Exploring silk based biomaterials functionalized with antimicrobial peptides to prevent surgical site infections

Surgical site infections (SSI) often occur after invasive surgery, which is as a serious health problem, making it important to develop new biomaterials to prevent infections. Spider silk is a natural biomaterial with excellent biocompatibility, low immunogenicity and controllable biodegradability. Through recombinant DNA technology, spider silk-based materials can be bioengineered and functionalized with antimicrobial (AM) peptides 1. The aim of this study is to develop new materials by combining spider silk chimeric proteins with AM properties and silk fibroin extracted from Bombyx mori cocoons to prevent microbial infection. Here, spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6 mer and 15 mer) were fused with the AM peptides Hepcidin and Human Neutrophil peptide 1 (HNP1). The spider silk domain maintained its self-assembly features allowing the formation of beta-sheets to lock in structures without any chemical cross-linking. The AM properties of the developed chimeric proteins showed that 6 mer + HNP1 protein had a broad microbicidal activity against pathogens. The 6 mer + HNP-1 protein was then assembled with different percentages of silk fibroin into multifunctional films. In vitro cell studies with a human fibroblasts cell line (MRC5) showed nontoxic and cytocompatible behavior of the films. The positive cellular response, together with structural properties, suggests that this new fusion protein plus silk fibroin may be good candidates as multifunctional materials to prevent SSI.

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering

Effects of residues on the degradation of PHA produced from mixed microbial cultures and processed in extrusion

Thermal degradation upon melting is one of the major drawbacks reported for polyhydroxyalkanoates (PHA). However, the role of residues originating from the fermentation and the extraction steps on the thermal stability of this class of biopolymers still needs to be clarified. In the particular case of PHA produced from mixed microbial cultures (MMC), this topic is even less documented in the literature. Here, two polyhydroxy(butyrate-co-valerate) (PHBV) produced from MMC enriched in PHA accumulating organisms and fed with cheese whey were studied. A micro extrusion line is used to produce filaments and assess the processability and the degradation of processed PHBV. The prototype micro extrusion line allows for studying grams of materials. The two PHBV contain 18 mol% HV. PHBV was recovered with 11 wt% residues, and further submitted to a purification procedure resulting in a second biopolyester containing less than 2 wt% impurities. The thermorheological characterization of the two PHBV is first presented, together with their semicrystalline properties. Then the processing windows of the two biopolyesters are presented. Finally, the properties of extruded filaments are reported and the thermomechanical degradation of PHBV is extensively studied. The structure was assessed by wide angle X-ray diffraction, mechanical and rheological properties are reported, thermal properties are studied with differential scanning calorimetry and thermogravimetric analysis, whereas Fourier Transform Infrared spectroscopy was used to assess the impact of the extrusion on PHBV chemical structure. All results obtained with the two PHBV are compared to assess the effects of residues on both PHBV processability and degradation.

Biofunctionalization of titanium surfaces for dental implants: osteogenic, anti-microbial and tribocorrosion resistant surfaces

PhD thesis in Biomedical Engineering

Proteins on microbial identification: past achievements, present state-of-the-art, and future challenges

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

Advances in microbial diagnosis: using mass spectrometry for proteomics and genomics applications

Since the last two decades mass spectrometry (MS) has been applied to analyse the chemical cellular components of microorganisms, providing rapid and discriminatory proteomic profiles for their species identification and, in some cases, subtyping. The application of MS for the microbial diagnosis is currently well-established. The remarkable reproducibility and objectivity of this method is based on the measurement of constantly expressed and highly abundant proteins, mainly important conservative ribosomal proteins, which are used as markers to generate a cellular fingerprint. Mass spectrometry based on matrix-assisted laser desorption ionization-time of flight (MALDI- TOF) technique has been an important tool for the microbial diagnostic. However, some technical limitation concerning both MALDI-TOF and its used protocols for sample preparation have fostered the research of new mass spectrometry systems (e.g. LC MS/MS). LC MS/MS is able to generate online mass spectra of specific ions with further online sequencing of these ions, which include both specific proteins and DNA fragments. In this work a set of data for yeasts and filamentous fungi diagnostic obtained through an international collaboration project involving partners from Argentina, Brazil, Chile and Portugal will be presented and discussed.

Tuberculosis and HIV co-infection

Tuberculosis (TB) and human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) constitute the main burden of infectious disease in resource-limited countries. In the individual host, the two pathogens, Mycobacterium tuberculosis and HIV, potentiate one another, accelerating the deterioration of immunological functions. In high-burden settings, HIV coinfection is the most important risk factor for developing active TB, which increases the susceptibility to primary infection or reinfection and also the risk of TB reactivation for patients with latent TB. M. tuberculosis infection also has a negative impact on the immune response to HIV, accelerating the progression from HIV infection to AIDS. The clinical management of HIV-associated TB includes the integration of effective anti-TB treatment, use of concurrent antiretroviral therapy (ART), prevention of HIV-related comorbidities, management of drug cytotoxicity, and prevention/treatment of immune reconstitution inflammatory syndrome (IRIS).

To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.

Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"

The relevance of bacterial interactions on oral health

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

HPV DNA testing with cytology triage in cervical cancer screening: Influence of revealing HPV infection status

BACKGROUND: Knowledge of cervical human papillomavirus (HPV) status might influence a cytotechnician's assessment of cellular abnormalities. The authors compared original cytotechnicians' Papanicolaou (Pap) readings for which HPV status was concealed with Pap rereads for which HPV status was revealed separately for 3 screening populations. METHODS: Previously collected cervical Pap smears and clinical data were obtained from the Canadian Cervical Cancer Screening Trial (study A), the Democratic Republic of Congo Community-Based Screening Study (study B), and the Brazilian Investigation into Nutrition and Cervical Cancer Prevention (study C). Smears were reread with knowledge of HPV status for all HPV-positive women as well as a sample of HPV-negative women. Diagnostic performance of Pap cytology was compared between original readings and rereads. RESULTS: A total of 1767 Pap tests were reread. Among 915 rereads for HPV-positive women, the contrast between "revealed" and "concealed" Pap readings demonstrated revisions from negative to positive results for 109 women (cutoff was atypical squamous cells of undetermined significance or worse) and 124 women (cutoff was low-grade squamous intraepithelial lesions [LSIL] or worse). For a disease threshold of cervical intraepithelial neoplasia of grade 2 or worse, specificity significantly declined at the atypical squamous cells of undetermined significance cutoff for studies A (86.6% to 75.3%) and C (42.5% to 15.5%), and at the LSIL cutoff for study C (61.9% to 37.6%). Sensitivity remained nearly unchanged between readings, except in study C, in which reread performance was superior (91.3% vs 71.9% for the LSIL cutoff). CONCLUSIONS: A reduction in the diagnostic accuracy of Pap cytology was observed when revealing patients' cervical HPV status, possibly due to a heightened awareness of potential abnormalities, which led to more false-positive results. Cancer (Cancer Cytopathol) 2015. (c) 2015 American Cancer Society.

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...)

Biotechnological valorization of waste cooking oils: lipase and microbial lipids production by Yarrowia lipolytica

[Excerpt] Waste cooking oils (WCO) generated from vegetable oils used at high temperatures in food frying, cause environmental problems and must be reutilized. New strategies to valorize these wastes are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. A microbial platform can be established to convert low-value hydrophobic substrates, such as waste cooking oils, to microbial lipids (single cell oil, SCO) and other value-added bioproducts, such as lipase. (...)