Photodynamic therapy (PDT) is based on the production of oxidative species by a photosensitizer, which is a molecule capable of converting specific light energy into chemical potential. The oxidative stress generated by this therapy can induce immunogenic cell death (ICD) and trigger an immune response. This type of death generates different molecular patterns associated with damage (DAMPs) for the immune system, which can result in the activation of dendritic cells, specialized in antigen presentation. Therefore, PDT emerges as an alternative for cancer treatment, as it plays an important role in activating the immune system and consists of a noninvasive local treatment. However, the use of this therapy still faces some limitations, such as low long-term photosensitivity and low bioavailability of photosensitizers. To promote greater therapeutic efficacy and immune system activation, the current working group has developed a new third-generation photosensitizer system composed of aluminum chloro phthalocyanine associated with solid lipid nanoparticles based on Amazonian butter (ALPcSLN). Experiments were conducted to evaluate its therapeutic potential, aiming to verify if the established protocol can induce immunogenic cell death and dendritic cell activation. For in vitro assays, the B16-F10 melanoma cell line and precursor bone marrow dendritic cells from C57BL/6 mice were used. For treatment, melanoma cells were exposed to the nanocarrier for 15 minutes, then kept in the dark or irradiated for 10 minutes with LED light (660 nm at 25.88 J/cm2 energy density). Mitoxantrone was used as a positive control for immunogenic cell death. Cell viability assays showed that the nanocarrier performed well in its cytotoxic function when excited by light, leading to the production of reactive oxygen species. Immunostaining for confocal microscopy and transmission electron microscopy demonstrated that the treatment induced the production of DAMPs. After treatment of B16-F10 cells, the production of autophagosomes and cytokines such as IL-12 was observed, playing a significant role in the immunological response. Evaluation of dendritic cells by microscopy showed morphological changes after co-culture with treated tumor cells. Additionally, dendritic cells exhibited increased expression of membrane components such as MHCII and CD86, as well as production of cytokines such as IL-12 and IFN-γ, which are important factors for immune system activation. In conclusion, the results indicate that the established protocol with the nanostructure has the potential to improve PDT efficacy by promoting immunogenic cell d

At the end of 2019, a new coronavirus capable of infecting humans was identified: SARS-CoV-2, which is the etiological agent of COVID-19. SARS-CoV-2 quickly spread throughout the world, causing a global pandemic, with disastrous social and economic consequences. SARS-CoV-2 uses Angiotensin Converting Enzyme 2 (ACE2) as a cellular receptor. The SARS-CoV-2 Protein S, formed by a trimer where each monomer is around 180 kDa, is responsible for mediating the connection with ACE2 and thus the entry of the virus into the cell. Protein S is composed of two subunits, called S1 and S2. The S1 subunit contains the RBD (Receptor Binding Domain), which is the part of the glycoprotein that interacts directly with the cellular receptor. In the S2 subunit there is the S2' cleavage site and immediately after this site is the Fusion Peptide (FP) region, which together with the S2' site assists in the entry of the virus into the host cell. During the pandemic, therapeutic monoclonal antibodies were developed that targeted the S protein of SARS-CoV-2, especially RDB, with the aim of blocking the entry of the virus into the host cell by blocking the interaction between the S protein and the cellular receptor. However, variants of SARS-CoV-2 emerged and accumulated mutations in Protein S and mainly in the RBD, which reduced the effectiveness of antibodies against this region. In this work, we report the development of two antibody fragments from a combinatorial phage library, reactive with the FP of SARS-CoV-2, which, unlike the RBD, remained extremely conserved even among the SARS-CoV-2 variants. We demonstrated the ability of these antibody fragments, in scFv format, to bind to Protein S of the Gamma and Delta variants and also to the original Wuhan virus. These new antibodies are now being tested as therapeutic alternatives for treating SARS-CoV-2 infections.

: Alzheimer's Disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia in the elderly population worldwide. Considering the high prevalence of the disease and its socioeconomic costs to society as a whole, AD is considered a significant public health problem. Its causes are still uncertain, but many authors report the amyloid cascade hypothesis and the phosphorylated tau protein hypothesis as its main culprits, leading to neurodegeneration, neuronal death, and dementia. Currently, AD has few treatments, mostly symptomatic, that do not prevent or slow down the progression of the disease, making new treatments urgently needed. In this scenario, compounds from animal venoms have been observed as an innovative platform for discovering new treatments due to their high specificity for Central Nervous System targets. The peptides occidentalina and neurovespina, isolated from the wasp Polybia Occidentalis, have demonstrated neuroprotective action in neurodegenerative disease models, capable of crossing the blood-brain barrier and inhibiting calcium currents from Cav1.2-type channels. Furthermore, preliminary data support that neurovespina may inhibit the glutamatergic pathway, which is pathologically altered in AD. From the neurovespina peptide, the modified peptide octovespina was developed, showing in vivo and in vitro neuroprotective activity in a mouse model of Alzheimer's disease. It can prevent β-amyloid aggregation and reverse memory deficits caused by β-amyloid. Thus, octovespina may be a promising compound for use as a model drug in the development of new treatments for neurodegenerative diseases. To facilitate the delivery of octovespina to the Central Nervous System (CNS), an analogue named neurocidentallis was developed by removing an aromatic ring (phenylalanine) to reduce hydrophobicity for better solubilization in aqueous media. However, one of the challenges in using peptides in the clinical context is delivering them to the CNS via parenteral administration, overcoming obstacles such as peptide removal by endogenous proteases and unfavorable physicochemical properties of the drugs. To address these challenges and improve the delivery of neurocidentallis to the CNS, it was encapsulated in nanoemulsions, acting as drug delivery systems capable of carrying and delivering the compound to brain tissue. The nanoparticles used are conjugates of neurocidentallis peptide + chitosan or PEG nanoemulsions, both of which have been extensively studied and demonstrate good safety profiles, both in vivo and in vitro. This project aims to deepen studies on the bioinspired neurocidentallis peptide in Alzheimer's Disease through behavioral assays such as the Morris Water Maze, Novel Object Recognition Test, and Open Field, as well as immunohistochemical analyses, to demonstrate its potential as a tool in the study and treatment of the disease.

Heart failure (HF) secondary to non-ischemic dilated cardiomyopathy (NIDCM) leads the indications for heart transplantation (HT) in Brazil, however its study mainly focuses on idiopathic dilated cardiomyopathy (IDC). Chronic Chagas cardiomyopathy (CCC), highly prevalent in Latin America, presents greater severity and worse outcome compared to other cardiomyopathies. Since the molecular events of HF that drive the distinct clinical course of patients with NIMDC are still poorly understood, the present study characterizes and discriminates the protein and metabololite profiles in plasma of 15 patients with advanced HF that underwent HT – 8 patients with CCC and 7 with IDC – by mass spectrometry. Compared to 12 heart donors, included to experimentally reproduce physiological conditions (CTRL group), patients with advanced HF exhibited a greater abundance of proteins associated with antioxidant defense, with emphasis on hydrogen peroxide catabolism and nitric oxide transport, and a global metabolic imbalance indicative mainly of an accumulation of fatty acids, amino acids and components of the Krebs cycle. Among the etiologies, 6 proteins were discriminating between patients with CCC and IDC, in addition to the positive regulation of pro-inflammatory immune response and reverse cholesterol transport effector proteins in chagasic NIMDC. CCC vs. CDI analyses also revealed a metabolic disparity between pathological conditions, with 12 metabolites most representative of CCC and 11 of CDI. Disturbances were mainly related to amino acid metabolism profile. Although mitochondrial dysfunction may be a central mechanistic event in predisposing to HF, the differential protein regulation and metabolic impairment associated differ between CCC and IDC populations, corroborating clinical observations regarding the prognosis of patients with Chagas disease.

COVID-19 (coronavirus disease 2019), is a disease whose severe form is characterized by Severe Acute Respiratory Syndrome, is caused by the new betacoronavirus SARSCoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). first case in the city of Wuhan, Hubei province, China, in December 2019, SARS-CoV-2 has spread rapidly in the world. On January 30, 2020, the World Health Organization (WHO) classified the disease as an international health emergency and on March 11th it was declared a pandemic, with approximately 118,000 cases in 114 countries and territories (WHO, 2020). The current COVID-19 pandemic has required the development of various biotechnological products and services for detection, treatment and prevention of infections and diseases caused by this virus. In addition to molecular tools for detection of this virus, such as quantitative polymerase chain reaction with reverse transcriptase (RT-qPCR), technical serological tests were developed and have been widely used to assess the seroconversion of infected and immunized patients. In this work, we used the expression of the SARS-CoV-2 nucleocapsid (N) protein in insect cells, followed by its purification by affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used to infect Lepidoptera cells (Sf9) adapted to the high density suspension. Using Tris-HCl buffer pH 8.0 as the mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg of N protein can be purified from each 50 mg of total protein from the extract of soluble proteins from infected insect cells. The antigen was tested in samples obtained from patients confirmed for COVID-19 by RTqPCR at Hospital Regional de Santa Maria –DF and applied in an indirect ELISA model – in house. The results showed satisfactory rates for the use of the antigen in the detection of IgG in infected patients in different periods of the disease. In-house indirect ELISA can be used as a tool for quantitative IgG detection. In this work, the analyzes obtained showed that the N protein has immunogenic potential, and that the ELISA prototype - in-house, can be a tool used in the diagnosis of COVID-19, in view of the data obtained in the statistical analysis, where they presented a sensitivity of 94.4%, specificity of 92.1% and accuracy of 92.5% in the samples with discount of background reactions (nonspecific reactions), using the ROC curve and with a confidence interval of 95% (CI95% ) in patients who presented symptoms less than seven days after infection. Samples with an interval of >7 to 14 days reached 100% sensitivity and 94.4% specificity using the ROC curve. Another observed factor indicates that analyzes without discounting background reactions (nonspecific reactions) decrease the specificity sensitivity in both analyzes (NBS and NBF)

Horizontal gene transfer (HGT) plays an important role in the evolutionary plasticity of organisms to new environments and conditions, and may be favored by several ecological relationships, such as parasitism. Convincing examples of HGT have been demonstrated in different parasites and hosts, as is the case with Trypanosoma cruzi. Several methodologies are used to infer the presence of HGT among unrelated organisms, each with its own set of characteristics and limitations. Recently, fluorescent labeling techniques have been used to verify the integration and localization of HGT candidate genes in the host genome. In this regard, several studies have already used CRISPR/dCas9 (deactivated Cas9) system, linked to GFP, to investigate chromosomal organization and visualize chromosomal dynamics and genomic loci. Although no study has yet used the technique to detect HGT events, the system shows promise for this, since it makes it possible to evaluate the spatial-temporal organization of these sequences in the genome. In this work, we demonstrate lateral gene transfer in the host-parasite relationship by CRISPR/dCas9 methodology, using T. cruzi-infected cells as a model. Initially, the technique was validated with several controls, which showed specific label and absence of off-targets. Then, HEK293 cells infected by the parasite were transfected with guide RNA (gRNA) for T. cruzi kDNA minicircle after different periods of infection. kDNA sequences were tagged in all analyzed periods. Also, aiming to demonstrate that kDNA transfer is independent of the presence of the parasite, the cells were treated with benznidazole and analyzed 30 days after the onset of infection. In another experimental group, the cells were placed in co-culture with the parasite using transwell systems. In both situations, the CRISPR/dCas9 system demonstrated the presence of a T. cruzi kDNA minicircle in the cell genome. Analysis of the samples by real-time PCR (qPCR) confirmed the absence of parasite nuclear DNA and the presence of kDNA. The results show that the CRISPR/dCas9 system was able to identify the DNA sequence from a HGT event in the study model. The findings support the use of the technique as a complementary methodology in the identification and study of horizontally transferred sequences between different organisms

Chagas disease (CD) is a parasitic disease caused by the protozoan Trypanosoma cruzi. Considered a neglected tropical disease, CD affects millions of people in the 21 Latin American countries where it is endemic and in the countries receiving immigrants from that region. Due to the scarcity of drugs available for its treatment, there is great interest in knowing the homeostatic components of the trypanosomatid Trypanosoma cruzi, in order to seek possible pharmacological targets. In this sense, considering the already known importance of kinesins in several diseases due to their role in the regulation of cytokinesis and maintenance of cell morphology, we sought in this work to characterize the motor protein kinesin C (TcKIN-C) of T. cruzi and its importance in homeostasis cell phone. Initially, an in silico survey of the characteristics of TcKIN-C, an orphan kinesin present only in T. cruzi, expressed by the TcCLB.504427.260 and TcCLB.508043.40 alleles, was carried out. Additionally, using the premise of metagenomics, the CRISPR/Cas9 biotechnological tool was used in order to cause an interruption in TcKIN-C expression, resulting in truncated expression of this protein and possible loss of its functionality. Through the in silico analysis, a significant difference was observed in the sequence of the two alleles, so an analysis of both alleles was performed using bioinformatics tools, from alignments, prediction of post-translational modifications (MPTs), interaction networks to the 3D structure of both alleles. Then, a knockout was performed using the aforementioned technique, where two strategies were outlined: an initial knockout (called Strategy I) without selection to observe the effects at the population level and a second knockout (called Strategy II), where the transgenic population was selected and the effects of TcKIN-C deletion were studied in more detail. As of the date of this publication, no other work has been published that makes the functional characterization of a kinesin in Trypanosoma cruzi. In short, as observed in Trypanosoma brucei, this work corroborates the notion that kinesins are of central importance for the survival of these parasites. Furthermore, it was also possible to achieve the objective of selecting transgenic populations, allowing further studies.

Malaria is a disease whose etiological agents are protozoa of the genus Plasmodium, and P. falciparum is the species responsible for the most severe clinical cases. In 2020, 241 million cases of malaria were registered, and the African Region, where P. falciparum is dominant, was responsible for about 95% of the total cases registered. Therefore, studying the parasite becomes necessary, mainly due to the growing records of resistance to current antimalarials. To better understand one of the essential organelles for the survival of the parasite, the present work aimed to use APEX2 to label P. falciparum mitochondrial proteins through biotinylation. This methodology, through the use of substrate (biotin-phenol) and oxidizing agent (hydrogen peroxide), allows the biotinylation of proteins from cellular compartments of interest to occur, allowing a detailed and reliable analysis of the proteomic content, through the enrichment of the biotinylated proteins. After obtaining parasites transfected with the episomal expression plasmid containing the coding sequence for APEX2 fused to a putative mitochondrial targeting signal, by Western-blot and streptavidin-blot, it was observed that both HAAPEX2 expression and biotinylation labeling occurred. However, cytolocalization of APEX2 by immunofluorescence occurred diffusely throughout the parasite's cell body. Therefore, HA-APEX2 was not specifically targeted in P. falciparum mitochondrion. In addition to mitochondrion, P. falciparum has another plastid organelle with plant-like characteristics, called apicoplast. For some reason still unknown, both organelles have a biochemical interrelationship in the biosynthesis of the heme group. For this reason, in silico prediction and data mining strategies for mitochondrial and apicoplast proteins were employed to compose the predicted subproteomes of both organelles. In total, were predicted 708 mitochondrial proteins and 781 apicoplast proteins of P. falciparum. After comparing the datasets, an overlap of 108 proteins was observed. Despite being a mere bioinformatics analysis, this fact reinforces the widely known evidence about the biology of the mitochondrion and apicoplast of P. falciparum. Due to the nonspecific labeling of mitochondrion by HA-APEX2, a new sequence is in the process of being cloned for further transfection. The main purpose of making predictions for both organelles is based on the already exposed characteristics of both organelles. Thus, a detailed overview of the biochemical characteristics of both organelles has already been established, to assist in future proteomic analyses.

Infection with the new SARS-CoV-2 Coronavirus, responsible for the Severe Acute Respiratory Syndrome known as Covid-19, resulted in a major pandemic as of March 2020, with more than 670 million confirmed cases and about 6.8 million of deaths. The disease is associated with respiratory complications, but there are also studies that have already proven lesions in different organs, even showing that some patients have sequelae even years later. Among the treatment strategies the development of new monoclonal antibodies stands out for its high affinity, greater specificity, pharmacological safety, and can also be used in more efficient diagnostic kits. In this sense, the objective of this work was the development of specific antibodies that bind to SARS-CoV-2 with the potential to block the infection of cells by the virus .For this, we developed antibody fragments called ScFv (single chain variable fragment) using a library of developed from the plasma of convalescents,. We selected in 4 rounds, using two different types of antigens, the host receptor recognition subunit (S1), and the domain of this protein that is responsible for receptor binding (RBD). The strategy was to separately immobilize the antigens on ELISA plates and add the phage library. The analysis of the selection progress revealed that there was enrichment of antibodies specific to the viral antigen, and 13 of them were produced at high levels in a heterologous system of bacterial cells , and purified by affinity chromatography on a Nickel column. It was shown that the recombinant antibodies selected and produced showed the ability to bind to the viral antigen. It was also shown that the antibodies were able to neutralize the infection by Sars-Cov-2 virus vitro. These data show the neutralizing potential of the human antibodies developed in this research and reveal their relevance in the development of efficient and safe methodologies for the treatment of Sars-Cov-2 virus infections.

The SARS-CoV-2 virus is the pathological agent of the COVID-19 pandemic, responsible for 635 million cases and 6 million deaths by November 2022. As the pandemic progresses, research into antiviral drugs has begun, with a focus on viral proteases due to their essential role in replication. Among the targets of SARS-CoV-2, the Main protease - Mpro, is a 33 kDa cysteine protease, responsible for cleaving 16 sites of the polyprotein and common in several species of coronavirus, as well as having no similarity to any human proteins. Another target, papain-like protease - PLpro, is a 36 kDa cysteine protease responsible for cleaving 3 viral proteins and 2 regulators of the host's immunological action.  The aim of this study was to provide new data on the expression and in vitro activity of two important proteases from the Coronaviridae family, as well as to assess their inhibition by natural compounds. With regard to Mpro, different forms of induction described in the literature were tested and good results were achieved with inductions lasting 5 and 18 hours at 18°C ± 2, with subsequent purification on a Ni+NTA column, confirmed by the Western blot technique. With regard to PLpro, the conditions described in the literature were tested, converging on the best condition at 18°C for 18 hours with 0.1 mM IPTG and 1 mM ZnCl2.The enzyme activity tests indicated that storage at -80°C was the most effective, regardless of the presence of glycerol. It was also observed that among the buffers evaluated, both the HEPES 10 mM pH 7.5 and DTT 5 µM buffer and the Tris-HCl 50 mM pH 7.6 and DTT 5 µM buffer provided the best enzymatic activity. The impact of adding different compounds was assessed, indicating that the use of DTT in the activity buffer showed an increase of around 68% in activity at a concentration of 10 µM. On the other hand, the addition of zinc chloride reduced enzyme activity by up to 95 per cent. The addition of sodium chloride also reduced activity by 85% at the concentration used in the purification buffers, as did imidazole, which reduced activity by up to 94% at a concentration of 500 µM. EDTA, on the other hand, was unable to alter enzyme activity. Among the 24 natural compounds tested, the essential oils of Litsea cubeba, Cananga odorata, Salvia sclarea and Citrus limon showed good inhibition results, reaching IC50s of between 53 and 76 µg/mL. Based on the literature, this study analysed the different induction patterns for two important COVID-19 targets, converging on a better expression pattern, as well as analysing different additives and buffers for enzyme activity tests, proposing an efficient composition for future analyses targeting coronavirus PLpro. Finally, at least three essential oils with good inhibitory activity were recommended for further pharmacological studies.

019, the SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2) was described as responsible for COVID-19. This disease presented with a heterogeneous scenario, with the most severe cases being characterized by hyperactivation of the immune system, causing a cytokine storm and damage to multiple organs. Due to their immunoregulatory and regenerative functions, mesenchymal stem cells (MSCs) began to be investigated as a therapeutic alternative for the treatment of severe cases of COVID-19. In this work, we initially sought to potentiate the immunosuppressive effect of MSCs derived from adipose tissue. Subsequently, we tested the ability of these cells to control the immune response of an in vitro model of lung cell inflammation generated by SARS-CoV-2 antigens. Initially, we tested different inflammatory and epigenetic modulation strategies to potentiate the immunosuppressive effect of MSCs. At this stage, we identified that licensing with INF-γ was able to enhance the anti-inflammatory profile of MSCs. Subsequently, we tested Calu-3 and A549 cells, for classic entry virus receptors, to establish a model of lung inflammation induced by SARS-CoV-2 antigens. Calu-3 cells showed higher expression of ACE2 and TMPRSS2 receptors and, when at an inflammatory environment and challenged with the Nucleocapsid/Spike (NS) antigen, showed high levels of inflammatory factors and entered a process of cell apoptosis. Finally, to understand the influence of virus-induced lung injury on the immune response, we submitted PBMCs to the supernatant of Calu-3 cells stimulated with NS antigen and IFN-γ. In parallel, with a therapeutic purpose, we added MSCs licensed with INF-γ to this model. We noticed that the MSCs controlled the inflammatory process, inhibiting the generation of memory T cells and reducing the levels of IL-6 and IL-10 cytokines. Furthermore, MSCs licensed with INF-γ were able to maintain T cell viability, indicating that the use of MSCs also promotes a regenerative role in the inflammatory process generated in the evaluated model. These results are relevant to the field of cell therapy, contributing to a better mechanistic understanding of MSCs by controlling the inflammatory response induced by SARS-CoV-2 components.

The increase in systemic fungal infections associated with the emerging need for new therapeutic alternatives imposes the need for knowledge of the control mechanisms of gene expression related to the regulation of the immune response. Literature data demonstrate that fungal infections represent a relevant cause of morbidity and mortality, especially regarding the growing universe of immunocompromised people. Understanding host immune system protective responses to fungal infections can help predict disease progression and directly influence patient treatment and prognosis. There is a great diversity of fungal species that affect humans and cause these infections. Fungi such as Paracoccidioides brasiliensis and Cryptococcus neoformans represent important pathogens, which are etiological agents of paracoccidioidomycosis and cryptococcosis, respectively. The main immune cells first interacting with pathogens are the macrophages and dendritic cells, which recognize and phagocytose the fungal cells, orchestrating the innate and adaptive immune response that contributes to infection eradication. The host's immune response to infections by microbial pathogens involves extensive gene reprogramming, which is finely controlled to effectively contain or eliminate the pathogen without causing excessive damage to the host. This study aimed to characterize the transcriptomic profile of immune response cells in response to infection models by P. brasiliensis and C. neoformans, to better understand the molecular bases of the host-pathogen interaction. The objective of the first work was on the analysis of the transcriptome of bone marrow-derived dendritic cells (BMDCs) from resistant (A/J) and susceptible (B10.A) mice in response to infection by P. brasiliensis. The second work was directed to the transcriptomic analysis of mouse bone marrow-derived macrophages (BMDMs) in response to capsular (B3501) and acapsular (Cap67) strains of C. neoformans. In this regard, we employed RNA sequencing to provide a global landscape of host gene expression in response to the two infection models. The main results showed that BMDCs from the susceptible mouse showed a stronger response to P. brasiliensis infection, suggesting that an early exaggerated immune reaction to the fungus may be linked to host susceptibility. On the other hand, the results of the C. neoformans infection model showed the adequate innate and adaptative immune response is delayed in the macrophages infected by encapsulated strain, affecting antigen presentation and macrophage activation. These findings could potentially give advanced insights into understanding the cellular and molecular mechanism of immune response to fungal infections

Escherichia coli consist highly versatile bacterium capable of acquiring various specific virulence factors, resulting in pathotypes that can cause a wide range of infectious diseases. Consequently, there is a need to identify new antimicrobial drugs considering the development of bacterial resistance to a broad spectrum of antibiotics. Mastoparans are cationic peptides with multifunctional pharmacological properties. Mastoparans-R1 and R4 were computationally designed based on native mastoparan-L from wasps and have improved therapeutic potential for the control of bacterial infections. Here we evaluated whether these peptides maintain their activity against Escherichia coli strains under a range of salt concentrations. We found that mastoparans-R1 and R4 preserved their activity under the conditions tested, including having antibacterial activities at physiological salt concentrations. The overall structure of the peptides was investigated using circular dichroism spectroscopy in a range of solvents. No significant changes in secondary structure were observed (random coil in aqueous solutions and α-helix in hydrophobic and anionic environments). The three-dimensional structures of mastoparan-R1 and R4 were elucidated through nuclear magnetic resonance spectroscopy, revealing amphipathic α-helical segments for Leu3-Ile13 (mastoparan-R1) and Leu3-Ile14 (mastoparan-R4). Possible membrane-association mechanisms for mastoparan-R1 and R4 were investigated through surface plasmon resonance and leakage studies of carboxyfluoroscein with lipid bilayers and synthetic vesicles of POPC and POPC/POPG (4:1). Mastoparan-L had the highest affinity for both membrane systems, whereas the two analogs had weaker association, but improved selectivity for lysing anionic membranes. This finding was also supported by molecular dynamics simulations, in which mastoparan-R1 and R4 were found to have greater interactions with bacteria-like membranes compared to model mammalian membranes. Despite having a few differences in their functional and structural profiles, the mastoparan-R1 analog stood out for the highest activity, greater bacteriostatic and bactericidal potential, and selectivity for lysing anionic membranes. This study reinforces the potential of mastoparan-R1 as a drug candidate, as well as a model for the study and development of more selective antimicrobial peptides

Resumo em inglês: The human alphaherpesvirus 1 (HHV-1) is a sexually transmitted viral infection and presents a global health concern. Despite being a well-known virus with established treatment protocols, the infection remains incurable, and the emergence of viral resistance is a worrisome factor, especially for immunocompromised patients. For this reason, several studies have demonstrated the potential of antimicrobial peptides derived from arthropod venom, such as mastoparan peptides derived from Vespula lewisii venom. These peptides have exhibited antibacterial, antifungal, and antiviral activities. Using bioinformatics tools, two peptides were designed based on mastoparan L, namely [I5 , R8 ] mastoparan and mastoparan-MO. This study aimed to determine the antiviral potential of synthetic mastoparans derived from mastoparan L. Initially, a CD and NMR analysis revealed that [I5 , R8 ] mastoparan peptide adopts an αhelix structure in SDS25. Subsequently, in vitro assays demonstrated that the tested mastoparans exhibited low cytotoxicity against Vero cells and up to 90% inhibition of HHV-1 through plaque assays at a concentration of 50 µg.ml-1. Furthermore, [I5 , R8 ] mastoparan inhibits viral replication in a dose-dependent manner, possesses virucidal activity, and interferes with early infection stages, resulting in up to 99% inhibition. On the other hand, mastoparan-MO exhibited inhibition above 90% only at a concentration of 50 µg.ml-1 . Subsequent mechanism assays revealed that the peptide could inhibit the viral penetration step, achieving up to 99% inhibition. Additionally, experiments using fluorescent probes demonstrated that [I5 , R8 ] mastoparan does not significantly impact membrane fluidity in model membranes. However, these experiments were insufficient to determine whether this peptide interacts with membranes. Overall, the obtained results indicate that the tested mastoparans may be antiviral medications.

INTRODUCTION: The Zika virus is a flavivirus transmitted by mosquitoes that can cause congenital infection, microcephaly and Guillain-Barré syndrome in humans. The nuclear transcription factor NF-κB is a key regulator of inflammation, cell survival and the immune response, which can be activated by different canonical and non-canonical pathways. Some natural or synthetic compounds, such as atazanavir, curcumin and lopinavir, have demonstrated modulating effects on the NF-κB pathway, which may interfere with the progression of the immune response of peripheral blood mononuclear cells in pregnant women and neuroblastoma cells. Atazanavir and lopinavir are antivirals that inhibit HIV protease and curcumin is a pigment extracted from turmeric, which has anti-inflammatory properties, being able to inhibit NF-κB activation in various types of cells. These compounds may represent potential therapeutic agents for NFκB-related diseases and infections such as the Zika virus. OBJECTIVES: The aim of this study was to evaluate the effect of atazanavir, curcumin and lopinavir on the canonical (which controls the inflammatory response) and non-canonical pathways of NF-kB activation in neuroblastoma cells and peripheral blood mononuclear cells (PBMC) of pregnant and non-pregnant women incubated in vitro with the Zika virus. MATERIALS AND METHODS: This study investigated by flow cytometry the effects of in vitro incubation with the Zika virus and in vitro treatment with atazanavir, curcumin and lopinavir on the expression of TNF receptors (LTXR, TNFR1 and TNFR2) and molecules involved in the NFkB pathway in SKnBe neuroblastoma cells and peripheral blood mononuclear cells (PBMC) from normal control women and pregnant women. The production of inflammatory and immunological cytokines was quantified by flow cytometry. The following study groups were evaluated in neuroblastomas and PBMC: 1) Control (Cont), 2) incubated with the Zika virus (ZKV), 3) treated with atazanavir (Ataz), 4) treated with atazanavir and incubated with the Zika virus (Ataz+ZKV), 5) treated with curcumin (Curc), 6) treated with curcumin and incubated with the Zika virus (Curc+ZKV), 7) treated with lopinavir (Lopi) and 8) treated with lopinavir and incubated with the Zika virus (Lopi+ZKV). To evaluate cytokine kinetics, the following study groups were formed: 1) Zika, 2) atazanavir+Zika, 3) curcumin+Zika, and 4) lopinavir+Zika. The adsorption of the Zika virus was performed after 30 min of incubation of the plates containing the cells and the treatments started after 1 h of virus adsorption, remaining in treatment for 6 hours. Women in the pregnant or non-pregnant groups were normal with the body mass index (BMI) of the volunteers and the prenatal exams of the pregnant women within the normal range. It was evaluated by flow cytometry, the receptors for lymphotoxin (rLTXβ) and receptors for TNF (TNFR1 and TNFR2), the molecules FADD, TRAF, NIK, JNK, IkB, P50, P52, P65 relA, relB and c-Rel in the neuroblastoma cells and PBMC, and Immune cytokines: TNF, IFN-γ, IL-12p70, IL-2, IL-10, IL-4, IL-17A, and inflammatory ones: IL-8, IL-1, IL , IL10, TNF, IL-12p70 in supernatants of cultures of neuroblastomas and PBMC and the chemokines: CXCL8/IL-8, RANTES, CXCL9/MIG, CCL2/MCP-1, CXCL10/IP-10 in supernatants of cultures of CMSP. The results were analyzed by ANOVA or Kruskal-Wallis tests, ANOVA for repeated measures or Friedamn test, t test or Mann-Whitney test, according to indications and considered significant for p<0.05. RESULTS: By electron microscopy, it was observed the presence of the Zika virus particles in the cytoplasm of neuroblastomas. It was observed inhibition of Zika virus growth by atazanavir treatment within 6 to 24 hours after incubation. With curcumin treatment, there was a reduction in virus growth in the first 6 hours after incubation and with lopinavir there was a reduction in virus growth between the first periods after infection. Our results showed that neuroblastomas constitutively presented LTXβR, already showing higher expression of the receptor in the control group. In the groups incubated only with the Zika virus, and the atazanavir+zika, curcumin, curcumin+zika, lopinavir and lopinavir+zika groups, there was a higher expression of TNFR2. In PBMC, however, non-pregnant women showed higher expression of TNFR1 in all groups and pregnant women expressed LTXβR. In the evaluation of NFkB pathway molecules in neuroblastomas, the FADD molecule was more expressed than the TRAF molecule in all study groups, and this pattern was similar in the comparison between the expression of the NIK and JNK molecules, being the molecule with the highest expression of the final molecules of the pathway was c-Rel. In the evaluation of NFkB pathway molecules in PBMC both in pregnant and non-pregnant women, there were no significant differences within each group, and the expressed levels of each molecule were similar. In the evaluation of the expression of molecules linked to the NFkB pathways, in the control (baseline), neuroblastomas constitutively expressed LTXβR, FADD, NIK, IkB and c-Rel, with production of IL-8 and IL-6. In addition, in the PBMC of non-pregnant women, the highest expression receptor was TNFR1 and in pregnant women, LTXβR, as well as the production of IL-6, IL-8, IL-1β, TNF and the chemokines MCP1 and RANTES. In PBMC, there was no difference in the expression of other molecules in the pathway. In the incubation with the Zika virus, there was a higher expression of cRel in neuroblastomas and a lower expression of relB, with increased production of IL-8 and IL-6. In the PBMC there was a higher expression of relA in non-pregnant women, and a higher production of IL-8, TNF, MCP-1, and in the pregnant women there was a higher production of IL-8, IL-6, TNF and IL1β. In the treatment with atazanavir, in neuroblastomas, c-Rel continued to be the most expressed, but an increase in the expression of the P52 molecule was noted, with a reduction of IL-6, maintaining the levels of IL-8. In the PBMC, the nonpregnant women showed a higher expression of the P52 molecule, without variation in the expression of cytokines and in the pregnant women, there was a greater expression of the relA molecule, with an increase in the production of IL-8. In the atazanavir+zika group, for neuroblastomas, c-rel continued to be the most expressed molecule, however, P52 showed an increase and there was no difference in the expression of IL6 and IL-8, and in PBMC the most expressed molecule was relA for non-pregnant women, without alteration in the production of cytokines and also for pregnant women, with an increase in IL-8 and IL-1β. In the treatment with curcumin, c-Rel continued to be the molecule most expressed by neuroblastomas, but there was an increase in the P52 molecule and relA and a reduction in the production of IL-6 and IL-8, and in PBMC, both pregnant and non-pregnant women expressed relA and did not modify cytokine production. In the curcumin+Zika group, the neuroblastoma continued to express c-Rel, with an increase in relA and P52 and did not modify cytokine production, and PBMC showed greater expression of relA, both in nonpregnant women, who increased IL-8 and IL-1β and in pregnant women, who produced more IL-6, IL-8 and IL-1β. In the treatment with lopinavir, the neuroblastomas presented only the elevated c-Rel molecule, with a reduction of IL-6 and without alteration of IL-8. and the PBMC of the non-pregnant women, did not present difference in the expression of these molecules and the pregnant women continued to express relA higher than the others and in relation to cytokines, there was no change in production. In the lopinavir+Zika group, the neuroblastomas expressed c-Rel and higher expression of P52, without modifying the expression of cytokines and in PBMC, the non-pregnant women did not show differences in the expression of the molecules and in the production of cytokines and the pregnant women had the highest relA and increased IL-8. CONCLUSIONS Electron microscopy confirmed that the Zika virus infects neuroblastomas. Direct inhibition of virus growth has shown that atazanavir, curcumin and lopinavir can have a direct action in reducing the growth of Zika virus. Neuroblastomas showed a profile of cytokine responses to NfkB pathway molecules different from PBMC in pregnant and non-pregnant women, suggesting that the immunopathogenic mechanisms of the Zika virus were different. The NFkB activation profile was suggestive of the non-canonical pathway in neuroblastoma and in the PBMC of pregnant women, as they express LTXβR, but the molecules activated together with this receptor show a profile that tends towards the canonical pathway and incubation with the virus showed that neuroblastomas tend to respond by TNFR2, highlighting its canonical profile. Non-pregnant PBMC expressed TNFR1, with or without viral infection, which suggests a canonical response. The cytokines detected in this study, which also underwent variation, have a canonical activation profile. The studied compounds did not affect the type of response, but they acted on the responsive intensity of the cells. Studies on the immunopathogenesis of the Zika virus and that to verify the modulation by drugs of the intensity of responses of these cells to the viral infection and also to the gestational condition are necessary to define the use of these drugs for use in the treatment of Zika virus infection. The use of curcumin as a supplement during pregnancy and/or the use of atazanavir or lopinavir for treatment would aim to alleviate or protect the fetus from one of the worst consequences of Zika disease, which is microcephaly, but for this objective to be achieved, further studies of these compounds are required

Ulcerative colitis and Crohn&#39;s disease are part of the inflammatory bowel
diseases group, characterized by inflammation of the gastrointestinal tract (GIT). There is still
no cure for these diseases, and available treatments aim to reduce symptoms and contain the
spread of the disease. One of the most efficient clinical approaches is the use of TNF inhibitors,
such as the recombinant antibody Infliximab (Remicade®). However, immunotherapy with
monoclonal antibodies still presents a high cost and intrinsic toxicity, suggesting the need for
less toxic and more accessible alternatives. This work investigated the use of the gram-
negative bacterium Zymomonas mobilis as a potential vehicle for the expression of
recombinant anti-TNF and anti-CD3 antibodies in the GIT of mice to reduce the effects of DSS-
induced ulcerative colitis. For this, three Z. mobilis expression vectors were constructed
containing the genes that code for the variable regions of the infliximab mAb in single chain
format (scFv) and the murine anti-CD3 antibody 145-2c11 in the scFv and FvFc formats. With
the treatment, it was possible to observe that the administration of Z. mobilis containing or
not the plasmids of interest was effective in reducing inflammation, as indicated by stability in
weight loss, disease activity index and improvement in histopathological indices. In addition,
treatment with Z. mobilis restored the mucosal barrier in addition to increasing the expression

of the Muc3 and Ocln genes, potentiating a regulatory phenotype, inducing
immunomodulatory genes such as Tgfb, Il5, Il10 and Foxp3, and reducing the relative
expression of mRNA pro-inflammatory cytokines Tnf, Il6 and Il17. Microbiome analysis
suggests that Z. mobilis can alter the intestinal microbiota, increasing the abundance of
bacteria such as Akkermansia muciniphila and decreasing Escherichia coli. Finally, the data
suggest that Z. mobilis may alleviate disease progression and be considered a possible
probiotic adjuvant for the treatment of inflammatory bowel diseases.

Histoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, is a mycosis of great clinical importance due to its opportunistic and systemic character. The natural habitat of H. capsulatum is limited to environments that have birds or bats, such as caves, sheds, abandoned places and so on. The excrements of these animals are rich in nitrogen/phosphorus, which are crucial compounds for the metabolism and growth of the fungus. The infection begins with the inhalation of microconidia from the filamentous phase, which settles in the lungs’ alveoli and begins the process of cell differentiation into yeast. Yeasts use mechanisms to evade the immune system and can infect other organs such as the spleen and liver. In the most severe cases, it can lead to death, especially immunocompromised patients such as AIDS or in the transplanted. The qPCR technique was used to identify the HC100 gene that is specific to this fungus. Soil samples from caves in the Federal District, Goiás and Minas presented positive results. With this data, future visitors to these caves will know about the risk of contamination by H. capsulatum and signal to future academics where this fungus can be found for study. The qPCR technique for H. capsulatum is new and studies such as this one can help to better standardize it and test it in different samples from different origins

Cryptococcus neoformans is the major etiologic agent of cryptococcosis, mainly affecting immunocompromised individuals. The disease has the lung as its primary site, presenting tropism for the Central Nervous System where its main clinical manifestation is meningoencephalitis. This disease is an important public health issue, which is diagnosed late and its epidemiological data are underestimated. Capsule, melanin and laccase are outstanding virulence factors, being a promising field for understanding the pathogenesis and searching more suitable therapeutic targets. C. neoformans’ melanin protects it against the effector immune response and also hindering the action of antifungals. Our group studied the immunomodulatory effects of C. neoformans’ melanin on the interaction with murine macrophages through phagocytosis assays and also detection of LC3-associated phagocytosis (LAP). Two fungal strains were used, the wild type H99 and the laccase mutant LAC1. We verified that without melanization supplementation, the wild type was more phagocytosed than the mutant, which changed with the addition of melanin supplementation. Regarding the mutant, we observed effects on phagocytosis only after 48 hours of supplementation. As for the detection of LAP, without stimulation of melanin production, the mutant showed a higher occurrence of LAP than the wild type. When we added supplementation, we saw that the melanization of C. neoformans did not bring significant effects on the wild type and on the mutant, we only found variation after 48 hours of supplementation. These results together could suggest that there are other interferers than just melanin in the macrophage response

Familial hypercholesterolemia (FH) is a dyslipidemia with a genetic origin, which is caused by a functional impairment in the low-density lipoprotein receptor (LDLR), with subsequent disturbance in lipoprotein metabolism and cholesterol accumulation in the bloodstream. The high levels of circulating cholesterol existent in the low-density lipoprotein (LDL) induces harmful effects on cellular metabolism, tissue development and mitochondrial bioenergetics. Mitochondrial dysfunctions have already been described in several biological tissues in FH models, however, the vast majority of studies analyzed only male individuals. Considering that the sex differently impacts metabolic dysfunctions, it is paramount to study the impact of sexual dimorphism on FH model bioenergetics’ outcomes. Therefore, the aim of this study was to evaluate and to compare the effect of sexual dimorphism on the mitochondrial function of hippocampus, brown adipose tissue (BAT), liver and heart in C57Bl/6 wild-type (WT) and LDLR knockout (LDLR-/-) mice. Male and female C57Bl/6 and LDLR-/- mice approximately 6 months old were used. Animals were anesthetized and euthanized for blood collection, in which triglycerides and total cholesterol levels were measured. Hippocampi and BAT homogenates, as well as hepatic and cardiac isolated mitochondria, were used to assess oxygen consumption through high-resolution respirometry. Male and female LDLR-/- mice presented a significant increase in cholesterol and triglyceride levels, when compared to WT animals of the same sex. In male mice hippocampus, the genetic deletion of LDLR provoked a significant decrease in O2 consumption related to complex I+II activity, in oxidative phosphorylation and in maximal respiratory capacity, when compared to WT animals. Although no differences in these same hippocampal parameters were observed between LDLR-/- and C57Bl/6 females, they were significantly lower in WT females, when compared to males of the same genotype. In LDLR-/- males BAT, there was also a significant decrease in mitochondrial respiration related to UCP-1 activity, and in the respiratory reserve capacity, compared to WT animals. The BAT maximum respiratory capacity was significantly lower in WT females, when compared to WT males. On the other hand, LDLR-/- females presented a significant decrease in the hepatic O2 consumption related I+II complex activity, when compared to WT ones. Both sexes showed no significant differences in cardiac mitochondrial bioenergetics. The results suggest that LDLR deletion affects the mitochondrial bioenergetics of several tissues, with a greater impact on males than females, reinforcing the importance of investigating HF in both sexes, since sexual dimorphism impacts the outcomes of this disease.

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen, whose virulence and mechanism of interaction with the host are controlled by cell-density-dependent signaling molecules, called quorum sensing (QS). This pathogen has two types of QS signaling molecules, the N-Acyl-homoserine lactones and the 2-heptyl-3-hydroxy-4-quinolone signal. Mesenchymal stem cells (MSCs) have been explored to treat a variety of clinical conditions, especially immune disorders. In this sense, several studies have shown that priming MSCs with INF-γ increases the immunomodulatory property of these cells. In the present study, we investigated the influence of QS N-(3-oxododecanoyl)-L-homoserine (OdDHL) on the biological properties of MSCs subjected or not to INF-γ priming. Using MTT test, we demonstrated that, after 24h of cell culture, OdDHL (10 and 50µM) compromises the viability of MSCs, regardless of INF-γ priming. Through cell apoptosis assay with Annexin/PI, by Flow Cytometry, we observed that OdDHL (10 and 50µM) induces apoptosis in MSCs and that this effect seems to be more pronounced in MSCs licensed with INF-γ. In line, MSCs submitted to treatment with 50µM OdDHL showed greater release of the enzyme lactate dehydrogenase (LDH) in the extracellular medium, an effect that was enhanced by the priming of these cells with INF-γ. Interestingly, at non-toxic concentrations of 0.5 and 1µM, we observed that OdDHL enhanced the migratory potential of MSCs, mainly in unlicensed cells. However, at these same doses, we noticed that OdDHL decreased the immunosuppressive potential of MSCs. We also demonstrate, functionally, that MSCs directly inhibit the growth of Pseudomonas aeruginosa. As can be seen, in this study we demonstrated that licensing with INF-γ increases the immunosuppressive potential of MSCs, but does not protect these cells from the deleterious effects promoted by the high concentration of OdDHL. More importantly, licensing MSCs with INF-γ appears to significantly compromise the antimicrobial effect of MSCs when these cells are challenged to control the growth of Pseudomonas aeruginosa bacteria. Although our study was developed in vitro, our findings serve as an alert for the effects of QS molecules on the properties of MSCs, indicating that when in low doses these molecules can even potentiate important properties of MSCs, but at higher doses they compromise the viability of these cells, independent of licensing with INF-γ.