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1.
Bioeng Transl Med ; 8(3): e10504, 2023 May.
Article in English | MEDLINE | ID: mdl-37206216

ABSTRACT

Diabetic foot infection (DFI) is an important cause of morbidity and mortality. Antibiotics are fundamental for treating DFI, although bacterial biofilm formation and associated pathophysiology can reduce their effectiveness. Additionally, antibiotics are often associated with adverse reactions. Hence, improved antibiotic therapies are required for safer and effective DFI management. On this regard, drug delivery systems (DDSs) constitute a promising strategy. We propose a gellan gum (GG)-based spongy-like hydrogel as a topical and controlled DDS of vancomycin and clindamycin, for an improved dual antibiotic therapy against methicillin-resistant Staphylococcus aureus (MRSA) in DFI. The developed DDS presents suitable features for topical application, while promoting the controlled release of both antibiotics, resulting in a significant reduction of in vitro antibiotic-associated cytotoxicity without compromising antibacterial activity. The therapeutic potential of this DDS was further corroborated in vivo, in a diabetic mouse model of MRSA-infected wounds. A single DDS administration allowed a significant bacterial burden reduction in a short period of time, without exacerbating host inflammatory response. Taken together, these results suggest that the proposed DDS represents a promising strategy for the topical treatment of DFI, potentially overcoming limitations associated with systemic antibiotic administration and minimizing the frequency of administration.

2.
BMC Res Notes ; 15(1): 293, 2022 Sep 07.
Article in English | MEDLINE | ID: mdl-36071445

ABSTRACT

OBJECTIVE: Diabetic foot infection (DFI) represents a major healthcare burden, for which treatment is challenging owing to the pathophysiological alterations intrinsic to diabetes and the alarming increase of antimicrobial resistance. Novel therapies targeting DFI are therefore a pressing research need for which proper models of disease are required. RESULTS: Here, we present an optimized diabetic mouse model of methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds, that resemble key features of DFI, such as pathogen invasion through wound bed and surrounding tissue, necrosis, persistent inflammation and impaired wound healing. Thus, in a time-efficient manner and using simple techniques, this model represents a suitable approach for studying emerging therapies targeting DFI caused by MRSA.


Subject(s)
Diabetes Mellitus , Diabetic Foot , Methicillin-Resistant Staphylococcus aureus , Skin Diseases , Staphylococcal Infections , Animals , Diabetic Foot/therapy , Disease Models, Animal , Mice , Staphylococcal Infections/complications , Staphylococcal Infections/therapy , Staphylococcus aureus , Ulcer
3.
Res. Biomed. Eng. (Online) ; 32(1): 44-54, Jan.-Mar. 2016. tab, graf
Article in English | LILACS | ID: biblio-829463

ABSTRACT

Abstract Introduction Early detection of suspicious skin lesions is critical to prevent skin malignancies, particularly the melanoma, which is the most dangerous form of human skin cancer. In the last decade, image processing techniques have been an increasingly important tool for early detection and mathematical models play a relevant role in mapping the progression of lesions. Methods This work presents an algorithm to describe the evolution of the border of the skin lesion based on two main measurable markers: the symmetry and the geometric growth path of the lesion. The proposed methodology involves two dermoscopic images of the same melanocytic lesion obtained at different moments in time. By applying a mathematical model based on planar linear transformations, measurable parameters related to symmetry and growth are extracted. Results With this information one may compare the actual evolution in the lesion with the outcomes from the geometric model. First, this method was tested on predefined images whose growth was controlled and the symmetry known which were used for validation. Then the methodology was tested in real dermoscopic melanoma images in which the parameters of the mathematical model revealed symmetry and growth rates consistent with a typical melanoma behavior. Conclusions The method developed proved to show very accurate information about the target growth markers (variation on the growth along the border, the deformation and the symmetry of the lesion trough the time). All the results, validated by the expected phantom outputs, were similar to the ones on the real images.

4.
ACS Chem Biol ; 8(2): 432-42, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23148778

ABSTRACT

Cystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Hepatocyte Growth Factor/metabolism , Signal Transduction , rac1 GTP-Binding Protein/metabolism , Animals , Cells, Cultured , Cystic Fibrosis/drug therapy , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Mice , Models, Biological , Molecular Structure , Mutation , rac1 GTP-Binding Protein/genetics
5.
Mol Cell Biol ; 31(22): 4392-404, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21930781

ABSTRACT

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.


Subject(s)
Casein Kinase II/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Cricetinae , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Ion Channels/genetics , Ion Channels/metabolism , Mice , Mice, Inbred C57BL , Mutation , Phosphorylation/genetics , Protein Transport , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Syk Kinase , Xenopus laevis
6.
Mol Cell Biol ; 31(19): 4076-86, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21807898

ABSTRACT

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.


Subject(s)
Cell Membrane/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Cricetulus , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Molecular Sequence Data , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Syk Kinase , Tyrosine/metabolism
7.
Mol Genet Metab ; 102(4): 465-9, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21236712

ABSTRACT

Germline mutations in the WNK4 gene originate Gordon syndrome or pseudohypoaldosteronism type II, a familial form of hypertension with hyperkalemia and hypercalciuria. In order to elucidate the contribution of WNK4 genetic variants to hypertension and/or osteoporosis, we analyzed 271 control individuals and a cohort of 448 hypertensive and 372 osteoporosis patients from the Portuguese population. Ten genetic variants were detected in 4.3% of the population under study, none of which revealed any significant association to the hypertension phenotype. In contrast, a rare missense alteration within exon 17 in a highly conserved arginine residue showed a possible tendency for association to the osteoporosis group. Our data suggest that WNK4 polymorphism rs56116165 is a rare allelic variant in a candidate gene with a biological function in renal calcium homeostasis that may contribute to a genetic predisposition to osteoporosis.


Subject(s)
Hypertension/genetics , Osteoporosis/genetics , Protein Serine-Threonine Kinases/genetics , Adult , Aged , Amino Acid Sequence , Animals , Case-Control Studies , Exons , Female , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Hypertension/epidemiology , Introns , Male , Middle Aged , Molecular Sequence Data , Mutation, Missense , Osteoporosis/epidemiology , Polymorphism, Single Nucleotide , Portugal , Pregnancy
8.
J Biol Chem ; 285(50): 39117-26, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-20937822

ABSTRACT

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.


Subject(s)
GTPase-Activating Proteins/metabolism , Gene Expression Regulation , Glucose Transporter Type 1/metabolism , Protein Serine-Threonine Kinases/metabolism , rab GTP-Binding Proteins/metabolism , 14-3-3 Proteins/metabolism , Biological Transport , Exocytosis , Gene Silencing , Glucose/metabolism , Glucose Transporter Type 4/metabolism , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins , Minor Histocompatibility Antigens , Oligonucleotides/chemistry , RNA, Small Interfering/metabolism , WNK Lysine-Deficient Protein Kinase 1
9.
Ann Hematol ; 88(3): 229-34, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18762941

ABSTRACT

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 -9T-->A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5'UTR -25G-->A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C-->G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.


Subject(s)
Hemochromatosis/genetics , Membrane Proteins/genetics , Mutation/genetics , Adult , Aged , Female , Hemochromatosis/diagnosis , Hemochromatosis Protein , Histocompatibility Antigens Class I/genetics , Humans , Iron Metabolism Disorders/diagnosis , Iron Metabolism Disorders/genetics , Iron Overload/diagnosis , Iron Overload/genetics , Male , Middle Aged , Portugal , Receptors, Transferrin/genetics , Sequence Homology, Amino Acid , Young Adult
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