Purification and characterization of a thrombolytic enzyme produced by a new strain of Bacillus subtilis

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48°C and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.This work was supported by Direcao Regional da Ciencia e Tecnologia - DRCT-Acores (Medida 2.2.2/I/025/2008) . Duarte Toubarro received a post-doctoral grant (SFRH/BPD/77483/2011; M3.1.a/F/050/2016) from FundacAo para a Ciencia e Tecnologia, Portugal; Jorge Frias received a doctoral grant (SFRH/BD/131698/2017) . The authors are grateful to Dr. Teresa Sampaio and Nelia Arruda, from Dr. Forjaz Sampaio Medical Center, for helping with the plasma anticoagulation assays. Duarte Toubarro and Nelson Simoes designed the study and helped with the paper's redaction. Jorge Frias performed the experiments, analyzed the data and wrote the paper. Alexandra Fraga and Jorge Pedrosa performed thrombolytic assays in a rat model. Claudia Botelho and Jose Teixeira participated in the discussion and redaction of the paper.info:eu-repo/semantics/publishedVersio

Biomedical applications of human cathelicidin

[Excerpt] Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, represents the second world’s deadliest infectious disease, affecting around 9 million people worldwide in 2013. Of those, about 1.1 million died from the disease. The potential of cathelicin, a human AMP, in the treatment of mycobacteriosis and wound regeneration was assessed in pre-clinical trials. (...

The selective COX-2 inhibitor etoricoxib reduces acute inflammatory markers in a model of neurogenic laryngitis but loses its efficacy with prolonged treatment

The selective COX-2 inhibitor Etoricoxib reduces acute inflammatory markers in a model of neurogenic laryngitis but loses its efficacy with prolonged treatment.OBJECTIVE: A randomised experimental study was used to evaluate the therapeutic effect of a selective cyclooxygenase-2 (COX-2) inhibitor in neurogenic laryngitis. MATERIALS AND METHODS: Male Wistar Han rats were subjected to the nasogastric intubation model (NGI) of laryngitis for 1 and 2 weeks. The NGI animals were divided into three groups: (1) treated with COX-2 inhibitor Etoricoxib, (2) vehicle and (3) non-intubated animals. A fourth group of animals was submitted to NGI only. Laryngeal sections were immunostained for substance P (SP) and calcitonin gene-related peptide (CGRP) fibre-immunoreactivity (IR) and quantification of COX-2 positive cells through stereological analysis. The expression of COX-2, interleukins IL-1beta, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha) was determined by quantitative real time QRT-PCR. TREATMENT: Etoricoxib (6 mg/kg/day) was prepared in 0.9% sterile saline with 5% glucose (vehicle) and administered daily during 1 or 2 weeks. RESULTS: Treatment for 1 week with Etoricoxib attenuated the CGRP-IR fibre depletion, the COX-2-IR increased cell number and the TNF-alpha and COX-2 mRNA increased levels induced by NGI. Two weeks of treatment had no beneficial effect. CONCLUSIONS: Etoricoxib is effective in neurogenic laryngitis for limited periods of administration, indicating that selective COX-2 inhibitors should be evaluated in the future.This study was supported by Fundacao Calouste Gulbenkian Project No 74551 and Fundacao Grunenthal (Portugal)

Novel capsular depolymerases-based strategy to kill multidrug-resistant pathogenic bacteria

Multidrug resistant pathogens represent one of the greatest threats to human health of the new millennium. ESKAPE bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and other Enterobacteriaceae species) are the leading group among these socalled superbugs, which rapidly acquire resistances to several (and sometimes all) available antibiotics and cause a variety of nosocomial infections (e.g. bacteraemia and wound infections). Our research has been leading an innovative approach based on bacteriophage-derived enzymes (called capsular depolymerases) against A. baumannii (see video at ref 1). Previously, we found that some bacteriophages (i.e. viruses that specifically infect bacteria) acquired the ability to infect different Acinetobacter hosts through acquisition of different capsular depolymerases (2). These enzymes located at the bacteriophage tails bind and degrade specific bacterial capsules types (2). Recently, recombinantly expressed capsular depolymerases showed to be active in several environment conditions, non-nontoxic to mammalian cells and able to make A. baumannii fully susceptible to host complement effect, namely in i) Galleria mellonella caterpillar, ii) murine and iii) human serum models (3, 4). A single intraperitoneal injection of depolymerase protect 60% of mice from dead, with significant reduction of proinflammatory cytokine profile (4). We show that capsular depolymerases fit the new trend of antimicrobials needed, as they are highly specific, stable and refractory to resistance as they do not kill bacteria per se, instead they remove bacterial surface polysaccharides, diminishing bacterial virulence and exposing them to the host immune system. This innovative antimicrobial approach can be applied to other pathogenic bacteria.info:eu-repo/semantics/publishedVersio

Delivery of nanogel formulations with antimicrobial peptides for the treatment of mycobacteriosis

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Mycobacterium tuberculosis is the human pathogen that causes Tuberculosis (TB). In 2015, 10.4 million TB cases and 1.8 million deaths were reported, placing this disease alongside HIV/AIDS as the deadliest infectious diseases. Current treatments rely in the administration of a cocktail of four first-line antibiotics during 6 months and, in the worst case scenario, a long-lasting treatment (24 months) with second-line drugs. The overuse or misuse of antimicrobial agents decreases the success of treatments and increases emergence of Multi-drug resistant (MDR) strains. Therefore, the development of new strategies for TB therapy is urgently needed. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment since they present high spectrum of antimicrobial activity, high efficacy at low concentrations and low propensity for bacterial resistance. Nevertheless, the low capacity of AMPs to reach the infected site and the use of high concentrations to overcome this problem limits its clinical application - this can be circumvented using a drug delivery system [1]. [...]info:eu-repo/semantics/publishedVersio

Delivery of antimicrobial peptides for the treatment of mycobacteriosis

Mycobacterium tuberculosis, which resides inside macrophages, has always been recognized as one of the most “successful” pathogens. Standard treatments have already been used for decades and, therefore, resistances to the first-line medicines are increasing. Additionally, poor patient compliance with stringent therapies is often pointed out as a major reason leading to treatment failure. Antimicrobial peptides (AMPs), a promising new class of broad spectrum antibiotics, are less prone to result in pathogen resistances due to their target (cellular membranes) and rapid action. In our laboratory we search for AMPs with potent activity against mycobacteria and try to develop efficient delivery systems based on self-assembled colloidal nanocarriers. Additionally, this systems are expected to reduce peptide toxicity and enhance selective uptake on infected cells. Finally, the use of encapsulated drugs in mycobacterial therapy may help reducing drug administration schedules which would ultimately improve patient compliance

A new model of laryngitis: neuropeptide, cyclooxygenase, and cytokine profile

Objectives/Hypothesis: To develop and characterize a new model of laryngeal inflammation by analyzing the presence of neurogenic peptides and expression of cyclooxygenases (COX) and cytokines in the mucosa.Study Design: Laryngitis induced by nasogastric intubation (NGI) was evaluated by histopathologic changes of the mucosa, alterations in calcitonin gene related peptide (CGRP) and substance P (SP) neuropeptides in sensory fibers, and COX-1,2, and cytokine (interleukin [IL]-1, IL-6, IL-10, tumor necrosis factor [TNF]-alpha) expression in the laryngeal mucosa.Methods: Rats submitted to NGI for 1 to 5 weeks were compared with controls. Laryngeal sections were immunostained for stereologic analysis of SP and CGRP fiber density and number of mucosal cells expressing COX-2. Alterations in inflammatory mediators were evaluated by quantitative reverse-transcriptase polymerase chain reaction.Results: NGI induced metaplasia of the epithelium and narrowing of the laryngeal lumen because of hypertrophy of laryngeal glandules and edema. An initial decrease in CGRP- and SP-immunoreactive fibers in the laryngeal mucosa (1-3 wk) was reverted with time (5 wk). COX-2 expression in mucosal cells increased progressively, reaching a maximum level at 5 weeks, and was observed in mononuclear immune cells, which is indicative of a chronic inflammatory process. In regard to mRNA expression levels of inflammatory mediators, TNF-alpha was increased during the 5 week NGI, and IL-10 decreased during the 5 weeks,whereas IL-beta, IL-6, and COX-2 increased in the first 1 to 2 weeks and returned to baseline at 5 weeks.Conclusions: This NGI model results in laryngeal chronic inflammation without direct mechanical aggression of the mucosa and may contribute to the study of future therapeutic approaches to this pathology.FEDERFundação para a Ciência e Tecnologia (FCT) - POCTI/NSE/46399/2002Fundação Calouste Gulbenkian - projecto nº 7455

Processo de sobremoldação de vedante numa tampa plástica

O objeto da presente patente é o processo de sobremoldação de uma tampa Plástica com vedante incorporado, na qual é injetado o material de maior dureza sobre o De menor dureza. Este processo de sobremoldação é aplicado somente em bi-injecção (utilização de dois materiais ou mais), em tampas plásticas rígidas para embalagens de Forma redonda ou outras. A solução adotada passou por proteger o vedante (3) do fluxo (9) do material da tampa (2) de modo a evitar que este cause deformações no vedante (3) Durante o processo de enchimento. A forma de orientação precisa do fluxo (9) do sobre o Material de menor dureza (3) evita a deformação deste ultimo recorrendo aos diferenciais De pressão conjugados com a velocidade. Desta forma é possível injetar sobre um Material de menor dureza (3), um material de maior dureza (2)

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

Extracellular vesicles are membrane-enclosed secreted vesicles involved in cell-to-cell communication processes, identified in virtually all body fluids. Among extracellular vesicles, exosomes have gained increasing attention in recent years as they have unique biological origins and deliver different cargos, such as nucleic acids, proteins, and lipids, which might mediate various health processes. In particular, milk-derived exosomes are proposed as bioactive compounds of breast milk, which have been reported to resist gastric digestion and reach systemic circulation, thus being bioavailable after oral intake. In the present manuscript, we critically discuss the available evidence on the health benefits attributed to milk exosomes, and we provide an outlook for the potential future uses of these compounds. The use of milk exosomes as bioactive ingredients represents a novel avenue to explore in the context of human nutrition, and they might exert important beneficial effects at multiple levels, including but not limited to intestinal health, bone and muscle metabolism, immunity, modulation of the microbiota, growth, and development