Molecular determinants for brain targeting by peptides: a meta-analysis approach with experimental validation.

Blood-brain barrier (BBB) peptide-shuttles (BBBpS) are able to translocate the BBB and reach the brain. Despite the importance of brain targeting in pharmacology, BBBpS are poorly characterized. Currently, their development relies on the empiric assumption that cell-penetrating peptides (CPPs), with proven ability to traverse lipid membranes, will likewise behave as a BBBpS. The relationship between CPPs/BBBpS remains elusive and, to the best of our knowledge, has not hitherto been subject to thorough experimental scrutiny. In this work, we have identified/quantified the main physicochemical properties of BBBpS and then searched for CPPs with these properties, hence potential BBBpS. The specific features found for BBBpS are: (i) small size, (ii) none or few aromatic residues, (iii) hydrophobic, and (iv) slight cationic nature. Then, we selected the 10 scoring best in an ordinary least squares analysis, and tested them in vitro and in vivo. Overall, we identified the molecular determinants for brain targeting by peptides, devised a methodology that can be used to assist in the design of peptides with potential brain penetration from amino acid residue sequences, and found four new BBBpS within the CPP library.

The use of a selective, nontoxic dual-acting peptide for breast cancer patients with brain metastasis.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of commonly targeted receptors. Unspecific chemotherapy is currently the main therapeutic option, with poor results. Another major challenge is the frequent appearance of brain metastasis (BM) associated with a significant decrease in patient overall survival. The treatment of BM is even more challenging due to the presence of the blood-brain barrier (BBB). Here, we present a dual-acting peptide (PepH3-vCPP2319) designed to tackle TNBC/BM, in which a TNBC-specific anticancer peptide (ACP) motif (vCPP2319) is joined to a BBB peptide shuttle (BBBpS) motif (PepH3). PepH3-vCPP2319 demonstrated selectivity and efficiency in eliminating TNBC both in monolayers (IC50≈5.0 µM) and in spheroids (IC50≈25.0 µM), with no stringent toxicity toward noncancerous cell lines and red blood cells (RBCs). PepH3-vCPP2319 was also able to cross the BBB in vitro and penetrate the brain in vivo, and was stable in serum with a half-life above 120 min. Tumor cell-peptide interaction is fast, with quick peptide internalization via clathrin-mediated endocytosis without membrane disruption. Upon internalization, the peptide is detected in the nucleus and the cytoplasm, indicating a multi-targeted mechanism of action that ultimately induces irreversible cell damage and apoptosis. In conclusion, we have designed a dual-acting peptide capable of brain penetration and TNBC cell elimination, thus expanding the drug arsenal to fight this BC subtype and its BM.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Panobinostat-loaded folate targeted liposomes as a promising drug delivery system for treatment of canine B-cell lymphoma.

Introduction: Cancer is a major public health problem with over 19 million cases reported in 2020. Similarly to humans, dogs are also largely affected by cancer, with non-Hodgkin's lymphoma (NHL) among the most common cancers in both species. Comparative medicine has the potential to accelerate the development of new therapeutic options in oncology by leveraging commonalities between diseases affecting both humans and animals. Within this context, in the present study, we investigated the potential of panobinostat (Pan)-loaded folate-targeted PEGylated liposomes (FA-PEG-Pan-Lip) for the treatment of canine B-cell lymphoma, while contributing to new perspectives in comparative oncology. Methods and results: Two formulations were developed, namely: PEG-Pan-Lip and FA-PEG-Pan-Lip. Firstly, folate receptor expression in the CLBL-1 canine B-cell lymphoma cell line was assessed. After confirming receptor expression, both Pan-loaded formulations (PEG-Pan-Lip, FA-PEG-Pan-Lip) demonstrated dose-dependent inhibitory effects on CLBL-1 cell proliferation. The FA-PEG-Pan-Lip formulation (IC50 = 10.9 ± 0.03 nM) showed higher cytotoxicity than the non-targeted PEG-Pan-Lip formulation (IC50 = 12.9 ± 0.03 nM) and the free panobinostat (Pan) compound (IC50 = 18.32±0.03 nM). Moreover, mechanistically, both Pan-containing formulations induced acetylation of H3 histone and apoptosis. Flow cytometry and immunofluorescence analysis of intracellular uptake of rhodamine-labeled liposome formulations in CLBL-1 cells confirmed cellular internalization of PEG-Lip and FA-PEG-Lip formulations and higher uptake profile for the latter. Biodistribution studies of both radiolabeled formulations in CD1 and SCID mice revealed a rapid clearance from the major organs and a 1.6-fold enhancement of tumor uptake at 24 h for 111In-FA-PEG-Pan-Lip (2.2 ± 0.1 %ID/g of tumor) compared to 111In-PEG-Pan-Lip formulation (1.2±0.2 %ID/g of tumor). Discussion: In summary, our results provide new data validating Pan-loaded folate liposomes as a promising targeted drug delivery system for the treatment of canine B-cell lymphoma and open innovative perspectives for comparative oncology.

A Promising Approach: Magnetic Nanosystems for Alzheimer's Disease Theranostics.

Among central nervous system (CNS) disorders, Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and a major cause of dementia worldwide. The yet unclear etiology of AD and the high impenetrability of the blood-brain barrier (BBB) limit most therapeutic compounds from reaching the brain. Although many efforts have been made to effectively deliver drugs to the CNS, both invasive and noninvasive strategies employed often come with associated side effects. Nanotechnology-based approaches such as nanoparticles (NPs), which can act as multifunctional platforms in a single system, emerged as a potential solution for current AD theranostics. Among these, magnetic nanoparticles (MNPs) are an appealing strategy since they can act as contrast agents for magnetic resonance imaging (MRI) and as drug delivery systems. The nanocarrier functionalization with specific moieties, such as peptides, proteins, and antibodies, influences the particles' interaction with brain endothelial cell constituents, facilitating transport across the BBB and possibly increasing brain penetration. In this review, we introduce MNP-based systems, combining surface modifications with the particles' physical properties for molecular imaging, as a novel neuro-targeted strategy for AD theranostics. The main goal is to highlight the potential of multifunctional MNPs and their advances as a dual nanotechnological diagnosis and treatment platform for neurodegenerative disorders.

Biological response and cell death signaling pathways modulated by tetrahydroisoquinoline-based aldoximes in human cells.

The uncharged 3-hydroxy-2-pyridine aldoximes with protonatable tertiary amines are studied as antidotes in toxic organophosphates (OP) poisoning. Due to some of their specific structural features, we hypothesize that these compounds could exert diverse biological activity beyond their main scope of application. To examine this further, we performed an extensive cell-based assessment to determine their effects on human cells (SH-SY5Y, HEK293, HepG2, HK-2, myoblasts and myotubes) and possible mechanism of action. As our results indicated, aldoxime having a piperidine moiety did not induce significant toxicity up to 300 µM within 24 h, while those with a tetrahydroisoquinoline moiety, in the same concentration range, showed time-dependent effects and stimulated mitochondria-mediated activation of the intrinsic apoptosis pathway through ERK1/2 and p38-MAPK signaling and subsequent activation of initiator caspase 9 and executive caspase 3 accompanied with DNA damage as observed already after 4 h exposure. Mitochondria and fatty acid metabolism were also likely targets of 3-hydroxy-2-pyridine aldoximes with tetrahydroisoquinoline moiety, due to increased phosphorylation of acetyl-CoA carboxylase. In silico analysis predicted kinases as their most probable target class, while pharmacophores modeling additionally predicted the inhibition of a cytochrome P450cam. Overall, if the absence of significant toxicity for piperidine bearing aldoxime highlights the potential of its further studies in medical counter-measures, the observed biological activity of aldoximes with tetrahydroisoquinoline moiety could be indicative for future design of compounds either in a negative context in OP antidotes design, or in a positive one for design of compounds for the treatment of other phenomena like cell proliferating malignancies.

The SisaMob Information System: Implementation of Digital Data Collection as a Tool for Surveillance and Vector Control in the State of São Paulo.

Information systems are essential instruments in managing resources, in the evaluation of the epidemiological situation, and for decision-making at all hierarchical levels. Technological advances have allowed the development of systems that meet these premises. Therefore, it is recommended to consider the optimization of data entry and its immediate georeferencing in order to obtain information in real time. To meet this objective, we describe the application introduction process for the implementation of the digital collection of primary data and its integration with the database through synchronization with the SisaWeb platform (Information System for surveillance and control of Aedes aegypti), developed to meet the needs of the Arbovirus Surveillance and Control Program in the state of São Paulo, Brazil. For this purpose, the application-SisaMob-was conceived in the Android Studio development environment, Google®, following the same guidelines as the traditional collection method. Tablets equipped with the Android® operating system were used. To evaluate the implementation of the application, a semi-structured test was applied. The results highlighted that 774.9% (27) of the interviewees evaluated its use positively and, replacing the standard bulletin, 61.1% (22) of the users considered it regular to excellent. The automatic collection of geographic coordinates represented the greatest innovation in the use of the portable device, with reductions in errors and in the time taken to complete the report in the field. The integration to SisaWeb allowed obtaining information in real-time, being easily presented in tabular and graphic modes and spatially arranged through maps, making it possible to monitor the work at a distance, and allowing preliminary analyses during the data collection process. For the future, we must improve the mechanisms for assessing the effectiveness of information, increase the potential of the tool to produce more accurate analyses, which can direct actions more efficiently.

Rabbit derived VL single-domains as promising scaffolds to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.

DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna.

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.

Evaluation of Methods for Collecting Mosquitoes (Culicidae: Diptera) in Canopy and Ground Strata in the Brazilian Savanna.

The hand-net is the standard method for capturing mosquitoes with sylvatic diurnal activity in disease outbreaks in Brazil. However, occupational risks and biases related to the collectors' abilities and attractiveness are important limitations. In this study, we compared hand-nets with automatic traps (CDC) associated to CO2 and BG-Lure® in the Vassununga State Park, a Brazilian Savanna protection area. The collections carried out over 27 days on the ground and the forest canopy. A total of 1555 mosquitoes were obtained in 20 taxa. The diversity index ranged between 1.12 and 1.79 and the dominance index from 0.22 to 0.40. The dominant species on the ground was Aedes scapularis (46.0%), and in the canopy, Hg. janthinomys/capricornii (31.9%). Haemagogus leucocelaenus was rare (n = 2). The hand-net resulted in the greatest diversity and abundance of species in both strata, followed by the traps associated with CO2. A low degree of similarity was observed between the hand-net on the ground compared to the other capture methods. The use of BG-Lure® alone resulted in a low number of specimens. In conclusion, the hand-net is still the method of choice for collecting arbovirus vectors in the diurnal period, especially yellow fever vectors.

Selective Inhibition of Bruton's Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors.

Bruton's tyrosine kinase (BTK) is a member of the TEC-family kinases and crucial for the proliferation and differentiation of B-cells. We evaluated the therapeutic potential of a covalent inhibitor (JS25) with nanomolar potency against BTK and with a more desirable selectivity and inhibitory profile compared to the FDA-approved BTK inhibitors ibrutinib and acalabrutinib. Structural prediction of the BTK/JS25 complex revealed sequestration of Tyr551 that leads to BTK's inactivation. JS25 also inhibited the proliferation of myeloid and lymphoid B-cell cancer cell lines. Its therapeutic potential was further tested against ibrutinib in preclinical models of B-cell cancers. JS25 treatment induced a more pronounced cell death in a murine xenograft model of Burkitt's lymphoma, causing a 30-40% reduction of the subcutaneous tumor and an overall reduction in the percentage of metastasis and secondary tumor formation. In a patient model of diffuse large B-cell lymphoma, the drug response of JS25 was higher than that of ibrutinib, leading to a 64% "on-target" efficacy. Finally, in zebrafish patient-derived xenografts of chronic lymphocytic leukemia, JS25 was faster and more effective in decreasing tumor burden, producing superior therapeutic effects compared to ibrutinib. We expect JS25 to become therapeutically relevant as a BTK inhibitor and to find applications in the treatment of hematological cancers and other pathologies with unmet clinical treatment.

Targeting Zika Virus with New Brain- and Placenta-Crossing Peptide-Porphyrin Conjugates.

Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.

The Use of Antibody-Antibiotic Conjugates to Fight Bacterial Infections.

The emergence of antimicrobial resistance (AMR) is rapidly increasing and it is one of the significant twenty-first century's healthcare challenges. Unfortunately, the development of effective antimicrobial agents is a much slower and complex process compared to the spread of AMR. Consequently, the current options in the treatment of AMR are limited. One of the main alternatives to conventional antibiotics is the use of antibody-antibiotic conjugates (AACs). These innovative bioengineered agents take advantage of the selectivity, favorable pharmacokinetic (PK), and safety of antibodies, allowing the administration of more potent antibiotics with less off-target effects. Although AACs' development is challenging due to the complexity of the three components, namely, the antibody, the antibiotic, and the linker, some successful examples are currently under clinical studies.

Evaluation of Methods to Collect Diurnal Culicidae (Diptera) at Canopy and Ground Strata, in the Atlantic Forest Biome.

Hand-held insect nets are the standard method for capturing vector mosquitoes of sylvatic arboviruses; however, occupational risks and biases due to individual skill and attractiveness are important limitations. The use of chemical attractants and automatic traps could be an alternative to resolve these limitations. This study compares the yields achieved using nets with those employing electrical traps with CO2 and BG-Lure®, near the ground and in the canopy strata (6.0 and 8.0 m high). The study was conducted at the Cantareira State Park, which is in the Brazilian Atlantic Forest biome. In the 18 collections performed, 3570 specimens of 52 taxa were obtained. The most frequent species captured near the ground were Wyeomyia confusa and Limatus durhamii, whereas Sabethes albiprivus, Sabethes purpureus, and Haemagogus leucocelaenus were the most frequent in the canopy. The nets resulted in greater species richness and abundance, followed by the trap employing CO2. The combination of CO2 traps with BG-Lure® did not improve performance. The use of BG-Lure® alone resulted in low abundance and a low number of species. Our results demonstrate that the use of traps with CO2 can be complementary to collections with nets; however, for species of epidemiological interest such as those of the genera Haemagogus and Sabethes, especially in the canopy, the net remains the method of choice.

The HIV-1 Matrix Protein p17 Does Cross the Blood-Brain Barrier.

Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation in HIV-1-infected (HIV+) patients. Furthermore, detection of the HIV-1 matrix protein p17 (p17) in the central nervous system (CNS) and its ability to form toxic assemblies in the brain have been recently confirmed. Here, we show for the first time, using both an in vitro blood-brain barrier (BBB) model and in vivo biodistribution studies in healthy mice, that p17 can cross the BBB. There is rapid brain uptake with 0.35% ± 0.19% of injected activity per gram of tissue (IA/g) 2 min after administration, followed by brain accumulation with 0.28% ± 0.09% IA/g after 1 h. The interaction of p17 with chemokine receptor 2 (CXCR2) at the surface of brain endothelial cells triggers transcytosis. The present study supports the hypothesis of a direct role of free p17 in neuronal dysfunction in HAND by demonstrating its intrinsic ability to reach the CNS. IMPORTANCE The percentage of patients affected by HIV-1-associated neurocognitive disorder (HAND) ranges from 30% to 50% of HIV-infected (HIV+) patients. The mechanisms leading to HAND development need to be elucidated, but the roles of secreted viral proteins, chemokines, and proinflammatory molecules appear to be clear. In particular, the blood-brain barrier (BBB) represents a route for entry into the central nervous system (CNS) and thus plays an important role in HAND. Several findings suggest a key role for the HIV-1 matrix protein p17 (p17) as a microenvironmental factor capable of inducing neurocognitive disorders. Here, we show the ability of the p17 to cross the BBB and to reach the CNS, thus playing a crucial role in neuronal dysfunction in HAND.

The antimetastatic breast cancer activity of the viral protein-derived peptide vCPP2319 as revealed by cellular biomechanics.

The incidence of metastatic breast cancer (MBC) is increasing and the therapeutic arsenal available to fight it is insufficient. Brain metastases, in particular, represent a major challenge for chemotherapy as the impermeable nature of the blood-brain barrier (BBB) prevents most drugs from targeting cells in the brain. For their ability to transpose biological membranes and transport a broad spectrum of bioactive cargoes, cell-penetrating peptides (CPPs) have been hailed as ideal candidates to deliver drugs across biological barriers. A more ambitious approach is to have the CPP as a drug itself, capable of both killing cancer cells and interacting with the blood/brain interface, therefore blocking the onset of brain metastases. vCPP2319, a viral protein-derived CPP, has both properties as it: (a) is selective toward human breast cancer cells (MDA-MB-231) and increases cell stiffness compared to breast epithelial cells (MCF 10A) hindering the progression of metastases; and (b) adsorbs at the surface of human brain endothelial cells potentially counteracting metastatic cells from reaching the brain. Overall, the results reveal the selective anticancer activity of the peptide vCPP2319, which is also able to reside at the blood-brain interface, therefore counteracting brain penetration by metastatic cancer cells.

Development of Breast Cancer Spheroids to Evaluate Cytotoxic Response to an Anticancer Peptide.

Breast cancer (BC) is the most commonly diagnosed cancer in women and one of the most common causes of cancer-related deaths. Despite intense research efforts, BC treatment still remains challenging. Improved drug development strategies are needed for impactful benefit to patients. Current preclinical studies rely mostly on cell-based screenings, using two-dimensional (2D) cell monolayers that do not mimic in vivo tumors properly. Herein, we explored the development and characterization of three-dimensional (3D) models, named spheroids, of the most aggressive BC subtypes (triple-negative breast cancer-TNBC; and human-epidermal growth receptor-2-HER2+), using the liquid overlay technique with several selected cell lines. In these cell line-derived spheroids, we studied cell density, proliferation, ultrastructure, apoptosis, reactive oxygen species (ROS) production, and cell permeabilization (live/dead). The results showed a formation of compact and homogeneous spheroids on day 7 after seeding 2000 cells/well for MDA-MB-231 and 5000 cells/well for BT-20 and BT-474. Next, we compared the efficacy of a model anticancer peptide (ACP) in cell monolayers and spheroids. Overall, the results demonstrated spheroids to be less sensitive to treatment than cell monolayers, revealing the need for more robust models in drug development.

Updating Ecological and Behavioral Aspects of the Sandfly Fauna in the Vale do Ribeira Region, São Paulo State, Brazil.

Some ecological parameters and the distribution of vectors in the municipality of Eldorado, Vale do Ribeira Region, São Paulo, were studied. Entomological surveys were carried out from September 2019 to March 2021. It was observed that a few ecological parameters, including richness, abundance, diversity, and equitability, were typical of a modified environment, where artificial ecotopes maintain the presence of sandflies throughout the year. A total of 11,668 sandflies were captured. The presence of five taxa were observed in Eldorado, with low diversity and high dominance of Nyssomyia intermedia next to Ny. neivai, which are sympatric species. The results presented reinforce the importance of these species in anthropized areas in the transmission of cutaneous leishmaniasis (CL) agents and the need for entomological monitoring. Psathyromyia pascalei was encountered for the first time in the municipality, expanding the known area of distribution of this species in a modified environment.

Exosomes and Brain Metastases: A Review on Their Role and Potential Applications.

Brain metastases (BM) are a frequent complication in patients with advanced stages of cancer, associated with impairment of the neurological function, quality of life, prognosis, and survival. BM treatment consists of a combination of the available cancer therapies, such as surgery, radiotherapy, chemotherapy, immunotherapy and targeted therapies. Even so, cancer patients with BM are still linked to poor prognosis, with overall survival being reported as 12 months or less. Intercellular communication has a pivotal role in the development of metastases, therefore, it has been extensively studied not only to better understand the metastization process, but also to further develop new therapeutic strategies. Exosomes have emerged as key players in intercellular communication being potential therapeutic targets, drug delivery systems (DDS) or biomarkers. In this Review, we focus on the role of these extracellular vesicles (EVs) in BM formation and their promising application in the development of new BM therapeutic strategies.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.