), magnetic-resonance-imaging (MRI) lesion load and hemodynamic parameters. We enrolled 20 relapsing-remitting MS and 20 healthy subjects. CA was assessed by transfer function analysis parameters (coherence, gain and phase), as obtained in the very low, low and high-frequency domains (VLF, LF, HF, respectively). We evaluated the autonomic parameters heart rate variability and spontaneous baroreflex sensitivity (BRS). There were no significant differences in CA parameters between MS and controls (p>0.05). Lesion load was not correlated with any CA parameter. LF gain was positively correlated with BRS in both groups (MS: p=0.017; controls: p=0.025). Brainstem lesion load in MS was associated with higher systolic blood pressure (SBP; p=0.009). Our findings suggest that CA is preserved in our MS cohort. On the other hand, AD in MS patients with brainstem lesions could contribute to the increase of supine SBP. Whether this systemic deregulation could contribute to disease burden remains to be investigated.

Abstract 1611: Epithelial-mesenchymal-epithelial transition induced by long term exposure to TGFB1 creates cellular heterogeneity

Reversible and dynamic transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression and dissemination. Whereas EMT facilitates initial steps of tumour cell detachment, MET is likely required for colonization at distant sites. Although MET is generally perceived as mirroring EMT, we hypothesize that MET entails its own set of novel and/or differentially active molecular circuitries, generating cells with features distinct from the original epithelial state. Using an in vitro TGFβ1-induced EMT/MET model, we demonstrated that MET generates co-existing heterogeneous cell populations (Reverted-Epithelial or RE-cells) with novel phenotypic and functional properties, such as increased self-renewal, in vivo increased tumourigenicity and distinct chemoresistance properties. Overall, our results indicate that MET is a permissive process, driving cellular plasticity towards heterogeneity and with it, creating novel biological signatures of relevance for cancer growth. Citation Format: Patricia Oliveira, Joana Carvalho, Sara Rocha, Mafalda Azevedo, Andre F. Vieira, Daniel Ferreira, Nuno Mendes, Ines Reis, Joao Vinagre, Alireza Heravi-Moussavi, Joana B. Nunes, Jorge Lima, Valdemar Maximo, Angela Burleigh, Calvin Roskelley, Joana Paredes, Fatima Carneiro, David Huntsman, Carla Oliveira. Epithelial-mesenchymal-epithelial transition induced by long term exposure to TGFB1 creates cellular heterogeneity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1611.