Proteomic Analysis of Trypanosoma cruzi Response to Ionizing Radiation Stress

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.     

System Dynamics Modeling in the Evaluation of Delays of Care in ST-Segment Elevation Myocardial Infarction Patients within a Tiered Health System

Background

Mortality rates amongst ST segment elevation myocardial infarction (STEMI) patients remain high, especially in developing countries. The aim of this study was to evaluate the factors related with delays in the treatment of STEMI patients to support a strategic plan toward structural and personnel modifications in a primary hospital aligning its process with international guidelines.

Methods and Findings

The study was conducted in a primary hospital localized in Foz do Iguaçu, Brazil. We utilized a qualitative and quantitative integrated analysis including on-site observations, interviews, medical records analysis, Qualitative Comparative Analysis (QCA) and System Dynamics Modeling (SD). Main cause of delays were categorized into three themes: a) professional, b) equipment and c) transportation logistics. QCA analysis confirmed four main stages of delay to STEMI patient’s care in relation to the ‘Door-in-Door-out’ time at the primary hospital. These stages and their average delays in minutes were: a) First Medical Contact (From Door-In to the first contact with the nurse and/or physician): 7 minutes; b) Electrocardiogram acquisition and review by a physician: 28 minutes; c) ECG transmission and Percutaneous Coronary Intervention Center team feedback time: 76 minutes; and d) Patient’s Transfer Waiting Time: 78 minutes. SD baseline model confirmed the system’s behavior with all occurring delays and the need of improvements. Moreover, after model validation and sensitivity analysis, results suggested that an overall improvement of 40% to 50% in each of these identified stages would reduce the delay.

Conclusions

This evaluation suggests that investment in health personnel training, diminution of bureaucracy, and management of guidelines might lead to important improvements decreasing the delay of STEMI patients’ care. In addition, this work provides evidence that SD modeling may highlight areas where health system managers can implement and evaluate the necessary changes in order to improve the process of care.     

Vitamin D Deficiency Aggravates Nephrotoxicity,Hypertension and Dyslipidemia Caused by Tenofovir: Role of Oxidative Stress and Renin-Angiotensin System

Vitamin D deficiency (VDD) is prevalent among HIV-infected individuals. Vitamin D has been associated with renal and cardiovascular diseases because of its effects on oxidative stress, lipid metabolism and renin-angiotensin-aldosterone system (RAAS). Tenofovir disoproxil fumarate (TDF), a widely used component of antiretroviral regimens for HIV treatment, can induce renal injury. The aim of this study was to investigate the effects of VDD on TDF-induced nephrotoxicity. Wistar rats were divided into four groups: control, receiving a standard diet for 60 days; VDD, receiving a vitamin D-free diet for 60 days; TDF, receiving a standard diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days; and VDD+TDF receiving a vitamin D-free diet for 60 days with the addition of TDF for the last 30 days. TDF led to impaired renal function, hyperphosphaturia, hypophosphatemia, hypertension and increased renal vascular resistance due to downregulation of the sodium-phosphorus cotransporter and upregulation of angiotensin II and AT1 receptor. TDF also increased oxidative stress, as evidenced by higher TBARS and lower GSH levels, and induced dyslipidemia. Association of TDF and VDD aggravated renovascular effects and TDF-induced nephrotoxicity due to changes in the redox state and involvement of RAAS.     

Acute Chagas Disease: New Global Challenges for an Old Neglected Disease

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi, and although over 100 years have passed since the discovery of Chagas disease, it still presents an increasing problem for global public health. A plethora of information concerning the chronic phase of human Chagas disease, particularly the severe cardiac form, is available in the literature. However, information concerning events during the acute phase of the disease is scarce. In this review, we will discuss (1) the current status of acute Chagas disease cases globally, (2) the immunological findings related to the acute phase and their possible influence in disease outcome, and (3) reactivation of Chagas disease in immunocompromised individuals, a key point for transplantation and HIV infection management.     

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)^1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)⁴, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization^5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman^9,10; further field use is planned or in progress in other countries around the world.Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults^11,12.To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared^13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti ⁸, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.     

Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 μg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-X_L, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.Endostatin (ES) is a specific inhibitor of endothelial cell proliferation, migration, invasion, tube formation, angiogenesis, and tumor growth in animal models.^{1, 2} Treatment with ES does not produce side effects or induce drug resistance.¹ ES exerts its biological activities by binding to cell surface receptors, a process that triggers intracellular signaling cascades. Proteins such as nucleolin, matrix metalloproteinase 2, integrins, tropomyosin, glypicans, and laminin-1 are possible ES receptors that display binding affinities and that were described to be involved in the ES antiangiogenic function.^{3, 4, 5, 6, 7}The necessity to administer high ES levels on a daily basis (15–600 mg/m²/day),⁸ the need to adjust doses,⁹ and the low antitumoral effect observed in clinical assays in humans¹⁰ have limited the use of ES to treat human cancer. Therefore, modifying the ES structure might improve its proapoptotic activity and provide better therapeutic protocols for human patients with cancer.The B-cell lymphoma 2 (BCL-2) family of proteins constitutes regulators of the apoptosis intrinsic pathway.^{11, 12} The BCL-2 members can be divided into three main subclasses that are partly defined by the homology shared within four conserved regions. These regions, termed BCL-2 homology (BH) 1–4 domains, correspond to α-helices with similar sequences that dictate protein structure and function. The antiapoptotic subfamily members BCL-2, B-cell lymphoma-extra large (BCL-X_L), BCL-W, MCL-1, and A1 contain three or four BH domains. The apoptosis effectors BCL-2-associated X-protein (BAX) and BCL-2 antagonist/killer (BAK) are subfamily relatives that possess structures in the domains BH1 through BH3; they closely resemble their prosurvival cognates.^{13, 14} The proapoptotic ‘BH3-only'' proteins are related to the other members by the short signature BH3 domain, which is essential for their killing function.^{15, 16} The apoptotic switch operates through interactions between the proteins within the subfamilies. The structure of the prosurvival BCL-X_L monomer revealed that its BH1, BH2, and BH3 domains are in close proximity and create a hydrophobic pocket that can accommodate a BH3 domain of the BAK proapoptotic member.¹⁷ In viable cells, the multidomains BAX and BAK exist as inactive monomers. Inactive BAX resides in the cytosol or loosely attaches to membranes; its C-terminal α9 helix occupies its hydrophobic pocket.¹³ Upon receipt of a death signal by a triggering BH3 helix, BAX transforms into a fully activated monomer that can propagate its own activation.¹⁸ Activated BAX translocates to the mitochondria, forming a putative homo-oligomer and generating pores that irreversibly damages these organelles.¹⁹ Consequently, proapoptogenic factors are released, activating the executioner caspases.^{20, 21} The BAX BH3 domain confers BAX killing functionality. The minimal portion of BAK, critical for the heterodimerization and proapoptotic function, consists of a 15/16-amino acid peptide mapped to the BH3 domain.^{15, 17, 22}Impaired apoptosis is a critical step in tumor development. Enhanced levels of the prosurvival BCL-2 family members or, alternatively, the loss or inactivation of the pro-death relatives frequently occur in cancer.²³ Therefore, scientists have designed strategies to induce downstream apoptotic events that could overcome the inhibition of tumor cells apoptosis by either delivering proapoptotic BH3 peptides^{24, 25} or using compounds that function as cell permeable, small molecular mimics of the BH3 domain.²⁶ However, there is concern about the therapeutic use of proapoptotic BH3 or its mimetics because of the lack of specificity to tumor cells, possibly prompting to greater toxicity to normal cells. Inducing an imbalance in favor of cell death by raising the levels of the proapoptotic BH3 peptide, is an interesting strategy, especially in cells with normal levels of the antiapoptotic BCL-2 proteins, which is the case of cells of tumor vasculature.In the present study, we produced three chimerical recombinant proteins based on the core of the ES fused with the BH3 domains of the proapoptotic proteins BAK and BAX as a means to target these proteins. Such proteins display enhanced proapoptotic properties toward the tumor endothelium, avoiding damage to normal tissues. In addition, we determined if ES and ES-BAX interact with members of the BCL-2 family in endothelial cell lysates.     

Fullerene-Based Symmetry in Hibiscus rosa-sinensis Pollen

The fullerene molecule belongs to the so-called super materials. The compound is interesting due to its spherical configuration where atoms occupy positions forming a mechanically stable structure. We first demonstrate that pollen of Hibiscus rosa-sinensis has a strong symmetry regarding the distribution of its spines over the spherical grain. These spines form spherical hexagons and pentagons. The distance between atoms in fullerene is explained applying principles of flat, spherical, and spatial geometry, based on Euclid’s “Elements” book, as well as logic algorithms. Measurements of the pollen grain take into account that the true spine lengths, and consequently the real distances between them, are measured to the periphery of each grain. Algorithms are developed to recover the spatial effects lost in 2D photos. There is a clear correspondence between the position of atoms in the fullerene molecule and the position of spines in the pollen grain. In the fullerene the separation gives the idea of equal length bonds which implies perfectly distributed electron clouds while in the pollen grain we suggest that the spines being equally spaced carry an electrical charge originating in forces involved in the pollination process.     

Foreign Body Response to Subcutaneous Implants in Diabetic Rats

Implantation of synthetic matrices and biomedical devices in diabetic individuals has become a common procedure to repair and/or replace biological tissues. However, an adverse foreign body reaction that invariably occurs adjacent to implant devices impairing their function is poorly characterized in the diabetic environment. We investigated the influence of this condition on the abnormal tissue healing response in implants placed subcutaneously in normoglycemic and streptozotocin-induced diabetes in rats. In polyether-polyurethane sponge discs removed 10 days after implantation, the components of the fibrovascular tissue (angiogenesis, inflammation, fibrogenesis, and apoptosis) were assessed. Intra-implant levels of hemoglobin and vascular endothelial growth factor were not different after diabetes when compared with normoglycemic counterparts. However, there were a lower number of vessels in the fibrovascular tissue from diabetic rats when compared with vessel numbers in implants from non-diabetic animals. Overall, the inflammatory parameters (neutrophil accumulation - myeloperoxidase activity, tumor necrosis factor alpha, and monocyte chemotactic protein-1 levels and mast cell counting) increased in subcutaneous implants after diabetes induction. However, macrophage activation (N-acetyl-β-D-glucosaminidase activity) was lower in implants from diabetic rats when compared with those from normoglycemic animals. All fibrogenic markers (transforming growth factor beta 1 levels, collagen deposition, fibrous capsule thickness, and foreign body giant cells) decreased after diabetes, whereas apoptosis (TUNEL) increased. Our results showing that hyperglycemia down regulates the main features of the foreign body reaction induced by subcutaneous implants in rats may be relevant in understanding biomaterial integration and performance in diabetes.