Natural modulation of redox status throughout the ontogeny of Amazon frog Physalaemus ephippifer (Anura, Leptodactylidae).

During their development, amphibians undergo various physiological processes that may affect their susceptibility to environmental pollutants. Naturally occurring fluctuations caused by developmental events are often overlooked in ecotoxicological studies. Our aim is to investigate how biomarkers of oxidative stress are modulated at different stages of larval development in the Amazonian amphibian species, Physalaemus ephippifer. The premetamorphosis, prometamorphosis and metamorphic climax stages were used to analyze total antioxidant capacity (ACAP), glutathione S-transferase (GST) activity, lipid peroxidation (LPO) levels and the expression of genes nrf2, gst, gsr (glutathione reductase) and gclc (glycine-cysteine ligase, catalytic subunit). Although there was no difference in ACAP and the genes expression among the studied stages, individuals from the premetamorphosis and prometamorphosis showed higher GST activity than ones under the climax. LPO levels were highest in individuals from the metamorphic climax. The present study suggests that the oxidative status changes during ontogeny of P. ephippifer tadpoles, especially during the metamorphic climax, the most demanding developmental phase. Variations in the redox balance at different developmental stages may lead to a divergent response to pollution. Therefore, we recommend that studies using anuran larvae as biomonitors consider possible physiological differences during ontogeny in their respective analyses.

Exploring boundary conditions of the single-code/default strategy in pigeons.

To investigate the extent of adoption of more efficient coding strategies, pigeons learned, in three experiments, a symbolic matching-to-sample task that featured an asymmetric sample-comparison mapping. In all experiments, one comparison was correct following one of the samples (one-to-one mapping), and another comparison was correct following the remaining samples (many-to-one mapping). The experiments differed in sample number; Experiment 1 featured three samples, Experiment 2 five samples, and Experiment 3 seven samples. Our goal was to assess the adoption of a single-code/default coding strategy, which establishes two response rules: one rule specific to the sample mapped one-to-one (the single code), and another rule to be applied following any other sample (the default rule). Alternatively, the animals could establish more response rules, one per sample. Thus, the single-code/default strategy allows learning a task via a reduced number of response rules, and the more samples are mapped many-to-one, the greater the savings it allows. As such, the three experiments should progressively be more amenable to the adoption of this strategy. Overall, the adoption of a single-code/default strategy was not widespread. When taken together with previous results, the present study suggests that the amount of training may affect the coding strategy pigeons adopt. Additionally, our results underscore that individual differences are a fundamental aspect to consider when studying learning flexibility.

Entre baques e atraques: cenas de uma pesquisa cartográfica entre o Sistema Único de Saúde (SUS) e a saúde suplementar / Blows and clinches: scenes of a cartographic study between the Brazilian National Health System (SUS) and supplementary health / Entre caídas y golpes: escenas de una investigación cartográfica entre el Sistema Brasileño de Salud (SUS) y la Salud suplementaria

A pesquisa cartográfica se caracteriza pela imersão da pessoa pesquisadora no território afetivo-existencial a ser pesquisado. Este artigo põe em análise, por meio dessa proposta metodológica, as implicações e sobreimplicações de um pesquisador negro, bissexual e médico de Família e Comunidade em um contexto de trabalho e pesquisa entre uma Unidade Básica de Saúde (UBS) e uma clínica de atenção primária de um plano privado de saúde localizada no município de Natal, RN. Ele desvela, por meio da "escrevivência" de quatro cenas, a precarização do processo de trabalho, a deterioração de algumas ferramentas clínicas e a forte presença de um racismo estrutural e institucional.(AU)

In cartographic research, researchers immerse themselves in the affective-existential setting being studied. Using cartographic methods, this study analyzed the implications and superimplications of a black, bisexual family and community doctor working and conducting research in a public health center and private primary care clinic in Natal, Rio Grande do Norte. Based on "escrevivências" of four cenas-de-viragem (milestones), the analysis reveals worsening working terms and conditions, the deterioration of certain clinical tools and the strong presence of structural and institutional racism.(AU)

La investigación cartográfica se caracteriza por la inmersión de la persona investigadora en el territorio afectivo-existencial a investigar. Este artículo analiza por medio de esta propuesta metodológica las implicaciones y sobreimplicaciones de un investigador negro, bisexual, médico de familia y comunidad en un contexto de trabajo e investigación entre una Unidad Básica de Salud y una clínica de atención primaria de un plan privado de salud localizada en el municipio de Natal (Estado de Rio Grande do Norte). Desvela por medio de la "escritura-vivencia" de cuatro puntos de inflexión la precarización del proceso de trabajo, el deterioro de algunas herramientas clínicas y la fuerte presencia de un racismo estructural e institucional.(AU)

Chitosan-PEG Gels Loaded with Jatropha mollissima (Pohl) Baill. Ethanolic Extract: An Efficient and Effective Biomaterial in Hemorrhage Control.

A lack of control over blood loss can have catastrophic implications, including death. Although several hemostatic medications have been employed to reduce bleeding, a vast majority of them are ineffective, expensive, or pose health risks to the patient. To overcome these constraints, chitosan-polyethylene glycol (CS-PEG) hemostatic gels loaded with ethanolic extract of Jatropha mollissima sap (EES) were prepared and their hemostatic, physicochemical, and cytotoxic properties were evaluated. The gels were produced by mixing CS with PEG (an external plasticizer) and EES. The phytochemical analysis revealed a significant concentration of total polyphenols and tannins content in the extract and catechin was identified as one of the key compounds of EES. Infrared spectroscopy analysis revealed the presence of EES in the gels, as well as the chemical interaction between CS and PEG. The gels were thermally stable between 25 and 37 °C (ambient and human body temperature range), had pseudoplastic deformation behavior (rheological properties preserved after shearing), were simple to inject (compression force 30 N), and were biocompatible. In vivo experiments showed that both CS-PEG-EES gels exhibited greater hemostatic action in preventing tail hemorrhage in Wistar rats, with decreased bleeding time and blood weight compared with unloaded CS-PEG gels (control groups) and Hemostank, a commercial product. However, the gel prepared with acetic acid was more efficient in controlling bleeding. These findings reveal that CS-PEG-EES gels can reduce hemorrhages and are a potent, simple, and safe hemostatic agent.

X-linked hypophosphatemic rickets with advanced bone age treated with aromatase inhibitor.

Summary: We present an adolescent with X-linked hypophosphatemic rickets (XLH) with bone age advancement and its response to aromatase inhibitors (AIs). A male with XLH, confirmed with a deletion on the PHEX gene, received regular treatment since the first year of life with average growth velocity and height. He had bone age compatible with chronological age until 13 when he had a bone age advancement and a decrease in the predicted final height thought to be due to initiation of oral isotretinoin, which has been previously reported. Then, anastrozole was initiated and maintained concomitant to the rickets treatment for 2 years with bone age stabilization. He had no adverse effects or worsening of bone health markers. As a result, he maintained his height gain and improved his final height Z score compared with the predicted final height at initiating anastrozole. In conclusion, although AIs was a reasonable strategy to stabilize bone age and minimize height impairment, careful monitoring is mandatory to understand its benefits and effects on XLH patients. Learning points: Although X-linked hypophosphatemic rickets patients have normal puberty, they can be affected by metabolic and environmental factors that may advance their bone age and impair the predicted final height, similar to the general population. Isotretinoin may accelerate skeletal maturation during puberty in an adolescent with X-linked hypophosphatemic rickets. Aromatase inhibitors showed to be a reasonable strategy to stabilize bone age and minimize height impairment in an adolescent with X-linked hypophosphatemic rickets.

The effects of mercury exposure on Amazonian fishes: An investigation of potential biomarkers.

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum, presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 µg kg-1, respectively, and in their muscle tissue, 101 ± 1.30 µg kg-1 and 87.4 ± 0.900 µg kg-1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 µg kg-1) and muscle (71.4 ± 0.930 µg kg-1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 µg kg-1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish.

Structural evolution of an amphibian-specific globin: A computational evolutionary biochemistry approach.

Studies on the globin family are continuously revealing insights into the mechanisms of gene and protein evolution. The rise of a new globin gene type in Pelobatoidea and Neobatrachia (Amphibia:Anura) from an α-globin precursor provides the opportunity to investigate the genetic and physical mechanisms underlying the origin of new protein structural and functional properties. This amphibian-specific globin (globin A/GbA) discovered in the heart of Rana catesbeiana is a monomer. As the ancestral oligomeric state of α-globins is a homodimer, we inferred that the ancestral state was lost somewhere in the GbA lineage. Here, we combined computational molecular evolution with structural bioinformatics to determine the extent to which the loss of the homodimeric state is pervasive in the GbA clade. We also characterized the loci of GbA genes in Bufo bufo. We found two GbA clades in Neobatrachia. One was deleted in Ranidae, but retained and expanded to yield a new globin cluster in Bufonidae species. Loss of the ancestral oligomeric state seems to be pervasive in the GbA clade. However, a taxonomic sampling that includes more Pelobatoidea, as well as early Neobatrachia, lineages would be necessary to determine the oligomeric state of the last common ancestor of all GbA. The evidence presented here points out a possible loss of oligomerization in Pelobatoidea GbA as a result of amino acid substitutions that weaken the homodimeric state. In contrast, the loss of oligomerization in both Neobatrachia GbA clades was linked to independent deletions that disrupted many packing contacts at the homodimer interface.

Qual família e qual comunidade? Reconfigurações da Medicina de Família e Comunidade na saúde suplementar / ¿Qué familia y qué comunidad? Reconfiguraciones de la Medicina Familiar y Comunitaria en salud complementaria / Which family and which community? Reconfigurations of Family and Community Medicine in supplementary health

RESUMO: Trata-se de um estudo cartográfico que buscou analisar a atuação de médicos(as) de família e comunidade na Atenção Primária da saúde suplementar, realizado por meio de diários e entrevistas cartográficas entre março de 2021 e janeiro de 2022, processados semanalmente em reuniões de pesquisa. Tal estudo se deu com base nos analisadores: 'território', 'família' e 'comunidade'. Notou-se que a territorialização e a abordagem familiar ganham outros contornos na Medicina de Família e Comunidade praticada na saúde suplementar. Além disso, verificou-se que algumas das ferramentas típicas da Atenção Básica - como visita domiciliar, educação em saúde, genograma, ecomapa e vigilância em saúde - não eram utilizadas na atenção suplementar ou tiveram outras aplicabilidades dissonantes do modelo preconizado. Concluiu-se que a Medicina de Família e Comunidade na saúde suplementar se aproxima de uma atuação mais clínica, com perda da potência das linhas de força que constituem tal especialidade, tendendo a uma medicina menos familiar e comunitária.

RESUMEN: Se trata de un estudio cartográfico que buscó analizar el desempeño de los médicos de familia y comunidad en atención primaria de salud complementaria, realizado a través de diarios y entrevistas cartográficas entre marzo de 2021 y enero de 2022, que fueron procesados semanalmente en reuniones de investigación. Este estudio se basó en los analizadores: 'territorio', 'familia' y 'comunidad'. Se observó que la territorialización y el enfoque familiar adquieren otros contornos en la Medicina Familiar y Comunitaria practicada en salud complementaria. Además, se encontró que algunas de las herramientas típicas de la atención básica, como las visitas domiciliarias, la educación sanitaria, el genograma, el ecomap y la vigilancia sanitaria, no se utilizaron en la atención complementaria o tenían otra aplicabilidad disonante del modelo recomendado. Se concluyó que la Medicina Familiar y Comunitaria en salud complementaria se aproxima a una práctica más clínica, con pérdida de potencia de las líneas eléctricas que constituyen dicha especialidad, tendiendo a una medicina menos familiar y comunitaria.

ABSTRACT: This is a cartographic study that sought to analyze the performance of family and community physicians in primary care of supplementary health, carried out through diaries and cartographic interviews between March 2021 and January 2022, which were weekly processed in research meetings. This study was based on the analyzers: 'territory', 'family' and 'community'. It was noticed that territorialization and family approach gain other contours in Family and Community Medicine practiced in supplementary health. In addition, it was found that some of the typical tools of basic care - such as home visits, health education, genogram, ecomap and health surveillance - were not used in supplementary care or had other dissonant applicabilities of the recommended model. It was concluded that Family and Community Medicine in supplementary health approaches a more clinical practice, with loss of power from the power lines that constitute such specialty, tending to a less familiar and community medicine.

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson's disease. Currently, Parkinson's disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography-IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and -80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

Mercury metalloproteomic profile in muscle tissue of Arapaima gigas from the Brazilian Amazon.

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg-1 and 142 ± 1.50 mg kg-1 (total precipitation of protein fraction) and 139 ± 1.45 mg kg-1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00 mg kg-1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LC‒MS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases.

Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation.

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson's disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs' design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at -80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Unveiling the biopathway for the design of novel COMT inhibitors.

Catechol-O-methyltransferase (COMT) is an enzyme responsible for the O-methylation of biologically active catechol-based molecules. It has been associated with several neurological disorders, especially Parkinson's disease (PD), because of its involvement in catecholamine metabolism, and has been considered an important therapeutic target for central nervous system disorders. In this review, we summarize the biophysical, structural, and therapeutical relevance of COMT; the medicinal chemistry behind the development of COMT inhibitors and the application of computer-aided design to support the design of novel molecules; current methodologies for the biosynthesis, isolation, and purification of COMT; and revise existing bioanalytical approaches for the assessment of enzymatic activity in several biological matrices.

Arginine-Affinity Chromatography for Nucleic Acid (DNA and RNA) Isolation.

Nucleic acid-based therapy has been emerging as a new strategy with great potential for the treatment of numerous diseases, especially those caused by gene defects. In this context, biotechnology plays a critical role on establishing suitable processes for biopharmaceuticals manufacturing, while the purification step still imposes a major burden. Affinity chromatography using amino acids as specific ligands has been successfully applied for plasmid DNA purification. In this protocol, we describe the process for nucleic acids production and extraction, as well as the chromatographic matrix synthesis for separation between DNA and RNA. This novel arginine-macroporous support presents excellent binding capacity and great robustness for nucleic acids isolation.

Surgical treatment of spinal arachnoid web: Report of two cases and literature review.

BACKGROUND: Arachnoid webs (AWs) can cause cord compression and syringomyelia in the thoracic spine. Here, we describe two patients who underwent operative treatment for AW and reviewed the literature. CASE DESCRIPTION: Two patients underwent surgical treatment for thoracic AW. Both presented with spastic gait and numbness in the lower extremities. On MR, these lesions exhibited the "scalpel" sign (i.e. due to the accumulation of cerebrospinal fluid on the dorsal aspect of the spinal cord). Operative intervention, consisting of fenestration and web resection, resulted in symptom resolution. CONCLUSION: Thoracic AWs are rare lesions that should be considered among the differential diagnosis of spinal compressive syndromes. Surgical fenestration and resection of the AW correct the flow dynamics allowing for full symptoms resolution.

Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro.

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.

Fields of Forel Brain Stimulation Improves Levodopa-Unresponsive Gait and Balance Disorders in Parkinson's Disease.

BACKGROUND: Gait and balance disturbance are challenging symptoms in advanced Parkinson's disease (PD). Anatomic and clinical data suggest that the fields of Forel may be a potential surgical target to treat these symptoms. OBJECTIVE: To test whether bilateral stimulation centered at the fields of Forel improves levodopa unresponsive freezing of gait (FOG), balance problems, postural instability, and falls in PD. METHODS: A total of 13 patients with levodopa-unresponsive gait disturbance (Hoehn and Yahr stage ≥3) were included. Patients were evaluated before (on-medication condition) and 1 yr after surgery (on-medication-on-stimulation condition). Motor symptoms and quality of life were assessed with the Unified Parkinson's Disease Rating scale (UPDRS III) and Quality of Life scale (PDQ-39). Clinical and instrumented analyses assessed gait, balance, postural instability, and falls. RESULTS: Surgery improved balance by 43% (95% confidence interval [CI]: 21.2-36.4 to 35.2-47.1; P = .0012), reduced FOG by 35% (95% CI: 15.1-20.3 to 8.1-15.3; P = .0021), and the monthly number of falls by 82.2% (95% CI: 2.2-6.9 to -0.2-1.7; P = .0039). Anticipatory postural adjustments, velocity to turn, and postural sway measurements also improved 1 yr after deep brain stimulation (DBS). UPDRS III motor scores were reduced by 27.2% postoperatively (95% CI: 42.6-54.3 to 30.2-40.5; P < .0001). Quality of life improved 27.5% (95% CI: 34.6-48.8 to 22.4-37.9; P = .0100). CONCLUSION: Our results suggest that DBS of the fields of Forel improved motor symptoms in PD, as well as the FOG, falls, balance, postural instability, and quality of life.

The Chlorate-Nitrous Acid-Iodine-Iodide Oscillating Reaction.

This work presents a new oscillating reaction based on chlorate and observed in a CSTR at room temperature. This can be the first member of a new family of oscillating reactions. In addition, it is also the first oscillating reaction to use nitrous acid as a reactant. Four different behaviors were observed: simple oscillations, mixed mode oscillations, bursts, and quasiperiodicity. The period of oscillations is very short, which is around 1 s. Together with the fact that it also shows fast bursts, it opens the possibility that it can be used to simulate fast biological events, like the neuron's communications signals.

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment.

Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.

Conception of Plasmid DNA and Polyethylenimine Delivery Systems with Potential Application in DNA Vaccines Field.

In DNA-based therapy research, the conception of a suitable vector to promote the target gene carriage, protection, and delivery to the cell is imperative. Exploring the interactions between polyethylenimine (PEI) and a plasmid DNA can give rise to the formation of suitable complexes for gene release and concomitant protein production. The nanosystems formulation method, based on coprecipitation, seems to be adequate for the conception of nanoparticles with suitable properties (morphology, size, surface charge, and pDNA complexation capacity) for intracellular applications. The developed systems are able of cell uptake, intracellular trafficking, and gene expression, in an extent depending on the ratio of nitrogen to phosphate groups (N/P). It comes that the transfection process can be tailored by this parameter and, therefore, also the therapeutic outcomes. This knowledge contributes for progresses in the development of suitable delivery systems with potential application in DNA vaccines field.