Peptide-based biosensing approaches for targeting breast cancer-derived exosomes.

Exosomes are nanovesicles present in all the biological fluids, making them attractive as non-invasive biomarkers for diseases like cancer, among many others. However, exosomes are complex to separate and detect, requiring comprehensive molecular characterization for their routine use in diagnostics. This study explores the use of peptides as cost-effective and stable alternatives to antibodies for exosome binding. To achieve that, phage display technology was employed to select peptides with high specificity for target molecules in exosomes. Specifically, a selected peptide was evaluated for its ability to selectively bind breast cancer-derived exosomes. Proteomic analysis identified 38 protein candidates targeted by the peptide on exosome membranes. The binding of the peptide to breast cancer-derived exosomes was successfully demonstrated by flow cytometry and magneto-actuated immunoassays. Furthermore, an electrochemical biosensor was also tested for breast cancer-derived exosome detection and quantification. The peptide demonstrated effective binding to exosomes from aggressive cancer cell lines, offering promising results in terms of specificity and recovery. This research shows potential for developing rapid, accessible diagnostic tools for breast cancer, especially in low-resource healthcare settings.

A new copper(II) complex containing long-chain aliphatic hydrazide and 1,10-phenanthroline upregulates ADP hydrolysis in triple-negative breast cancer cells.

Copper can be opportunely complexed to modulate oncogenic pathways, being a promising strategy for cancer treatment. Herein, three new copper(II) complexes containing long-chain aliphatic hydrazides and 1,10-phenanthroline (1,10-phen), namely, [Cu(octh)(1,10-phen)(H2O)](NO3)21, [Cu(dech)(1,10-phen)(H2O)](NO3)22 and [Cu(dodh)(1,10-phen)(H2O)](NO3)2.H2O 3 (where octh = octanoic hydrazide, dech = decanoic hydrazide, dodh = dodecanoic hydrazide) were successfully prepared and characterized by several physical-chemical methods. Furthermore, X-ray structural analysis of complex 2 indicated that the geometry around the copper(II) ion is distorted square-pyramidal, in which hydrazide and 1,10-phenanthroline act as bidentate ligands. A water molecule in the apical position completes the coordination sphere of the metal ion. All new copper(II) complexes were cytotoxic to breast cancer cell lines (MCF7, MDA-MB-453, MDA-MB-231, and MDA-MB-157) and selective when compared to the non tumor lineage MCF-10A. In particular, complex 2 showed half-maximal inhibitory concentration (IC50) values ranging between 2.7 and 13.4 µM in MDA-MB231 cells after 24 and 48 h of treatment, respectively. Furthermore, this complex proved to be more selective for tumor cell lines when compared to doxorubicin and docetaxel. Complex 2 inhibited the clonogenicity of MDA-MB231 cells, increasing adenosine diphosphate (ADP) hydrolysis and upregulating ecto-nucleoside triphosphate diphosphohydrolase 1 (ENTPD1) transcriptional levels. In this sense, we suggest that the inhibitory effect on cell proliferation may be related to the modulation of adenosine monophosphate (AMP) levels. Thus, a novel copper(II) complex with increased cytotoxic effects and selectivity against breast cancer cells was obtained, contributing to medicinal chemistry efforts toward the development of new chemotherapeutic agents.

Tailoring Potential Natural Compounds for the Treatment of Luminal Breast Cancer.

Breast cancer (BC) is the most diagnosed cancer worldwide, mainly affecting the epithelial cells from the mammary glands. When it expresses the estrogen receptor (ER), the tumor is called luminal BC, which is eligible for endocrine therapy with hormone signaling blockade. Hormone therapy is essential for the survival of patients, but therapeutic resistance has been shown to be worrying, significantly compromising the prognosis. In this context, the need to explore new compounds emerges, especially compounds of plant origin, since they are biologically active and particularly promising. Natural products are being continuously screened for treating cancer due to their chemical diversity, reduced toxicity, lower side effects, and low price. This review summarizes natural compounds for the treatment of luminal BC, emphasizing the activities of these compounds in ER-positive cells. Moreover, their potential as an alternative to endocrine resistance is explored, opening new opportunities for the design of optimized therapies.

Glyphosate and Aminomethylphosphonic Acid (AMPA) Modulate Glutathione S-Transferase in Non-Tumorigenic Prostate Cells.

Glyphosate (GLY) was developed in the early 1970s and has become the most used broad-spectrum herbicide in the world so far. Its main metabolite is aminomethylphosphonic acid (AMPA), and the accumulation of GLY and its derivative compounds raises some concerns regarding possible health outcomes. In this study, we aimed to evaluate the effects of GLY and AMPA on prostate cell lines by evaluating cell viability, proliferation, gene and protein expression, and cellular pathways involved in the response to oxidative stress. Our results indicated that GLY and AMPA reduced the cell viability of tumorigenic and non-tumorigenic prostate cell lines only at higher concentrations (10 mM GLY and 20 mM AMPA). In contrast, both compounds increased the clonogenicity of non-tumorigenic PNT2 cells, mainly at concentrations below the IC50 (5 mM GLY and 10 mM AMPA). Moreover, treatment of non-tumorigenic cells with low concentrations of GLY or AMPA for 48 h increased GSTM3 expression at both mRNA and protein levels. In contrast, the treatments decrease the GST activity and induced an increase in oxidative stress, mainly at lower concentrations. Therefore, both compounds can cause cellular damage even at lower concentrations in non-tumorigenic PNT2 cells, mainly affecting cell proliferation and oxidative stress.

4-Nerolidylcatechol (4-NC) and Docetaxel Synergize in Controlling Androgen- independent Prostate Cancer Cells.

BACKGROUND: Effective cancer treatment still challenges medicine since the strategies employed so far are not sufficiently safe and capable of specifically eliminating tumor cells. Prostate cancer (PCa) is a highly incident malignant neoplasm, and the outcome of patients, especially those with advanced castration-resistant PCa (CRPC), depends directly on the efficacy of the therapeutic agents, such as docetaxel (DOC). OBJECTIVES: This study investigated the synergistic potentiation of 4-nerolidylcatechol (4-NC) with DOC in inhibiting androgen-independent PCa cells. METHODS: The cytotoxic effect of 4-NC was evaluated against non-tumorigenic (RWPE-01) and PCa cell lines (LNCaP and PC-3), and the antiproliferative potential of 4-NC was assessed by flow cytometry and colony formation. The Chou-Talalay method was applied to detect the synergistic effect of 4-NC and DOC, and the mechanism of anticancer activities of this combination was investigated by analyzing players in epithelial-mesenchymal transition (EMT). RESULTS: 4-NC significantly reduced the viability of PC-3 cells in a dose-dependent manner, decreasing colony formation and proliferation. The combination of 4-NC and DOC was synergistic in the androgen-independent cells and allowed the reduction of DOC concentration, with increased cytotoxicity and induction of apoptosis when compared to compounds alone. Furthermore, when 4- NC was co-administered with DOC, higher expression levels of proteins associated with the epithelial phenotype were observed, controlling EMT in PC-3 cells. CONCLUSION: Collectively, these data demonstrated, for the first time, that the combination of 4-NC with reduced doses of DOC could be especially valuable in the suppression of oncogenic mechanisms of androgen-independent PCa cells.

Copper(II) Complex Containing 4-Fluorophenoxyacetic Acid Hydrazide and 1,10-Phenanthroline: A Prostate Cancer Cell-Selective and Low-Toxic Copper(II) Compound.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. The treatment of advanced cases is based on chemotherapy, which lacks specificity and efficacy, due to severe side effects and resistance to the traditional drugs. Copper complexes have shown antitumoral efficacy and low toxicity, being considered a promising class of metal-based drugs for the treatment of malignant neoplasms. Thus, the present study aimed to evaluate the cellular effects of a copper(II) complex with 4-fluorophenoxyacetic acid hydrazide and 1,10-phenanthroline (1) on PCa cell lines, as well as the mutagenic/recombinogenic and anticarcinogenic potential of 1 in Drosophila melanogaster. PNT-2 (non-tumorigenic), LNCaP (hormone-responsive PCa) and PC-3 (androgen-independent PCa) cells were cultured, and cytotoxicity was assessed using the MTT assay. The expression levels of the proliferation markers Ki-67 and Cyclin D1 were analyzed by flow cytometry. Furthermore, the Somatic Mutation and Recombination Test (SMART) and the Epithelial Tumor Test (ETT) were performed. Complex 1 was selective to LNCaP cells, significantly reducing Ki-67 and Cyclin D1 expression levels. Sub-toxic concentrations of complex 1 were defined by the toxicity test in D. melanogaster, and no mutagenic/recombinogenic/carcinogenic effects were observed. Anticarcinogenic potential was observed in D. melanogaster, suggesting modulating activity of the complex 1 against Doxorubicin, a drug used as control by its carcinogenic properties. Therefore, complex 1 is a possible starting point for the development of new antitumor agents for the treatment of PCa.

Interleukin-6 Signaling in Triple Negative Breast Cancer Cells Elicits the Annexin A1/Formyl Peptide Receptor 1 Axis and Affects the Tumor Microenvironment.

Annexin A1 (AnxA1) is a pleiotropic protein that exerts essential roles in breast cancer (BC) growth and aggressiveness. In our previous work, we described the autocrine signaling of AnxA1 through formyl peptide receptor 1 (FPR1) in the triple-negative (TN) BC cell line, MDA-MB-231. Here, we aimed to describe the interaction between the AnxA1/FPR1 and the Interleukin-6 (IL-6) signaling pathways and their role in the tumor microenvironment (TME). First, we demonstrated that AnxA1 and IL-6 expression levels are correlated in BC tissue samples. In three TNBC cell lines, overexpression of both AnxA1 and IL-6 was also identified. Next, we inhibited FPR1, the IL-6 receptor and STAT3 in both MDA-MB-231 and MDA-MB-157 cells. The FPR1 inhibition led to increased levels of IL-6 and secreted AnxA1 in both cell lines. On the other side, inhibition of the IL-6 receptor or STAT3 led to the impairment of AnxA1 secretion, suggesting the essential role of the IL-6 signaling cascade in the activation of the AnxA1/FPR1 autocrine axis. Finally, we described the interaction between IL-6 and the AnxA1/FPR1 pathways and their role on the TME by analyzing the effect of supernatants derived from MDA-MB-231 and MDA-MB-157 cells under the inhibition of FPR1 or IL-6 signaling on fibroblast cell motility.

Is the regulation by miRNAs of NTPDase1 and ecto-5'-nucleotidase genes involved with the different profiles of breast cancer subtypes?

Breast cancer (BC) is a public health problem worldwide, causing suffering and premature death among women. As a heterogeneous disease, BC-specific diagnosis and treatment are challenging. Ectonucleotidases are related to tumor development and their expression may vary among BC. miRNAs may participate in epigenetic events and may regulate ectonucleotidases in BC. This study aimed to evaluate the expression of ectonucleotidases according to BC subtypes and to predict if there is post-transcriptional regulation of them by miRNAs. MCF 10A (non-tumorigenic), MCF7 (luminal BC), and MDA-MB-231 (triple-negative BC - TNBC) breast cell lines were used and ENTPD1 (the gene encoding for NTPDase1) and NT5E (the gene encoding for ecto-5'-nucleotidase) gene expression was determined. Interestingly, the expression of ENTPD1 was only observed in MCF7 and NT5E was lower in MCF7 compared to MDA-MB-231 cell line. ATP, ADP, and AMP hydrolysis were observed on the surface of all cell lines, being higher in MDA-MB-231. Like qPCR, the activity of AMP hydrolysis was also lower in the MCF7 cells, which may represent a striking feature of this BC subtype. In silico analyses confirmed that the miRNAs miR-101-3p, miR-141-3p, and miR-340-5p were higher expressed in MCF7 cells and targeted NT5E mRNA. Altogether, data suggest that the regulation of NT5E by miRNAs in MCF7 lineage may direct the molecular profile of luminal BC. Thus, we suggest that the roles of ecto-5'-nucleotidase and the aforementioned miRNAs must be unraveled in TNBC to be possibly defined as diagnostic and therapeutic targets.

A selective CuII complex with 4-fluorophenoxyacetic acid hydrazide and phenanthroline displays DNA-cleaving and pro-apoptotic properties in cancer cells.

The thin line between efficacy and toxicity has challenged cancer therapy. As copper is an essential micronutrient and is important to tumor biology, CuII complexes emerged as an alternative to chemotherapy; however, its biological properties need to be better understood. Thus, we report in vitro the antitumor effects of two CuII complexes named [Cu(4-fh)(phen)(ClO4)2] (complex 1) and [Cu(4-nh)(phen)(ClO4)2]·H2O (complex 2), in which 4-fh = 4-fluorophenoxyacetic acid hydrazide; 4-nh = 4-nitrobenzoic hydrazide and phen = 1,10-phenanthroline. Both complexes presented cytotoxic activity against tumor cells, but only complex 1 showed significant selectivity. Complex 1 also induced DNA-damage, led to G0/G1 arrest and triggered apoptosis, which was initiated by an autophagy dysfunction. The significant in vitro selectivity and the action mechanism of complex 1 are noteworthy and reveal this prodrug as promising for anticancer therapy.

Annexin A1 as a Regulator of Immune Response in Cancer.

Annexin A1 is a 37 kDa phospholipid-binding protein that is expressed in many tissues and cell types, including leukocytes, lymphocytes and epithelial cells. Although Annexin A1 has been extensively studied for its anti-inflammatory activity, it has been shown that, in the cancer context, its activity switches from anti-inflammatory to pro-inflammatory. Remarkably, Annexin A1 shows pro-invasive and pro-tumoral properties in several cancers either by eliciting autocrine signaling in cancer cells or by inducing a favorable tumor microenvironment. Indeed, the signaling of the N-terminal peptide of AnxA1 has been described to promote the switching of macrophages to the pro-tumoral M2 phenotype. Moreover, AnxA1 has been described to prevent the induction of antigen-specific cytotoxic T cell response and to play an essential role in the induction of regulatory T lymphocytes. In this way, Annexin A1 inhibits the anti-tumor immunity and supports the formation of an immunosuppressed tumor microenvironment that promotes tumor growth and metastasis. For these reasons, in this review we aim to describe the role of Annexin A1 in the establishment of the tumor microenvironment, focusing on the immunosuppressive and immunomodulatory activities of Annexin A1 and on its interaction with the epidermal growth factor receptor.

Phospholipase A2 Drives Tumorigenesis and Cancer Aggressiveness through Its Interaction with Annexin A1.

Phospholipids are suggested to drive tumorigenesis through their essential role in inflammation. Phospholipase A2 (PLA2) is a phospholipid metabolizing enzyme that releases free fatty acids, mostly arachidonic acid, and lysophospholipids, which contribute to the development of the tumor microenvironment (TME), promoting immune evasion, angiogenesis, tumor growth, and invasiveness. The mechanisms mediated by PLA2 are not fully understood, especially because an important inhibitory molecule, Annexin A1, is present in the TME but does not exert its action. Here, we will discuss how Annexin A1 in cancer does not inhibit PLA2 leading to both pro-inflammatory and pro-tumoral signaling pathways. Moreover, Annexin A1 promotes the release of cancer-derived exosomes, which also lead to the enrichment of PLA2 and COX-1 and COX-2 enzymes, contributing to TME formation. In this review, we aim to describe the role of PLA2 in the establishment of TME, focusing on cancer-derived exosomes, and modulatory activities of Annexin A1. Unraveling how these proteins interact in the cancer context can reveal new strategies for the treatment of different tumors. We will also describe the possible strategies to inhibit PLA2 and the approaches that could be used in order to resume the anti-PLA2 function of Annexin A1.

Ruthenium (II) complex cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6-hmxbato induces ROS-mediated apoptosis in lung tumor cells producing selective cytotoxicity.

Ruthenium complexes have been extensively explored as potential molecules for cancer treatment. Considering our previous findings on the remarkable cytotoxic activity exhibited by the ruthenium (II) complex 3-hydroxy-4-methoxybenzoate (hmxbato)-cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6 against Leishmania promastigotes and also the similar metabolic characteristics between trypanosomatids and tumor cells, the present study aimed to analyze the anticancer potential of hmxbato against lung tumor cells, as well as the partial death mechanisms involved. Hmxbato demonstrated selective cytotoxicity against A549 lung tumor cells. In addition, this complex at a concentration of 3.8 µM was able to expressively increase the generation of reactive oxygen species (ROS) in tumor cells, causing an oxidative stress that may culminate in: (1) reduction in cellular proliferation; (2) changes in cell morphology and organization patterns of the actin cytoskeleton; (3) cell arrest in the G2/M phase of the cell cycle; (4) apoptosis; (5) changes in the mitochondrial membrane potential and (6) initial DNA damage. Furthermore, we demonstrated that the induction of programmed cell death can occur by the intrinsic apoptotic pathway through the activation of caspases. It is also worth highlighting that hmxbato exhibited predominant actions on A549 tumor cells in comparison to BEAS-2B normal bronchial epithelium cells, which makes this complex an interesting candidate for the design of new drugs against lung cancer.

Annexin A1 promotes the nuclear localization of the epidermal growth factor receptor in castration-resistant prostate cancer.

Epidermal growth factor receptor is a cancer driver whose nuclear localization has been associated with the progression of prostate cancer to the castration-resistant phenotype. Previous reports indicated a functional interaction between this receptor and the protein Annexin A1, which has also been associated with aggressive tumors. The molecular pathogenesis of castration-resistant prostate cancer remains largely unresolved, and herein we have demonstrated the correlation between the expression levels and localization of the epidermal growth factor receptor and Annexin A1 in prostate cancer samples and cell lines. Interestingly, a higher expression of both proteins was detected in castration-resistant prostate cancer cell lines and the strongest correlation was seen at the nuclear level. We verified that Annexin A1 interacts with the epidermal growth factor receptor, and by using prostate cancer cell lines knocked down for Annexin A1, we succeeded in demonstrating that Annexin A1 promotes the nuclear localization of epidermal growth factor receptor. Finally, we showed that Annexin A1 activates an autocrine signaling in castration-resistant prostate cells through the formyl peptide receptor 1. The inhibition of such signaling by Cyclosporin H inhibits the nuclear localization of epidermal growth factor receptor and its downstream signaling. The present work sheds light on the functional interaction between nuclear epidermal growth factor receptor and nuclear Annexin A1 in castration-resistant prostate cancer. Therefore, strategies to inhibit the nuclear localization of epidermal growth factor receptor through the suppression of the Annexin A1 autocrine loop could represent an important intervention strategy for castration-resistant prostate cancer.

Parthenolide and its Analogues: A New Potential Strategy for the Treatment of Triple-Negative Breast Tumors.

Triple Negative Breast Cancers (TNBC) are heterogeneous and aggressive pathologies, with distinct morphological and clinical characteristics associated with their genetic diversity, epigenetics, transcriptional changes and aberrant molecular patterns. Treatment with anti-neoplastic drugs exerts systemic effects with low specificity, and incipient improvement in overall survival due to chemoresistance and recurrence. New alternatives for TNBC treatment are urgent and parthenolide or its analogues have been explored. Parthenolide is a sesquiterpene lactone with promising antitumor effects against TNBC cell lines. This review highlights the importance of parthenolide and its analogue drugs in TNBC treatment.

Gene surgery: Potential applications for human diseases.

Gene therapy became in last decade a new emerging therapeutic era showing promising results against different diseases such as cancer, cardiovascular diseases, diabetes, and neurological disorders. Recently, the genome editing technique for eukaryotic cells called CRISPR-Cas (Clustered Regulatory Interspaced Short Palindromic Repeats) has enriched the field of gene surgery with enhanced applications. In the present review, we summarized the different applications of gene surgery for treating human diseases such as cancer, diabetes, nervous, and cardiovascular diseases, besides the molecular mechanisms involved in these important effects. Several studies support the important therapeutic applications of gene surgery in a large number of health disorders and diseases including ß-thalassemia, cancer, immunodeficiencies, diabetes, and neurological disorders. In diabetes, gene surgery was shown to be effective in type 1 diabetes by triggering different signaling pathways. Furthermore, gene surgery, especially that using CRISPR-Cas possessed important application on diagnosis, screening and treatment of several cancers such as lung, liver, pancreatic and colorectal cancer. Nevertheless, gene surgery still presents some limitations such as the design difficulties and costs regarding ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases) use, off-target effects, low transfection efficiency, in vivo delivery-safety and ethical issues.

Inhibition of Triple-Negative Breast Cancer Cell Aggressiveness by Cathepsin D Blockage: Role of Annexin A1.

Triple-negative breast cancers (TNBCs) are more aggressive than other breast cancer (BC) subtypes and lack effective therapeutic options. Unraveling marker events of TNBCs may provide new directions for development of strategies for targeted TNBC therapy. Herein, we reported that Annexin A1 (AnxA1) and Cathepsin D (CatD) are highly expressed in MDA-MB-231 (TNBC lineage), compared to MCF-10A and MCF-7. Since the proposed concept was that CatD has protumorigenic activity associated with its ability to cleave AnxA1 (generating a 35.5 KDa fragment), we investigated this mechanism more deeply using the inhibitor of CatD, Pepstatin A (PepA). Fourier Transform Infrared (FTIR) spectroscopy demonstrated that PepA inhibits CatD activity by occupying its active site; the OH bond from PepA interacts with a CO bond from carboxylic acids of CatD catalytic aspartate dyad, favoring the deprotonation of Asp33 and consequently inhibiting CatD. Treatment of MDA-MB-231 cells with PepA induced apoptosis and autophagy processes while reducing the proliferation, invasion, and migration. Finally, in silico molecular docking demonstrated that the catalytic inhibition comprises Asp231 protonated and Asp33 deprotonated, proving all functional results obtained. Our findings elucidated critical CatD activity in TNBC cell trough AnxA1 cleavage, indicating the inhibition of CatD as a possible strategy for TNBC treatment.

Biowar: o desafio no ensino de Biossegurança / Biowar: challenges in Biosafety education

A biossegurança se torna importante, tanto no controle dos riscos ocupacionais quanto no controle dos riscos de prejuízo ambiental provenientes das novas tecnologias científicas. O presente trabalho objetivou proporcionar uma prática docente caracterizada pela ênfase na autonomia, na liberdade e na criatividade, como pressupostos para a construção do conhecimento universitário. O jogo proposto, denominado Biowar, concentrou-se em fornecer e disseminar o conhecimento educativo entre os alunos, abordando temas multidisciplinares embasados em biossegurança. Com o elevado índice de aceitação dos alunos do curso de Biotecnologia da Universidade Federal de Uberlândia, o jogo mostrou-se eficiente ao abordar temas científicos recentes, funcionando como um recurso eficaz e método alternativo de ensino na formação de cientistas cidadãos devidamente alicerçados nos conceitos de biossegurança

Biosecurity becomes important in the control of occupational hazards and in controlling the risk of environmental damage from new scientific technologies. This study aimed to provide a teaching practice characterized by emphasis on autonomy, freedom and creativity, as prerequisites for the construction of university knowledge. The proposed game called Biowar, aimed on providing and disseminating educational knowledge among students, addressing multidisciplinary issues based in biosafety. With high acceptance rate of students of biotechnology at the Universidade Federal de Uberlândia, the game Biowar was efficient in addressing recent scientific topics, with an effective resource as alternative teaching methods in forming properly grounded citizen scientists in biosafety concepts

Unraveling Antibody Display: Systems Biology and Personalized Medicine.

BACKGROUND: The identification of combinatorial antibodies against many different targets in oncology, autoimmune, inflammatory and infectious diseases has uncovered novel strategies to control and prevent diseases' onset and progression, and represents the fastest growing market for the pharmaceutical industry. Phage Display has been successfully used in the identification of unknown targets, which combines shotgun approaches with high throughput selection schemes. METHODS: This specific review covers many aspects of combinatorial phage display technology starting from antibody selection strategies to its redesign for application purposes. Emphasis is specifically directed to how these biotherapeutics function on specific targets with an interactome view, especially within complex diseases. CONCLUSION: Novel combinatorial antibodies will lead to improved interventions in cancer, autoimmune and infectious diseases; however, the very large genetic diversity associated with environmental variations highlight the importance of the personalized medicine using a system's biology approach. Therefore, combined therapies are expected in the near future.

Association of vitamin D receptor variants with clinical parameters in prostate cancer.

PURPOSE: Prostate Cancer (PCa) is one of the most common cancers in men and its early detection can provide a high chance of cure. The detection of Vitamin D Receptor (VDR) gene polymorphisms may be useful as a molecular indicator of clinical outcome, once VDR is implicated in a wide variety of biological processes including modulation of the immune response and inhibition of cancer cell growth, angiogenesis and metastasis. In this study we explored the Single Nucleotide Polymorphisms (SNPs) FokI, BsmI, ApaI and TaqI, to evaluate the susceptibility locus for PCa and verify its correlation with clinical parameters. METHODS: VDR polymorphisms were detected by PCR followed by Restriction Fragment Length Polymorphism (PCR-RFLP). DNA samples were extracted from peripheral blood of 342 patients: 132 PCa, 41 Benign Prostatic Hyperplasia and 169 young healthy volunteers. RESULTS: Statistical analysis showed a noteworthy correlation among SNPs and clinical pathological features. CC genotype (TaqI) was correlated with the age at diagnosis (>58 years old), and GG (BsmI) was associated to lower Prostate-Specific Antigen (PSA) levels (<10 ng/mL). Moreover, when PCa patients were subgrouped, G allele (BsmI) significantly increased the estimated chance for PSA < 10 ng/mL, and GG/GG genotype (BsmI/ApaI) provided a 9.75 fold increased chance of patients with PCa to present lower PSA levels. CONCLUSIONS: The polymorphisms of VDR gene showed a genotype-phenotype association and presented new correlations with different parameters as age and PSA levels.

Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis.

We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.