Molecular modelling studies and in vitro enzymatic assays identified A 4-(nitrobenzyl)guanidine derivative as inhibitor of SARS-CoV-2 Mpro.

Scientists and researchers have been searching for drugs targeting the main protease (Mpro) of SARS-CoV-2, which is crucial for virus replication. This study employed a virtual screening based on molecular docking to identify benzoylguanidines from an in-house chemical library that can inhibit Mpro on the active site and three allosteric sites. Molecular docking was performed on the LaSMMed Chemical Library using 88 benzoylguanidine compounds. Based on their RMSD values and conserved pose, three potential inhibitors (BZG1, BZG2, and BZG3) were selected. These results indicate that BZG1 and BZG3 may bind to the active site, while BZG2 may bind to allosteric sites. Molecular dynamics data suggest that BZG2 selectively targets allosteric site 3. In vitro tests were performed to measure the proteolytic activity of rMpro. The tests showed that BZG2 has uncompetitive inhibitory activity, with an IC50 value of 77 µM. These findings suggest that benzoylguanidines possess potential as Mpro inhibitors and pave the way towards combating SARS-Cov-2 effectively.

Mioclonías positivas y negativas en paciente con enfermedad renal crónica / Positive and negative myoclonus in a patient with end stage renal disease

Introducción: Las mioclonías son contracciones musculares paroxísticas de corta duración o pérdida abrupta del tono muscular, denominadas mioclonías positivas y negativas, respectivamente. Se presenta un caso clínico de mioclonías positivas y negativas generalizadas y se pretende describir los múltiples mecanismos fisiopatológicos y etiologías que lo desencadenan. Presentación del caso: Hombre de 35 años, con diabetes mellitus tipo 1 complicada con enfermedad renal diabética en hemodiálisis, desarrolló una bacteriemia asociada a catéter por Staphylococcus aureus y presentó mioclonías positivas y negativas. Se identificaron como posibles desencadenantes la uremia, la infección y los fármacos con potencial promioclónico; el hallazgo incidental de una lesión isquémica en núcleo caudado no explicaba la semiología encontrada en el paciente. Se hizo el control y retiro de todos los factores promioclónicos enunciados, junto a manejo farmacológico con levetiracetam, y con ello se logró el control de los síntomas. Discusión: Los pacientes con enfermedad renal crónica son susceptibles a la acumulación de productos tóxicos de tipo guanidinas, que tienen potencial para producir mioclonías. Además, las infecciones, el uso de fármacos con potencial promioclónico y lesiones estructurales como las isquemias corticales son etiologías que deben considerarse en el diagnóstico diferencial. El mayor impacto en los síntomas se observa con el control del factor desencadenante, y, en caso de persistir, la terapia farmacológica proporciona buenos resultados. Conclusión: Las mioclonías son trastornos del movimiento relativamente comunes en la enfermedad renal crónica. La identificación del desencadenante es crucial para su manejo junto al uso de fármacos con actividad antimioclónica.

Introduction: Myoclonus are paroxysmal muscle contractions of short duration or abrupt loss of muscle tone, called positive and negative myoclonus respectively. A clinical case of generalized positive and negative myoclonus is presented and the aim is to describe the multiple pathophysiological mechanisms and etiologies that trigger it. Case presentation: A 35-year-old man with type 1 diabetes mellitus complicated by diabetic kidney disease on hemodialysis developed catheter-associated bacteremia due to Staphylococcus aureus and presented positive and negative myoclonus. Uremia, infection, and drugs with pro-myoclonic potential were identified as possible triggers; The incidental finding of an ischemic lesion in the caudate nucleus did not explain the semiology found in the patient. The control and removal of all the pro-myoclonic factors mentioned was carried out, along with pharmacological management with levetiracetam, thus achieving control of the symptoms. Discussion: Patients with chronic kidney disease are susceptible to the accumulation of guanidine-type toxic products, which have the potential to produce myoclonus. Furthermore, infections, the use of drugs with pro-myoclonic potential and structural lesions such as cortical ischemia are etiologies that should be considered in the differential diagnosis. The greatest impact on symptoms is observed with the control of the triggering factor and if it persists, pharmacological therapy provides good results. Conclusion: Myoclonus are relatively common movement disorders in chronic kidney disease. Identification of the trigger is crucial for its management along with the use of drugs with anti-myoclonic activity.

Polyhexamethylene guanidine hydrochloride as promising active ingredient for oral antiseptic products to eliminate microorganisms threatening the health of endangered wild cats: a comparative study with chlorhexidine digluconate.

The aim of this study was to evaluate the antimicrobial efficacy of polyhexamethylene hydrochloride guanidine (PHMGH) compared to chlorhexidine digluconate (CLX) for use as an oral antiseptic during dental procedures in wild cats. This research is crucial due to limited information on the diversity of oral microorganisms in wild cats and the detrimental local and systemic effects of oral diseases, which highlights the importance of improving prevention and treatment strategies. Samples were collected from the oral cavities of four Puma concolor, one Panthera onca, and one Panthera leo, and the number of colony-forming units per milliliter (CFU/mL) was counted and semi-automatically identified. The antimicrobial susceptibility profile of bacterial isolates was determined using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill kinetics of PHMGH and CLX. A total of 16 bacterial isolates were identified, consisting of six Gram-positive and 10 Gram-negative. PHMGH displayed MIC and MBC from 0.24 to 125.00 µg/mL, lower than those of CLX against three isolates. Time-kill kinetics showed that PHMGH reduced the microbial load by over 90% for all microorganisms within 30 min, whereas CLX did not. Only two Gram-positive isolates exposed to the polymer showed incomplete elimination after 60 min of contact. The results could aid in the development of effective prevention and treatment strategies for oral diseases in large felids. PHMGH showed promising potential at low concentrations and short contact times compared to the commercial product CLX, making it a possible active ingredient in oral antiseptic products for veterinary use in the future.

Evidence of Guanidines Potential against Leishmania (Viannia) braziliensis: Exploring In Vitro Effectiveness, Toxicities and of Innate Immunity Response Effects.

Leishmaniasis is a complex group of infectious and parasitic diseases that afflict many thousands of individuals across five continents. Leishmaniasis treatment remains a challenge because it relies on drugsknown for their high toxicity and limited efficacy, making itimperative to identify new molecules that offer greater effectiveness and safety. This study sought to explore the impact of seven synthetic guanidine derivatives (LQOF-G1, LQOF-G2, LQOF-G6, LQOF-G7, LQOF-G32, LQOF-G35 and LQOF-G36) onthe parasite Leishmania (Viannia) braziliensis and in vitro macrophage infection by this parasite, as well as cytotoxic approaches in vitro models of mammalian host cells and tissues. The synthesized compounds showed purity ≥ 99.65% and effectively inhibited parasite growth. LQOF-G1 proved the most potent, yielding the best half-maximal inhibitory concentration (IC50) values against promastigotes (4.62 µmol/L), axenic amastigotes (4.27 µmol/L), and intracellular amastigotes (3.65 µmol/L). Notably, the antileishmanial activity of LQOF-G1, LQOF-G2, and LQOF-G6 was related to immunomodulatory effects, evidenced by alterations in TNF-α, IL-12, IL-10, nitric oxide (NO), and reactive oxygen species (ROS) levels in the supernatant of culture macrophages infected with L. (V.) braziliensis and coincubated with these compounds. LQOF-G2 and LQOF-G36 compounds exhibited vasodilator and spasmolytic effects at higher concentrations (≥100 µmol/L). Generally, LQOF-G1, LQOF-G2, and LQOF-G32 compounds were found to be nontoxic to assessed organs and cells. No toxic effects were observed in human cell lines, such as HEK-293, CaCo-2 and A549, at concentrations ≥ 500 µmol/L. Collectively, data have shown unequivocal evidence of the effectiveness of these compounds against L. (V.) braziliensis parasite, one of the causative agents of Tegumentary Leishmaniasis and Mucocutaneous Leishmaniasis in America.

Novel Selective and Low-Toxic Inhibitor of LmCPB2.8ΔCTE (CPB) One Important Cysteine Protease for Leishmania Virulence.

Leishmaniasis is a highly prevalent, yet neglected disease caused by protozoan parasites of the genus Leishmania. In the search for newer, safer, and more effective antileishmanial compounds, we herein present a study of the mode of action in addition to a detailed structural and biological characterization of LQOF-G6 [N-benzoyl-N'-benzyl-N″-(4-tertbutylphenyl)guanidine]. X-ray crystallography and extensive NMR experiments revealed that LQOF-G6 nearly exclusively adopts the Z conformation stabilized by an intramolecular hydrogen bond. The investigated guanidine showed selective inhibitory activity on Leishmania major cysteine protease LmCPB2.8ΔCTE (CPB) with ~73% inhibition and an IC50-CPB of 6.0 µM. This compound did not show any activity against the mammalian homologues cathepsin L and B. LQOF-G6 has been found to be nontoxic toward both organs and several cell lines, and no signs of hepatotoxicity or nephrotoxicity were observed from the analysis of biochemical clinical plasma markers in the treated mice. Docking simulations and experimental NMR measurements showed a clear contribution of the conformational parameters to the strength of the binding in the active site of the enzyme, and thus fit the differences in the inhibition values of LQOF-G6 compared to the other guanidines. Furthermore, the resulting data render LQOF-G6 suitable for further development as an antileishmanial drug.

Physicochemical and biological properties of experimental dental adhesives doped with a guanidine-based polymer: an in vitro study.

OBJECTIVES: The objective of this study is to formulate experimental dental adhesives with different polyhexamethylene guanidine hydrochloride concentrations (PHMGH) and evaluate their physical, chemical, and biological properties. MATERIALS AND METHODS: The experimental adhesives were formulated with 0 (control, GCTRL), 0.5 (G0.5%), 1 (G1%), or 2 (G2%) wt.% into the adhesive. The adhesives were analyzed for degree of conversion (DC%), softening in solvent (ΔKHN%), ultimate tensile strength (UTS), microtensile bond strength (µTBS) immediately and after 1 year of aging, antibacterial activity, and cytotoxicity. RESULTS: There were no differences among groups for DC%, ΔKHN%, and UTS (p > 0.05%). There were no differences between each PHMGH-doped adhesive compared to GCTRL in the immediate µ-TBS (p > 0.05). Adhesives with at least 1 wt.% of PHMGH presented better stability of µ-TBS. PHMGH-doped adhesives showed improved longitudinal µ-TBS compared to GCTRL (p < 0.05). Lower Streptococcus mutans biofilm formation was observed for PHMGH-doped adhesives (p < 0.05). There was lower viability of planktonic S. mutans in the media in contact with the samples when at least 1 wt.% of PHGMGH was incorporated (p < 0.05). The formulated adhesives showed no cytotoxicity against pulp cells (p > 0.05). CONCLUSIONS: The adhesive with 2 wt.% of PHMGH showed the highest antibacterial activity, without affecting the physicochemical properties and cytotoxicity, besides conferring stability for the dental adhesion. CLINICAL RELEVANCE: PHMGH, a positively charged polymer, conveyed antibacterial activity to dental adhesives. Furthermore, it did not negatively affect the essential physicochemical and biocompatibility properties of the adhesives. More importantly, the incorporation of PHMGH provided stability for the µ-TBS compared to the control group without this additive.

Chronic exposure to environmentally relevant concentrations of guanylurea induces neurotoxicity of Danio rerio adults.

Recent studies have shown guanylurea (GUA) alters the growth and development of fish, induces oxidative stress, and disrupts the levels and expression of several genes, metabolites, and proteins related to the overall fitness of fish. Nonetheless, up to date, no study has assessed the potential neurotoxic effects that GUA may induce in non-target organisms. To fill the current knowledge gaps about the effects of this metabolite in the central nervous system of fish, we aimed to determine whether or not environmentally relevant concentrations of this metabolite may disrupt the behavior, redox status, AChE activity in Danio rerio adults. In addition, we also meant to assess if 25, 50, and 200 µg/L of GUA can alter the expression of several antioxidant defenses-, apoptosis-, AMPK pathway-, and neuronal communication-related genes in the brain of fish exposed for four months to GUA. Our results demonstrated that chronic exposure to GUA altered the swimming behavior of D. rerio, as fish remained more time frozen and traveled less distance in the tank compared to the control group. Moreover, this metabolite significantly increased the levels of oxidative damage biomarkers and inhibited the activity of acetylcholinesterase of fish in a concentration-dependent manner. Concerning gene expression, environmentally relevant concentrations of GUA downregulated the expression GRID2IP, PCDH17, and PCDH19, but upregulated Nrf1, Nrf2, p53, BAX, CASP3, PRKAA1, PRKAA2, and APP in fish after four months of exposure. Collectively, we can conclude that GUA may alter the homeostasis of several essential brain biomarkers, generating anxiety-like behavior in fish.

Oxidative stress as a potential mechanism by which guanylurea disrupts the embryogenesis of Danio rerio.

Metformin is one the most prescribed drug to treat type 2 diabetes. In wastewater treatment plants, this drug is bacterially transformed to guanylurea, which occurs at higher concentrations in the aquatic environments than its parent compound. Since there is a huge knowledge gap about the toxicity of this metabolite on aquatic organisms, we aimed to investigate the impact of guanylurea on the embryonic development and oxidative stress biomarkers of zebrafish (Danio rerio). For this effect, zebrafish embryos (4 h post fertilization) were exposed to 25, 50, 100, 200, 250, 25,000, 50,000, 75,000 µg/L guanylurea until 96 h post fertilization. Guanylurea led to a significant delay in the hatching process in all exposure groups. Furthermore, this transformation product affected the embryonic development of fish, inducing severe body alterations and consequently leading to their death. The most pronounced malformations were malformation of tail, scoliosis, pericardial edema, yolk deformation and craniofacial malformation. Concerning oxidative stress response, we demonstrated that guanylurea induced the antioxidant activity of superoxide dismutase, catalase, and glutathione peroxidase in zebrafish embryos. In addition, the levels of lipid peroxidation, protein carbonyl and hydroperoxide content were also increased in the embryos exposed to this transformation product. However, the integrated biomarker response (IBR) analysis carried out in this study demonstrated that oxidative damage biomarkers got more influence over the embryos than antioxidant enzymes. Thus, we can conclude that guanylurea induces oxidative stress in zebrafish embryos, and that this transformation product impair the normal development of this freshwater organism.

Development of an Improved Guanidine-Based Rac1 Inhibitor with in†vivo Activity against Non-Small Cell Lung Cancer.

The Rho GTPase Rac1 is involved in the control of cytoskeleton reorganization and other fundamental cellular functions. Aberrant activity of Rac1 and its regulators is common in human cancer. In particular, deregulated expression/activity of Rac GEFs, responsible for Rac1 activation, has been associated to a metastatic phenotype and drug resistance. Thus, the development of novel Rac1-GEF interaction inhibitors is a promising strategy for finding new preclinical candidates. Here, we studied structure-activity relationships within a new family of N,N'-disubstituted guanidine as Rac1 inhibitors. We found that compound 1D-142, presents superior antiproliferative activity in human cancer cell lines and higher potency as Rac1-GEF interaction inhibitor in vitro than parental compounds. In addition, 1D-142 reduces Rac1-mediated TNFα-induced NF-κB nuclear translocation during cell proliferation and migration in NSCLC. Notably, 1D-142 allowed us to show for the first time the application of a Rac1 inhibitor in a lung cancer animal model.

Avaliação de soluções antifúngicas de guanidina para resina base de dentadura: um estudo in vitro / Evaluation of guanidine antifungal solutions for denture base resin: an in vitro study

Background: The purposes of this study were: (1) to formulate polyhexamethylene guanidine hydrochloride (PHMGH) solutions at different concentrations; (2) to evaluate their antifungal activity against a mature biofilm of Candida albicans on acrylic resins; (3) to evaluate possible effects on acrylic resins flexural strength and surface roughness. Methods: PHMGH solutions were formulated with distilled water and 0.125, 0.250, or 0.5 wt.% of PHMGH. One group without PHMGH was used as control. For antifungal activity analysis, acrylic resin specimens were contaminated with C. albicans. Specimens were immersed in PHMGH solutions or distilled water for 5 or 10 min. Ultimate flexural strength and surface roughness of acrylic resins were evaluated. Results: All PHMGH solutions at 5 or 10 min showed antifungal activity compared to the control group (p<0.05). The group with 0.5 wt.% of PHMGH showed no countable colonies regardless of the time of contact with the acrylic resins. After 10 min, all PHMGH solutions had antifungal effect without differences from 0.125 to 0.5 wt.% of PHMGH. All groups showed high flexural strength after contact with the solutions compatible with ISO 20795-1:2013 recommendation. The values of surface roughness always remained low, from 0.01 to 0.04 µm for all groups. Conclusion: Within the limitations of this study, it was concluded that the use of a solution composed by distilled water and 0.5 wt.% of PHMGH for 5 min was effective as a disinfectant agent against mature biofilm of C. albicans , maintaining acceptable roughness and flexural strength.

Antecedentes: Os objetivos deste estudo foram: (1) formular soluções de cloridrato de polihexametileno guanidina (PHM-GH) em diferentes concentrações; (2) avaliar sua atividade antifúngica contra biofilme maduro de Candida albicans em resinas acrílicas; (3) avaliar efeitos na resistência à flexão e rugosidade da superfície. Métodos: Soluções de PHMGH foram formuladas com água destilada e 0,125, 0,250 ou 0,5% em peso de PHMGH. Um grupo sem PHMGH foi usado como controle. Para análise da atividade antifúngica, amostras de resina acrílica foram contaminadas com C. albicans, e imersas em soluções de PHMGH ou água destilada por 5 ou 10 min. Resistência à flexão final e rugosidade da superfície foram avaliadas. Resultados: Todas as soluções de PHMGH aos 5 ou 10 minutos apresentaram atividade antifúngica em comparação ao grupo controle (p <0,05). O grupo com 0,5% em peso de PHMGH não mostrou colônias contáveis, independentemente do tempo. Após 10 minutos, todas as soluções de PHMGH tiveram efeito antifúngico sem diferenças de 0,125 a 0,5% em peso de PHM-GH. Os grupos apresentaram alta resistência à flexão após o contato com as soluções compatíveis com a recomendação ISO 20795-1: 2013. Os valores de rugosidade da superfície permaneceram baixos, de 0,01 a 0,04 µm para todos os grupos. Conclusão: Dentro das limitações deste estudo, concluiu-se que o uso de uma solução composta por água destilada e 0,5% em massa de PHMGH por 5 min foi eficaz como agente desinfetante contra o biofilme maduro de C. albicans, mantendo rugosidade e resistência à flexão aceitáveis.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.

Guanidine- and purine-functionalized ligands of FeIIIZnII complexes: effects on the hydrolysis of DNA.

In this paper, the catalytic effects of aminoguanidine and aminopurine groups in the second sphere of a FeIIIZnII complex that mimics the active site of the metallohydrolase purple acid phosphatase (PAP) are investigated, with a particular view on DNA as substrate. The ligand 3-(((3-((bis(2-(pyridin-2-yl)ethyl)amino)methyl)-2-hydroxy-5-methylbenzyl)(pyridin-2-ylmethyl)amino)meth-yl)-2 hydroxy-5-methylbenzaldehyde-(H2L1bpea) was synthesized and its complex [(OH)FeIII(µ-OH)ZnII(H2O)(L1bpea)](ClO4) was used as a base for comparison with similar complexes previously published in the literature. Subsequent modifications were conducted in the aldehyde group, where aminoguanidine (amig) and aminopurine (apur) were used as side chain derivatives. The complexes [(OH)FeIII(µ-OH)ZnII(H2O)(L1bpea)](ClO4) (1), [(OH)FeIII(µ-OH)ZnII(H2O)(L1bpea-amig)](ClO4) (2) and [(OH)FeIII(µ-OH)ZnII(H2O)(L1bpea-apur)](ClO4) (3) were characterized by spectroscopic methods (infrared, UV-Vis) and ESI-MS spectrometry. Density functional theory (DFT) was also used to better understand the structure of the complexes. The hydrolytic activity of complexes 1, 2 and 3 was analyzed using both the model substrate 2,4-BDNPP (bis-(2,4-dinitrophenyl)phosphate) and DNA. Complexes 2 and 3, containing the derivatized ligands, have a significantly higher association constant (Kassoc≅ 1/KM) for the activated substrate 2,4-BDNPP compared to complex 1. The catalytic efficiency (kcat/KM) is also higher due to hydrogen bonds and/or π-stacking interactions between the substrate and the aminoguanidine or aminopurine groups present in 2 and 3, respectively. In the DNA cleavage assays, all complexes were able to cleave DNA, with 1 and 2 having higher catalytic activity than 3. In addition, when compared to previously analyzed complexes, complex 2 is one of the most active, having a kcat of 0.21 h-1.

Biochemical Properties and Catalytic Specificity of a Novel Neutral Serine Peptidase Secreted by Fungus Pyrenochaetopsis sp.

The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45 °C, it tolerated a wide range of pH conditions and temperatures lower than 45 °C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.

Evidence for the involvement of TNF-α, IL-1ß and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents.

BACKGROUND: Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. METHODS: Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. RESULTS: LQM01 inhibits TNF-α and IL-1ß production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. CONCLUSIONS: LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. GENERAL SIGNIFICANCE: Beneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of 'dysfunctional pain' and inflammatory diseases.

Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels.

Guanidinium toxins, such as saxitoxin (STX), tetrodotoxin (TTX) and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV). Members of the STX group, known collectively as paralytic shellfish toxins (PSTs), are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards development of pharmaceuticals and therapeutic applications.

Stability of different influenza subtypes: How can high hydrostatic pressure be a useful tool for vaccine development?

BACKGROUND: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. METHODS: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. RESULTS: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. CONCLUSIONS: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

Peptide presentation on primate erythroparvovirus 1 virus-like particles: In vitro assembly, stability and immunological properties.

Virus-like particles (VLPs) have demonstrated to be valuable scaffolds for the display of heterologous peptides for vaccine development and other specific interactions. VLPs of primate erythroparvovirus 1, generally referred as parvovirus B19 (B19V), have already been produced in-vivo and in-vitro from the recombinant VP2 protein of this virus. In this study, chimeric forms of B19V VP2 were constructed, and their ability to assemble into VLPs was evaluated. Chimeras were composed of the VP2 protein fused, at its N-terminus, with two peptides derived from the fusion glycoprotein (F) of the respiratory syncytial virus (RSV). The chimeric proteins self-assembled into VLPs morphologically similar to B19V virions. Stability of these VLPs was analyzed under denaturation conditions with guanidinium chloride (GdnHCl). Our results indicate that the presence of the heterologous fragments increased the stability of VLPs assembled by any of the VP2 chimeras. Specific proteolysis assays shown that a fraction of the N-termini of the chimeric proteins is located on the outer surface of the VLPs. Immunogenicity of VLPs against RSV was evaluated and the results indicate that the particles can elicit a humoral immune response, although these antibodies did not cross-react with RSV in ELISA tests. These results provide novel insights into the localization of the N-termini of B19V VP2 protein after in vitro assembly into VLPs, and point them to be attractive sites to display peptides or proteins without compromise the assembly or stability of VLPs.

Avaliação in vitro do efeito de diferentes processos de alisamento químico/térmico na fibra capilar / In vitro evaluation of the straightening effect by different chemical/thermal processes in the hair fiber

A aparência dos cabelos é de fundamental importância na sociedade atual. Estando em moda, cabelos mais lisos e com menos volume, os consumidores que antes os alisavam com produtos químicos e força mecânica, passaram a utilizar um tratamento térmico, além do secador de cabelos: as piastras ("chapinhas") que atuam em valores de temperatura ao redor de 230°C. Esse procedimento ocasiona além dos danos mecânicos e químicos também dano térmico, tornando os cabelos ainda mais fragilizados. O escopo deste estudo foi avaliar o dano na fibra capilar, de amostras não tratadas e nas que receberam aplicação de alisantes/relaxantes tradicionais e alternativos. O estudo foi dividido em cinco capítulos que avaliam: aplicação dos alisantes/relaxantes com ingredientes ativos distintos; danos mecânicos, perda Protéica; análise térmica e microscopia eletrônica de varredura. As amostras de cabelo utilizadas em todos os estudos foram tratadas como descrito no primeiro capítulo. Foram aplicados produtos comerciais contendo os seguintes ingredientes ativos: Hidróxido de Sódio, Tioglicolato de Amônio, Hidróxido de Guanidina (reação de hidróxido de cálcio com carbonato de guanidina), formaldeído e ácido glioxílico isolado e em combinação com carbocisteína. O uso de formaldeído e ácido glioxilico em formulações de alisantes/relaxantes está proibido pela Agência Nacional de Vigilância Sanitária. Todos os produtos aplicados alisaram os cabelos; os procedimentos que utilizaram a piastra tornaram os fios mais lisos. Os alisantes/relaxantes à base de ácido glioxilico e formaldeído reduziram de forma expressiva a tensão de ruptura dos cabelos tornando-os mais frágeis. A maior perda protéica foi observada na amostra tratada com carbocisteína (1,74 mg/g cabelo). Nos estudos de análise térmica, na fase de desidratação a amostra tratada com carbocisteína apresentou maior perda de massa (15,17%); na fase de denaturação da proteína, a tratada com hidróxido de sódio (51,06%); e na fase de eliminação do material carbonáceo, todas as amostras apresentaram perda de massa maior que a amostra não tratada; as menores temperaturas de pico foram as das amostras sem tratamento alisante (630°C) e ácido glioxílico (640°C). Observando-se as imagens de microscopia eletrônica nota-se modificação nas bordas das cutículas das amostras indicando que sofreram agressão; o hidróxido de guanidina deixou adicionalmente resíduo; as amostras tratadas com ácido glioxílico e formaldeído apresentaram a formação de filme superficial como um "envelopamento" da fibra. Os resultados sugerem que não há predominância de um procedimento mais danoso que os demais; porém os que utilizaram a piastra (alisamentos/relaxamento ácidos) acentuaram os danos

The appearance of the hair is of fundamental importance in today's society. Being in fashion, hair straight and with less volume, consumers that before straighted hair with chemicals products and mechanical strength began to use a heat treatment, in addition to hair dryers: the hot plates ("chapinhas") acting on temperature values around 230°C. This procedure causes not only mechanical and chemical damage but also thermal one, making the hair more fragile. The scope of this study was to evaluate the damage to the hair fiber, in untreated samples and these receiving straighteners/relaxers application of traditional and alternative products.The study was divided into five chapters that evaluated: application of straighteners/relaxers with different active ingredients; mechanical damage, protein loss; thermal analysis and scanning electron microscopy. The hair samples used in all studies were treated as described in the chapter one. Commercial products containing the following active ingredients were used: Sodium Hydroxide, Ammonium Thioglycolate, Guanidine Hydroxide (calcium hydroxide reaction with guanidine carbonate), Formaldehyde and Glyoxylic Acid alone and in combination with Carbocysteine. The use of Formaldehyde andGlyoxylicAcid in straightening/relaxing formulations are prohibited by the National Agency for Sanitary Vigilance. All applied products, straight the hair samples; the procedures that used the hot plates become the hair more straight. The straightening/relaxing based on Glyoxylic Acid and Formaldehyde reduced significantly the hair break point making them more fragile. Most protein loss was observed in the sample treated with Carbocysteine (1.74mg/g hair).In the thermal analysis studies at the dewatering stage, Carbocystein treated samples showed greater weight loss(15.17%), at the protein denaturation stage this treated with Sodium Hydroxide (51.06%) and in the carbonaceous material elimination phase all samples showed mass loss greater than the untreated sample;. The lower peak temperatures were observed in the samples without treatment (630°C) and with Glyoxylic Acid (640°C). Observing the images of electron microscopy is noted the change in the cuticle aspect of the samples showing that the edges were damaged, Guanidine Hydroxide, left further residue: the samples treated with Glyoxylic Acid and Formaldehyde showed the formation of surface film as an "enveloping" fiber. The results suggest that there is not a predominance of a more harmful treatment than other, but those using hot plates(straightening/relaxing acids) emphasize the damage

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach.

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.