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Breast cancer is a relevant cause of mortality in women and its triple-negative subtype (TNBC) is usually associated with poor prognosis. During tumor progression to metastasis, angiogenesis is triggered by the sprouting of endothelial cells from pre-existing vessels by a dynamic chain of events including VE-cadherin downregulation, actin protrusion, and integrin-mediated adhesion, allowing for migration and proliferation. The binding of tumoral and tumor-associated stromal cells with the extracellular matrix through integrins mediates angiogenic processes and certain integrin subtypes, such as the αvß3 integrin, are upregulated in hypoxic TNBC models. Integrin αvß3 inhibition by the high-affinity binding disintegrin DisBa-01 was previously demonstrated to induce anti-tumoral and anti-angiogenic responses in traditional 2D cell assays. Here, we investigate the effects of integrin αvß3 blockage in endothelial and TNBC cells by DisBa-01 in 3D cultures under two oxygen conditions (1% and 20%). 3D cultures created using non-adhesive micromolds with Matrigel were submitted to migration assay in Boyden chambers and fluorescence analysis. DisBa-01 inhibited cell migration in normoxia and hypoxia in both MDA-MB-231 and HUVEC spheroids. Protein levels of integrin αvß3 were overexpressed in HUVEC spheroids compared to MDA-MB-231 spheroids. In HUVEC 3D cultures, sprouting assays in collagen type I were decreased in normoxia upon DisBa-01 treatment, and VE-cadherin levels were diminished in HUVEC spheroids in hypoxia and upon DisBa-01 treatment. In conclusion, the blockage of integrin αvß3 by DisBa-01 inhibits cell migration in 3D culture and interferes with tumor-derived responses in different oxygen settings, implicating its crucial role in angiogenesis and tumor progression.

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This work aimed to assess the influence of different structured substrates with hydrophilic and hydrophobic properties on micro and nano topographies developed on titanium alloys over pre-osteoblastic cell behavior. Nano topography influences small dimension levels of cell morphology by inducing filopodia formation in cell membranes, irrespectively to the wettability behavior of the surface. Therefore, micro and nanostructured surfaces of titanium-based samples using different techniques of surface modification processing, such as chemical treatments, micro-arc anodic oxidation (MAO), and MAO combined to laser irradiation were developed. Isotropic and anisotropic texture morphologies, wettability, topological parameters and compositional alterations were measured after the surface treatments. Finally, cell viability, adhesion and morphological responses were assessed to investigate the influence of distinct topologies on osteoblastic cells aiming to encounter the conditions to better promote mineralization events. Our study demonstrated that the hydrophilic behavior improves cell adhesion, amplified when effective surface area increases. Surfaces presenting nano topography have a direct influence on cell morphology and play a key role for filopodia formation.

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3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the ß-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.

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Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O2) and normoxic (20% O2) conditions, using phenotypical and proteomic approaches. SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O2-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.

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A deficit of estrogen is associated with energy substrate imbalance, raising the risk of metabolic diseases. Physical training (PT) is a potent metabolic regulator through oxidation and storage of substrates transported by GLUT4 and FAT CD36 in skeletal muscle. However, little is known about the effects of PT on these carriers in an estrogen-deficit scenario. Thus, the aim of this study was to determine the influence of 12 weeks of PT on metabolic variables and GLUT4 and FAT CD36 expression in the skeletal muscle of animals energetically impaired by ovariectomy (OVX). The trained animals swam 30 min/day, 5 days/week, at 80% of the critical load intensity. Spontaneous physical activity was measured biweekly. After training, FAT CD36 and GLUT4 expressions were quantified by immunofluorescence in the soleus, as well as muscular glycogen and triglyceride of the soleus, gluteus maximus and gastrocnemius. OVX significantly reduced FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01), while PT significantly increased FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01). PT increased soleus glycogen, and OVX decreased muscular triglyceride of gluteus maximus. Therefore, OVX can cause energy disarray through reduction in GLUT4 and FAT CD36 and their muscle substrates and PT prevented these metabolic consequences, masking ovarian estrogen's absence.

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We aimed to investigate whether muscle fiber cross-sectional area (fCSA) and associated molecular processes could be differently affected at the group and individual level by manipulating resistance training (RT) variables. Twenty resistance-trained subjects had each leg randomly allocated to either a standard RT (RT-CON: without specific variables manipulations) or a variable RT (RT-VAR: manipulation of load, volume, muscle action, and rest interval at each RT session). Muscle fCSA, satellite cell (SC) pool, myonuclei content, and gene expression were assessed before and after training (chronic effect). Gene expression was assessed 24 h after the last training session (acute effect). RT-CON and RT-VAR increased fCSA and myonuclei domain in type I and II fibers after training (p < 0.05). SC and myonuclei content did not change for both conditions (p > 0.05). Pax-7, MyoD, MMP-2 and COL3A1 (chronic) and MGF, Pax-7, and MMP-9 (acute) increased similar for RT-CON and RT-VAR (p < 0.05). The increase in acute MyoG expression was significantly higher for the RT-VAR than RT-CON (p < 0.05). We found significant correlation between RT-CON and RT-VAR for the fCSA changes (r = 0.89). fCSA changes were also correlated to satellite cells (r = 0.42) and myonuclei (r = 0.50) changes. Heatmap analyses showed coupled changes in fCSA, SC, and myonuclei responses at the individual level, regardless of the RT protocol. The high between and low within-subject variability regardless of RT protocol suggests that the intrinsic biological factors seem to be more important to explain the magnitude of fCSA gains in resistance-trained subjects.

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Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.

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Vascularization is considered to be one of the key challenges in engineering functional 3D tissues. Engineering suturable vascular grafts containing pores with diameter of several tens of microns in tissue engineered constructs may provide an instantaneous blood perfusion through the grafts improving cell infiltration and thus, allowing rapid vascularization and vascular branching. The aim of this work was to develop suturable tubular scaffolds to be integrated in biofabricated constructs, enabling the direct connection of the biofabricated construct with the host blood stream, providing an immediate blood flow inside the construct. Here, tubular grafts with customizable shapes (tubes, Y-shape capillaries) and controlled diameter ranging from several hundreds of microns to few mm are fabricated based on poly(glycerol sebacate) (PGS)/poly(vinyl alcohol) (PVA) electrospun scaffolds. Furthermore, a network of pore channels of diameter in the order of 100µm was machined by laser femtosecond ablation in the tube wall. Both non-machined and laser machined tubular scaffolds elongated more than 100% of their original size have shown suture retention, being 5.85 and 3.96 N mm-2respectively. To demonstrate the potential of application, the laser machined porous grafts were embedded in gelatin methacryloyl (GelMA) hydrogels, resulting in elastomeric porous tubular graft/GelMA 3D constructs. These constructs were then co-seeded with osteoblast-like cells (MG-63) at the external side of the graft and human umbilical vein endothelial cells inside, forming a bone osteon model. The laser machined pore network allowed an immediate endothelial cell flow towards the osteoblasts enabling the osteoblasts and endothelial cells to interact and form 3D structures. This rapid vascularization approach could be applied, not only for bone tissue regeneration, but also for a variety of tissues and organs.

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Snake venom metalloproteinases (SVMPs) represent a diverse group of multi-domain proteins with several biological activities such as the ability to induce hemorrhage, proteolytic degradation of fibrinogen and fibrin, induction of apoptosis and inhibition of platelet aggregation. Due to these activities, SVMPs are responsible for many of the well-known pathological phenotypes in snake envenomations caused particularly by species from the Viperidae family and the Crotalinae subfamily. These proteins have been classified based on their size and domain structure into P-I, P-II and P-III classes. Comparatively, members of the P-I SVMPs possess the simplest structures, formed by the catalytic metalloproteinase domain only; the P-II SVMPs are moderately more complex, having the canonical disintegrin domain in addition to the metalloproteinase domain; members of the P-III class are more structurally varied, comprising the metalloproteinase, disintegrin-like, and cysteine-rich domains. Proteolytic cleavage, repeated domain loss and presence of other ancillary domains are responsible for structural diversities in the P-III class. However, studies continue to unveil the relationship between the structure and function of these proteins. In this review, we recovered evidences from literature on the structural peculiarities and functional classification of Snake Venom Metalloproteinases. In addition, we reflect on diversities that exist among each class while taking into account specific and up-to-date class-based activities.

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AIMS: We investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. MAIN METHODS: Male rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. KEY FINDINGS: Aging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). SIGNIFICANCE: RT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes.

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Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The αvß3 integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of αvß3 integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, αvß3 integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.

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Alternagin-C (ALT-C), a disintegrin-like protein obtained from the venom of Bothrops alternatus, is able to modulate cellular behaviors such as adhesion, migration and proliferation, as well as the production of various growth factors via α2ß1 integrin, important processes during inflammation, angiogenesis and fibrogenesis, which although appear as distinct events, act concomitantly in several chronic inflammatory diseases. Our objective was to investigate the effects of ALT-C on components of the sponge-induced inflammatory response in balb/c mice. The polyester-polyurethane sponges were implanted in mice's subcutaneous layer of the dorsal region and daily injected with saline (control group) or ALT-C (10, 100 or 1000 ng). Nine days after implantation the implants were removed and processed. ALT-C inhibited the inflammatory response, observed through mast cell reduction, NAG-activity and also by the inhibition of TNF-α, CXCL-1 and CCL2/JE/MCP-1 cytokines. ALT-C was also able to reduce hemoglobin content, number of vessels and the concentrations of VEGF and FGF cytokines. Finally, at its highest dose (1000 ng), ALT-C increased all evaluated markers associated with fibrogenesis (collagen production and TGF-ß1 levels). All these factors reveal that ALT-C is a strong candidate to be exploited in the development of anti-inflammatory and anti-angiogenic therapies in chronic inflammatory processes.

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The spread of cells from primary tumors toward distant tissues and organs, also known as metastasis, is responsible for most cancer-associated deaths. The metastasis cascade comprises a series of events, characterized by the displacement of tumor cells (TCs) from the primary tumor to distant organs by traveling through the bloodstream, and their subsequent colonization. The first step in metastasis involves loss of cell-cell and cell-matrix adhesions, increased invasiveness and migratory abilities, leading to intravasation of TCs into the blood or lymphatic vessels. Stationary TCs must undergo the process of epithelial-mesenchymal transition in order to achieve this migratory and invasive phenotype. Circulating tumor cells that have survived in the circulation and left the blood or lymphatic vessels will reach distant sites where they may stay dormant for many years or grow to form secondary tumors. To do this, cells need to go through the mesenchymal-epithelial transition to revert the phenotype in order to regain epithelial cell-to-cell junctions, grow and become a clinically relevant and detectable tumor mass. This work will review the main steps of the metastatic cascade and describe some strategies to inhibit metastasis by reducing cancer cell extravasation presenting recent studies in the context of breast cancer.

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A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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Scaffolds of poly(ε-caprolactone) (PCL) and their biocomposites with 0, 1, 3, and 5 wt.% Biosilicate® were fabricated by the generative manufacturing process coupled with a vertical miniscrew extrusion head to application for restoration of bone tissue. Their morphological characterization indicated the designed 0°/90° architecture range of pore sizes and their interconnectivity is feasible for tissue engineering applications. Mechanical compression tests revealed an up to 57% increase in the stiffness of the scaffold structures with the addition of 1 to 5 wt.% Biosilicate® to the biocomposite. No toxicity was detected in the scaffolds tested by in vitro cell viability with MC3T3-E1 preosteoblast cell line. The results highlighted the potential application of scaffolds fabricated with poly(ε-caprolactone)/Biosilicate® to tissue engineering.

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Exercise training (ET), anabolic androgenic steroids (AAS), and aging are potential factors that affect tendon homeostasis, particularly extracellular matrix (ECM) remodeling. The goal of this review is to aggregate findings regarding the effects of resistance training (RT), AAS, and aging on tendon homeostasis. Data were gathered from our studies regarding the impact of RT, AAS, and aging on the calcaneal tendon (CT) of rats. We demonstrated a series of detrimental effects of AAS and aging on functional and biomechanical parameters, including the volume density of blood vessel cells, adipose tissue cells, tendon calcification, collagen content, the regulation of the major proteins related to the metabolic/development processes of tendons, and ECM remodeling. Conversely, RT seems to mitigate age-related tendon dysfunction. Our results suggest that AAS combined with high-intensity RT exert harmful effects on ECM remodeling, and also instigate molecular and biomechanical adaptations in the CT. Moreover, we provide further information regarding the harmful effects of AAS on tendons at a transcriptional level, and demonstrate the beneficial effects of RT against the age-induced tendon adaptations of rats. Our studies might contribute in terms of clinical approaches in favor of the benefits of ET against tendinopathy conditions, and provide a warning on the harmful effects of the misuse of AAS on tendon development.

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BACKGROUND/OBJECTIVES: Regular exercise training is effective to altering many markers of metabolic syndrome and its effects are strongly influenced by the type of consumed diet. Nowadays, resistance training (RT) has been frequently associated with low-carbohydrate high-fat diet (LCD). After long term these diets causes body weight (BW) regain with deleterious effects on body composition and metabolic risk factors. The effects of RT associated with long-term LCD on these parameters remain unexplored. We aimed to investigate the effects of RT when associated with long-term LCD on BW, feed efficiency, body composition, glucose homeostasis, liver parameters and serum biochemical parameters during BW regain period in rats. SUBJECTS/METHODS: Male Sprague-Dawley rats were fed with LCD (LC groups) or standard diet (STD) (ST groups). After 10 weeks-diet animals were separated into sedentary (Sed-LC and Sed-ST) and resistance-trained (RT-LC and RT-ST) groups (N = 8/group). RT groups performed an 11-week climbing program on a ladder with progressive load. Dual x-ray absorptiometry, glucose tolerance tests and insulin tolerance tests were performed at weeks 10 and 20. Liver and serum were collected at week 21. RESULTS: RT reduced feed efficiency, BW gain, liver fat and total and LDL cholesterol, and improved body composition and glucose clearance in animals fed on STD. In those fed with LCD, RT reduced caloric intake, BW regain, liver fat and serum triglycerides levels. However, improvement in body composition was inhibited and bone mineral density and glucose clearance was further impaired in this association. CONCLUSIONS: The LCD nullifies the beneficial effects of RT on body composition, glucose homeostasis and impairs some health parameters. Our results do not support the association of RT with LCD in a long term period.

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BACKGROUND: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2ß1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2ß1 integrin. Herein, we used ALT-C as a α2ß1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. METHODS: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2ß1 integrin binding properties of ALT-C, its dissociation constant (Kd ) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. RESULTS: Our data demonstrate that ALT-C, after binding to α2ß1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. CONCLUSION: These results demonstrate that α2ß1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.

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