Obtention and evaluation of proteases from the nematophagous fungus Monacrosporium thaumasium from silver nanoparticles on egg masses of the mollusc Biomphalaria glabrata and their analysis by optical clamping

Fungos nematófagos têm sido utilizados amplamente para controle biológico, tanto em helmintos, quanto em hospedeiros intermediários. Dentre os agentes envolvidos na dispersão da doença esquistossomose, estão os caramujos aquáticos do gênero Biomphalaria, que são frequentemente acometidos pelo trematódeo Schistosoma mansoni e são responsáveis por grandes danos à saúde de animais e humanos, tornando-se um grande fator de importância médico-veterinária. Como alternativa para controlar esses helmintos, fungos predadores e ovicidas são considerados oponentes naturais e essenciais para tal controle. Este trabalho, portanto, tem por objetivo investigar o potencial fungo Monacrosporium thaumasium como agente de biossíntese de nanopartículas de prata (AgNPs) e interação de proteases à nível molecular e suas conformações na macromolécula de ácido desoxirribonucleico (DNA). Foram testadas as atividades do extrato bruto e da protease parcialmente purificada provenientes de um isolado de M. thaumasium (cepa NF34) associado com nitrato de prata (AgNO 3 ) sobre massas ovígeras de Biomphalaria glabrata, como modelo teste de embriotoxicidade. O experimento foi edificado em delineamento inteiramente casualizado em tratamentos contendo as AgNPs (proporções entre 0,5 mL e 1:1 mL + massas ovígeras expostas) e o grupo controle com água declorada. Ambos os grupos foram mantidos nas condições na temperatura de 25 ºC durante dez dias. Sobre a molécula de DNA, foi feita a purificação parcial do extrato bruto do fungo e aplicado à técnica de pinça óptica (PO), a fim de se de estudar sobre as possíveis interações dessas proteases à esta molécula. Obteve-se das caracterizações enzimáticas, maior atividade proteolítica até 60 ºC em pH 9 durante 25 minutos. As serino-proteases parcialmente purificadas foram verificadas em sistema de gel SDS-PAGE tricina na massa molecular de aproximadamente 40 kDa e as proteases se ligaram fortemente ao ácido desoxirribonucleico, formando o complexo serino protease - DNA. Como conclusão, essas proteases, apresentaram uma constante de associação de ligação de equilíbrio ~10 8 M -1 e um comportamento cooperativo positivo na forma de agregados ligados, com tendência a neutralizar cerca de 100% da carga negativa presente no esqueleto fosfato da dupla hélice, resultando em complexos DNA-proteínas neutras. Os resultados obtidos evidenciam que M. thaumasium é um organismo satisfatório na produção de biossíntese de nanopartículas e ocasionou nas massas ovígeras expostas à inibição de 100% dos caramujos. Logo, as proteases foram importantes nesse tipo de caracterização para elucidar os aspectos moleculares do mecanismo de ação dessa proteína, que pode trazer pistas sobre seu papel como agentes antipatógenos e, assim, contribuir para aprimorar o conhecimento no uso de tais macromoléculas biológicas e possíveis estudos futuros sobre aplicabilidades em programas de controle biológico. Palavras-chave: Biotecnologia. Controle biológico. Gastrópodes. Nanomateriais. Trematodíases. Pinça óptica.Nematophagous fungi have been widely used for biological control, both in helminths and in intermediate hosts. Among the agents involved in the spread of the schistosomiasis disease, there are the aquatic snails of the genus Biomphalaria, which are often affected by the trematode Schistosoma mansoni and are responsible for great damage to the health of field animals and humans, making it a major factor of medical-veterinary importance consider alternative and ecologically sustainable measures. As an alternative to control these helminths, predatory and ovicidal fungi are considered natural and essential opponents for such control. This work, therefore, aims to investigate the potential fungus Monacrosporium thaumasium as an agent of biosynthesis of silver nanoparticles (AgNPs) and interaction of proteases at the molecular level and their conformations in the deoxyribonucleic acid (DNA) macromolecule. The activities of the crude extract and the partially purified protease from an isolate of M. thaumasium (strain NF34) associated with silver nitrate (AgNO3) were tested on ovigerous masses of Biomphalaria glabrata, as a test model for embryotoxicity. The experiment was built in a completely randomized design with treatments containing AgNPs (proportions between 0.5 mL and 1:1 mL + exposed ovigerous masses) and the control group with dechlorinated water. Both groups were kept under the same conditions at 25°C for ten days. On the DNA molecule, a partial purification of the crude extract of the fungus was performed and applied to the optical tweezers (OT) technique, in order to study the possible interactions of these proteases with this molecule. It was obtained from the enzymatic characterizations, higher proteolytic activity up to 60º C at pH 9 for 25 minutes. The partially purified serine proteases were verified in a tricine SDS-PAGE gel system at a molecular mass of approximately 40 kDa and the proteases strongly bind to deoxyribonucleic acid, forming the serine protease - DNA complex. In conclusion, these proteases showed an equilibrium binding association constant ~10 8 M -1 and a positive cooperative behavior in the form of bound aggregates, with a tendency to neutralize about 100% of the negative charge present in the phosphate backbone of the double helix, resulting in neutral protein-DNA complexes. The results obtained show that M. thaumasium is a satisfactory organism in the production of nanoparticle biosynthesis and caused the ovigerous masses exposed to 100% inhibition of the snails. Therefore, proteases were important in this type of characterization to elucidate the molecular aspects of the mechanism of action of this protein, which may bring clues about its role as antipathogenic agents and, thus, contribute to improve knowledge in the use of such biological macromolecules and possible studies futures on applicability in biological control programs. Keywords: Biotechnology. Biological control. Gastropods. Nanomaterials. Trematodiasis. Optical tweezers

Prognostic Factors and Markers in Non-Small Cell Lung Cancer: Recent Progress and Future Challenges

Lung cancer is a highly aggressive neoplasm and, despite the development of recent therapies, tumor progression and recurrence following the initial response remains unsolved. Several questions remain unanswered about non-small cell lung cancer (NSCLC): (1) Which patients will actually benefit from therapy? (2) What are the predictive factors of response to MAbs and TKIs? (3) What are the best combination strategies with conventional treatments or new antineoplastic drugs? To answer these questions, an integrative literature review was carried out, searching articles in PUBMED, NCBI-PMC, Google Academic, and others. Here, we will examine the molecular genetics of lung cancer, emphasizing NSCLC, and delineate the primary categories of inhibitors based on their molecular targets, alongside the main treatment alternatives depending on the type of acquired resistance. We highlighted new therapies based on epigenetic information and a single-cell approach as a potential source of new biomarkers. The current and future of NSCLC management hinges upon genotyping correct prognostic markers, as well as on the evolution of precision medicine, which guarantees a tailored drug combination with precise targeting