Action of mycocins produced by Wickerhamomyces anomalus on Malassezia pachydermatis isolated from the ear canal of dogs.

This study aims to examine the effectiveness of mycocins produced by Wickerhamomyces anomalus in inhibiting Malassezia pachydermatis, a yeast commonly found in the ear canal of dogs. M. pachydermatis has a zoophilic origin and can be found in mammals, and frequently in dogs, where it mainly colonizes the ear canal region and the skin, leading to lesions that are difficult to treat. The antimicrobial mechanism was evaluated using dilutions of supernatant with enzymatic activity, which may include ß-glucanases, glycoproteins known to act on microorganism cell walls. However, it is important to note that this supernatant may contain other compounds as well. ß-glucanases in the mycocins supernatant were found at a concentration of 0.8 U/mg. The susceptibility of M. pachydermatis isolates was tested using the microdilution method. The isolates suffered 100% inhibition when tested with the culture supernatant containing mycocins. In the proteinases production test, 44% of the isolates tested were strong proteinases producers. Subsequently all these isolates suffered inhibition of their activity when tested in research medium containing mycocins supernatant at a subinhibitory concentration of ß-glucanases. This shows that mycocins can inhibit the production of proteinases, a virulence factor of M. pachydermatis. The viability test showed the antifungal action of mycocins in inhibiting the viability of M. pachydermatis cells after a period of 8  hours of contact. These results support the antimicrobial potential of mycocins and their promise as a therapeutic option.

Topical application of ozonated sunflower oil accelerates the healing of lesions of cutaneous leishmaniasis in mice under meglumine antimoniate treatment.

Besides being scarce, the drugs available for treating cutaneous leishmaniasis have many adverse effects. Ozone is an option to enhance the standard treatment due to the wound-healing activity reported in the literature. In this study, we evaluated the efficiency of ozonated sunflower oil as an adjuvant in treating cutaneous lesions caused by Leishmania amazonensis. BALB/c mice were infected with L. amazonensis, and after the lesions appeared, they were treated in four different schedules using the drug treatment with meglumine antimoniate (Glucantime®), with or without ozonated oil. After thirty days of treatment, the lesions' thickness and their parasitic burden, blood leukocytes, production of NO and cytokines from peritoneal macrophages and lymph node cells were analyzed. The group treated with ozonated oil plus meglumine antimoniate showed the best performance, improving the lesion significantly. The parasitic burden showed that ozonated oil enhanced the leishmanicidal activity of the treatment, eliminating the parasites in the lesion. Besides, a decrease in the TNF levels from peritoneal macrophages and blood leukocytes demonstrated an immunomodulatory action of ozone in the ozonated oil-treated animals compared to the untreated group. Thus, ozonated sunflower oil therapy has been shown as an adjuvant in treating Leishmania lesions since this treatment enhanced the leishmanicidal and wound healing effects of meglumine antimoniate.

Os desafios na conduta terapêutica em pacientes acometidos com feridas crônicas / Challenges in the therapeutic conduct in patients affected with chronic wounds / Retos en la conducta terapéutica en pacientes afectados de heridas crónicas

Considerado um grave problema em saúde pública, as feridas crônicas são patologias que desafiam o manejo terapêutico e infelizmente acometem milhares de pessoas em todo o mundo. Essa doença apresenta altos índices de morbidade impactando negativamente na qualidade de vida dos seus portadores, além de influenciar negativamente no domínio "bem-estar", principalmente quando associado aos fatores clínicos podendo estar relacionado há anos de tratamento sem cura da ferida. As feridas crônicas são caracterizadas por demora ou dificuldade nos processos de cicatrização e reparação ordenada da integridade anatômica e funcional da pele durante um período de no mínimo três meses. Porém, algumas lesões permanecem por anos e até décadas sem cicatrizar. Objetivo: O escopo dessa revisão é mostrar o limitado arsenal terapêutico bem como a dificuldade no manejo clínico e dessa forma proporcionar uma reflexão sobre sua fisiopatologia e a urgente necessidade de novas opções e condutas terapêuticas que possam auxiliar no tratamento desses pacientes. Metodologia: Trata-se de uma revisão integrativa da literatura sobre feridas crônicas, cujo critérios de inclusão foram artigos publicados no período de janeiro de 2005 a fevereiro de 2023. Conclusão: A problemática acerca dessa patologia é vasta, tratando de uma doença de difícil cura, com uma gama de fatores associados que dificultam a cura da lesão, estendendo essa doença a altos índices de morbidade. Novas condutas terapêuticas e novos fármacos, precisam ser desenvolvidos urgentemente. Destaca-se que o uso de probióticos e o emprego da nanotecnologia tem mostrado um grande potencial inovador no tratamento de pacientes portadores de feridas crônicas.

Considered a serious public health problem, chronic wounds are pathologies that defy therapeutic management and unfortunately affect thousands of people around the world. This disease has high morbidity rates, negatively impacting the quality of life of its patients, in addition to negatively influencing the "well-being" domain, especially when associated with clinical factors, which may be related to years of treatment without healing of the wound. Chronic wounds are characterized by delay or difficulty in healing processes and orderly repair of the anatomical and functional integrity of the skin over a period of at least three months. However, some injuries remain for years and even decades without healing. Objective: The scope of this review is to show the limited therapeutic arsenal as well as the difficulty in clinical management and thus provide a reflection on its pathophysiology and the urgent need for new options and therapeutic approaches that can help in the treatment of these patients. Methodology: This is an integrative review of the literature on chronic wounds, whose inclusion criteria were articles published from January 2005 to February 2023. Conclusion: The problem surrounding this pathology is vast, dealing with a difficult-to-cure disease, with a range of associated factors that make healing of the lesion difficult, extending this disease to high morbidity rates. New therapeutic approaches and new drugs need to be developed urgently. It is noteworthy that the use of probiotics and the use of nanotechnology have shown great innovative potential in the treatment of patients with chronic wounds.

Consideradas un grave problema de salud pública, las heridas crónicas son patologías que desafían el manejo terapéutico y que, lamentablemente, afectan a miles de personas en todo el mundo. Esta enfermedad presenta altas tasas de morbilidad, impactando negativamente en la calidad de vida de sus pacientes, además de influir negativamente en el dominio "bienestar", especialmente cuando se asocia a factores clínicos, que pueden estar relacionados con años de tratamiento sin curación de la herida. Las heridas crónicas se caracterizan por un retraso o dificultad en los procesos de cicatrización y reparación ordenada de la integridad anatómica y funcional de la piel durante un periodo de al menos tres meses. Sin embargo, algunas heridas permanecen durante años e incluso décadas sin cicatrizar. Objetivo: El alcance de esta revisión es mostrar el limitado arsenal terapéutico así como la dificultad en el manejo clínico y así aportar una reflexión sobre su fisiopatología y la urgente necesidad de nuevas opciones y enfoques terapéuticos que puedan ayudar en el tratamiento de estos pacientes. Metodología: Se trata de una revisión integradora de la literatura sobre heridas crónicas, cuyos criterios de inclusión fueron artículos publicados desde enero de 2005 hasta febrero de 2023. Conclusiones: La problemática que rodea a esta patología es amplia, tratándose de una enfermedad de difícil curación, con una serie de factores asociados que dificultan la cicatrización de la lesión, extendiendo esta enfermedad a altas tasas de morbilidad. Es urgente desarrollar nuevos enfoques terapéuticos y nuevos fármacos. Cabe destacar que el uso de probióticos y el empleo de nanotecnología han mostrado un gran potencial innovador en el tratamiento de pacientes con heridas crónicas.

Inhibition of Klebsiella pneumoniae carbapenemases by mycocins produced by Wickerhamomyces anomalus.

New alternatives have been under study as treatment due to the problem of multidrug-resistant bacteria. Among them, Wickerhamomyces anomalus mycocins have shown a great potential against several microorganisms since they have high antimicrobial activity, as well as they can be used as fast available nutrients and stand several extreme conditions. In this way, Klebsiella pneumoniae carbapenemases inhibition by mycocins produced by W. anomalus is important. Microdilution assays were carried out to evaluate strains inhibition in liquid medium and the test in solid medium were carried out. Toxicity was evaluated by both hemolysis and Artemia salina Leach tests. W. anomalus supernatant showed 2.36 U/mg ß-glucanases activity, and antimicrobial activity was evidenced in 100% Klebsiella pneumoniae carbapenemase strains up to 0.12 U/mg concentration. Besides, there was low toxicity in hemolysis and Artemia salina Leach tests. It is suggested that W. anomalus mycocins may be an alternative to develop new antimicrobial substances.

Aqueous ozone therapy improves the standard treatment of leishmaniasis lesions in animals leading to local and systemic alterations.

The current treatment of leishmaniasis presents some problems, such as cell toxicity, parenteral route, and time of treatment. Ozone emerges as an option to accelerate the standard treatment due to the immunomodulatory, antioxidant, and wound healing activity reported in the literature. This work aimed to evaluate the efficacy of aqueous ozone as an adjuvant to the standard treatment of cutaneous lesions caused by Leishmania amazonensis in an experimental model. For in vivo experiments, mice were randomly distributed in 6 groups, which were infected with L. amazonensis and treated in five different schedules using the standard treatment with Glucantime® with or without aqueous ozone. After the last day of treatment, the animals were euthanized and were analyzed: the thickness of lesions; collagen deposition, the parasitic burden of the lesions; blood leukocyte number; NO; and cytokine dosages and arginase activity from peritoneal macrophages. All treated groups showed a decrease in the lesion, but with a significative deposition of collagen in lesions with local ozone treatment. The parasite burden showed that ozone enhanced the leishmanicidal activity of the reference drug. The reduction of NO production and blood leukocyte count and increases in the arginase activity showed an immunomodulatory activity of ozone in the treated animals. Thus, ozone therapy has been shown to work as an adjuvant in the treatment of Leishmania lesions, enhancing leishmanicidal and wound healing activity of standard treatment.

Upregulation of the clpB gene in response to heat shock and beta-lactam antibiotics in Acinetobacter baumannii.

The role of the clpB gene encoding HSP/chaperone ClpB was evaluated in the multiresistant antibiotic cells of Acinetobacter baumannii (RS4 strain) under stress-induced heat shock and different beta-lactams. The expression of the clpB gene was assessed by qPCR during heat shock at 45 °C and subinhibitory concentrations of ampicillin (30 µg mL-1), amoxicillin + sulbactam (8/12 µg mL-1), cefepime (30 µg mL-1), sulfamethoxazole + trimethoprim (120/8 µg mL-1) and meropenem (18 µg mL-1). The results indicated a transient increase in clpB transcription in all treatments except cefepime. Both in the presence of ampicillin and amoxicillin/sulbactam for 20 min, the mRNA-clpB synthesis was 1.4 times higher than that of the control at time zero. Surprisingly, the mRNA-clpB levels were more than 30-fold higher after 10 min of incubation with meropenem and more than eightfold higher in the presence of trimethoprim/sulfamethoxazole. In addition, western blot assays showed that the RS4 strain treated with meropenem showed a marked increase in ClpB protein expression. Our data indicate that during exposure to beta-lactams, A. baumannii adjusts the transcription levels of the clpB mRNA and protein to respond to stress, suggesting that the chaperone may act as a key cellular component in the presence of antibiotics in this bacterium.

Antifungal efficiency of chemically and thermally-activated acrylic resins after surface treatment using poly (diallyldimethylammonium chloride).

INTRODUCTION: Acrylic resins are used in the preparation of facial prostheses and may be colonized by fungi. Here, we verified the antifungal efficacy of this material after surface treatment using poly (diallyldimethylammonium chloride). METHODS: Acrylic resin specimens with and without surface treatment were subjected to tests for fungistatic and fungicidal activities. Standard strains of Candida albicans and Aspergillus niger were used. RESULTS: After surface treatment, the fungistatic and fungicidal efficacies of the resins against C. albicans and fungistatic action against A. niger were verified. CONCLUSIONS: The surface treatment was a determinant of the antifungal activity of the material.

Natural history of experimental arthritis induced by Paracoccidioides brasiliensis in wistar rats.

INTRODUCTION: Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. This study aimed to evaluate the natural history of Paracoccidioides brasiliensis-induced experimental arthritis of the knee joints in Wistar rats. METHODS: Rats were randomly allocated to either an absolute control group, or 15-day, 45-day, or 90-day experimental (fungus-inoculated) groups. RESULTS: Experimental groups developed classic signs of articular PCM. Titers of anti-gp43 were observed to increase during the interval from 15 to 45 days post-inoculation. CONCLUSIONS: Articular arthritic lesions were induced and progressed during the study period in all experimental groups.

Natural history of experimental arthritis induced by Paracoccidioides brasiliensis in wistar rats

Abstract INTRODUCTION: Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. This study aimed to evaluate the natural history of Paracoccidioides brasiliensis-induced experimental arthritis of the knee joints in Wistar rats. METHODS: Rats were randomly allocated to either an absolute control group, or 15-day, 45-day, or 90-day experimental (fungus-inoculated) groups. RESULTS: Experimental groups developed classic signs of articular PCM. Titers of anti-gp43 were observed to increase during the interval from 15 to 45 days post-inoculation. CONCLUSIONS: Articular arthritic lesions were induced and progressed during the study period in all experimental groups.

Antifungal efficiency of chemically and thermally-activated acrylic resins after surface treatment using poly (diallyldimethylammonium chloride)

Abstract INTRODUCTION: Acrylic resins are used in the preparation of facial prostheses and may be colonized by fungi. Here, we verified the antifungal efficacy of this material after surface treatment using poly (diallyldimethylammonium chloride). METHODS: Acrylic resin specimens with and without surface treatment were subjected to tests for fungistatic and fungicidal activities. Standard strains of Candida albicans and Aspergillus niger were used. RESULTS: After surface treatment, the fungistatic and fungicidal efficacies of the resins against C. albicans and fungistatic action against A. niger were verified. CONCLUSIONS: The surface treatment was a determinant of the antifungal activity of the material.

Terapia fotodinâmica na artrite experimental induzida por Paracoccidioides brasiliensis / Photodynamic therapy in experimental arthritis induced by Paracoccidioides brasiliensis

OBJETIVOS: Avaliar a eficácia da terapia fotodinâmica com Brilliant Blue G no tratamento de um modelo experimental de artrite por Paracoccidioides brasiliensis (P. brasiliensis). MÉTODOS: Após a indução de artrite experimental com isolado de P. brasiliensis da linhagem Pb18 nos joelhos de ratos Wistar, os animais foram divididos em grupos e submetidos a terapia fotodinâmica com fotossensibilizador Brilliant Blue G intra-articular e a laserterapia apenas, sem o Brilliant Blue G. Todos os grupos receberam seus respectivos tratamentos do sétimo ao 11º dia. Para análise do edema foi mensurado o diâmetro latero-lateral do joelho de cada animal diariamente e após o período de tratamento os animais foram sacrificados para dissecação do joelho experimental e coleta de sangue para análise por ELISA, a fim de quantificar os níveis de anticorpos anti-P. brasiliensis. RESULTADOS: A aplicação da terapia fotodinâmica foi capaz de impedir a formação de edema quando comparado ao controle (p>0,005), bem como a produção de anticorpos anti-Gp-43 de P. brasiliensis (p=0,001). No exame anatomopatológico foi possível observar maior grau de sinovite e maior presença de granulomas com o fungo em seu interior no grupo que não recebeu tratamento quando comparado aos grupos que receberam a terapia fotodinâmica. CONCLUSÕES: A terapia fotodinâmica foi eficaz para atenuar a artrite experimental induzida por P. brasiliensis no modelo articular proposto.

AIMS: To evaluate the effectiveness of photodynamic therapy with Brilliant Blue G in the treatment of an experimental model of arthritis by Paracoccidioides brasiliensis (P. brasiliensis). METHODS: After the induction of experimental arthritis with isolated from P. brasiliensis of lineage Pb18 in the knees of Wistar rats, the animals were divided into groups and submitted to photodynamic therapy with intra-articular Brilliant Blue G photosensitizer and laser therapy only, without Brilliant Blue G. All groups received their respective treatments from the seventh to the 11th day. For edema analysis, the knee lateral-lateral diameter of each animal was measured daily and after the treatment period the animals were sacrificed for experimental knee dissection and blood collection for analysis by ELISA, in order to quantify levels of anti-P. brasiliensis antibodies. RESULTS: The results showed that the application of photodynamic therapy was able to prevent the formation of edema when compared to the control (p>0.005), as well as the production of anti-Gp-43 antibodies from P. brasiliensis (p=0.001). In the anatomopathological examination it was possible to observe a higher degree of synovitis and a greater presence of granulomas with the fungus inside the group that did not receive treatment when compared to the groups that received the photodynamic therapy. CONCLUSIONS: Photodynamic therapy was effective in attenuating the experimental arthritis induced by P. brasiliensis in the proposed joint model.

Saúde itinerante nos centros municipais de educação infantil do município de Guarapuava - PR; os desafios da promoção da saúde em crianças expostas a doenças parasitárias / Travelling health in the municipal kindergartens in the city of Guarapuava - PR; the challenges of health promotion in children exposed to parasitic diseases

A presença de enteroparasitoses em crianças em idade pré-escolar tem sido motivo de preocupação pelos órgãos de saúde principalmente devido ao impacto negativo sobre o crescimento e desenvolvimento intelectual dessas crianças. As infecções por parasitas intestinais podem levar a quadros de desnutrição e formas de gastroenterite crônica. O presente trabalho analisou crianças em idade pré-escolar frequentadoras de 11 unidades CMEIs (Centros Municipais de Educação Infantil) na cidade de Guarapuava-PR em 11 unidades. Foram estudadas 287 crianças e o índice de positividade para enteroparasitoses foi de 13,9%. A idade média das 287 crianças foi de 2,7 anos e a maioria das crianças analisadas foram domicialiadas em zona urbana 84,3% e somente 15,7% em zona rural, com média de filhos por família de 2,08 membros. A prevalência das parasitoses mostrou que G. duodenalis foi o protozoário mais isolado com 70,4%, seguido de Ascaris lumbricoides 6,8% o único helminto encontrado. Com relação à terapêutica empregada 20% das crianças tratadas com o fármaco albendazol continuaram apresentando exames positivos para os mesmos parasitas. Já com o fármaco benzoilmetronidazol todas as crianças apresentaram resultados negativos para presença de parasitas nas fezes. Os fatores de risco para enteroparasitoses indicaram que a presença da variável água tratada foi menor nos casos positivos. Nossos resultados mostraram que G. duodenalis foi o parasita mais isolado em crianças em idade pré-escolar.

The presence of enteroparasitoses in pre-school children has been a concern of health agencies mainly due to the negative impact on the growth and intellectual development of those children. Intestinal parasite infections can lead to malnutrition and chronic forms of gastroenteritis. The present study analyzed pre-school children attending 11 CMEIs (Municipal Kindergarten Schools) in the city of Guarapuava ­ PR. A total of 287 children were studied, resulting in a positive index for enteroparasitoses of 13.9%. The mean age of the 287 children was 2.7 years and most of the children analyzed were domiciled in urban areas (84.3%), with only 15.7% living in rural areas, with a mean number of children per family of 2.08. The parasite prevalence showed that G. duodenalis was the most frequently isolated protozoan, with 70.4%; followed by Ascaris lumbricoides with 6.8%, the only helminth found. Regarding therapy, 20% of the children treated with albendazole continued to present positive tests for the same parasites. When treated with benzoylmetronidazole, all children presented negative results for the presence of parasites in the faeces. The risk factors for enteroparasitoses showed that the variable treated water was lower in the positive cases. The results of this study showed that G. duodenalis was the most frequently parasite isolated in pre-school children.

Repression of Proteases and Hsp90 Chaperone Expression Induced by an Antiretroviral in Virulent Environmental Strains of Cryptococcus neoformans.

This study evaluated the effect of the antiretroviral ritonavir on protease secretion in different strains of Cryptococcus neoformans isolated from the environment and investigated the expression of heat shock protein (Hsp90), classically described virulence factors in other yeast in the presence of the same antiretroviral. The presence of the enzyme was detected by the formation of a degradation of the halo around the colonies. The results were classified as follows: level 1 (without proteases), level 2 (positive for proteases), and level 3 (strongly positive for proteases). Total protein extract isolated from the cell walls of the 12 strains incubated in the absence and presence of ritonavir (0.3125 mg mL-1) were resolved by SDS-PAGE and analyzed by Western blot assays using an antiserum against Hsp90 from Blastocladiella emersonii. All strains tested showed inhibition of proteinase activity in the presence of ritonavir at 0.3125 to 1.25 mg mL-1. High levels of Hsp90 were observed in the absence of ritonavir (0.3125 mg mL-1), except for the non-virulent control cells. In contrast, in the presence of the antiretroviral, a drastic reduction in the expression of the chaperone was observed. The data suggest that ritonavir, in addition to containing viral replication, could inhibit the expression of virulence factors in opportunistic yeast, as proteases and Hsp90. According to our current knowledge, this is the first time that the inhibition of Hsp90 by an antiretroviral was reported for environmental isolates of C. neoformans.

Susceptibility of Candida albicans Isolated from Blood to Wickerhamomyces anomalous Mycocins.

The occurrence of infections caused by Candida albicans in developed and developing countries and their resistance to some available antifungal drugs have been viewed as causing a great problem to human health worldwide. In order to find new researched molecules, there are some mycoses secreted by yeasts, especially mycocins produced by Wickerhamomyces anomalus with a broad antimicrobial spectrum of activity. Thus, this trial aimed at evaluating mycocins' activity obtained from environmental W. anomalus cell wall compared to thirty C. albicans strains isolated from blood. Mycocins were extracted from cell walls of three W. anomalus strains (WA40, WA45, and WA92). The 400 µg mL-1 concentration of WA40M1, WA45M2, and WA92M3 mycocin extracts showed the following respective activity results: 96.6, 96.6, and 90.0 % C. albicans strains. WA45M2 and WA92M3 mycocin extracts showed some activity in 3.3 % of C. albicans strains at 50 µg mL-1 concentration. Mycocins extracted from cell walls of three W. anomalus strains named as WA40, WA45, and WA92 showed antifungal activity compared to C. albicans and low degree of hemolysis.

Analysis of the xynB5 gene encoding a multifunctional GH3-BglX ß-glucosidase-ß-xylosidase-α-arabinosidase member in Caulobacter crescentus.

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted ß-glucosidase-ß-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), ß-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for ß-glucosidase (pNPG: p-nitrophenyl-ß-D-glucoside) ß-xylosidase (pNPX: p-nitrophenyl-ß-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for ß-glucosidase and α-L-arabinosidase and 60 °C for ß-xylosidase. BglX-V-Ara predominantly presented ß-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (Km 0.24 ± 0.0005 mM, Vmax 0.041 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.27 mM(-1) s(-1)), followed by ß-xylosidase (Km 0.64 ± 0.032 mM, Vmax 0.055 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.14 mM(-1)s(-1)) and finally α-L-arabinosidase (Km 1.45 ± 0.05 mM, Vmax 0.091 ± 0.0004 µmol min(-1) mg(-1) and Kcat/Km 0.1 mM(-1) s(-1)). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.

Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus.

Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for ß-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, ß-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 µM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

Dose response effect of Paracoccidioides brasiliensis in an experimental model of arthritis.

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS / Efeito da dose resposta de Paracoccidioides brasiliensis no modelo experimental de artrite

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

A paracoccidioidomicose (PCM) é causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb) e corresponde à micose sistêmica de maior prevalência na América Latina. O objetivo do presente trabalho foi avaliar a dose resposta de leveduras do fungo para padronização do modelo experimental de artrite séptica. Os experimentos foram realizados com grupos de 14 ratos que receberam doses de 103, 104 ou 105 células de P. brasiliensis (Pb18). Os fungos foram injetados em 50 µL de solução salina em tampão fosfatado (PBS) diretamente na articulação do joelho dos animais. Os seguintes parâmetros foram analisados neste trabalho: a formação de edema nos joelhos infundidos com as células das leveduras e alterações radiológicas, anatopalógicas além de titulação de anticorpos por Elisa. Após 15 dias de infecção, os sinais de inflamação foram evidentes. Aos 45 dias, algumas características de dano e necrose foram observadas na cartilagem articular. A disseminação sistêmica do fungo foi observada em 11% dos animais inoculados, concluiu-se que o modelo experimental é capaz de mimetizar a PCM articular em humanos e que a dose de 105 leveduras representa a dose padrão para o desenvolvimento do modelo.

Depletion of the xynB2 gene upregulates ß-xylosidase expression in C. crescentus.

Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode ß-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes ß-xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, Δ-xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. We demonstrated that C. crescentus cells lacking ß-xylosidase II upregulates the xynB genes inducing ß-xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the Δ-xynB2 cells, and high ß-xylosidase activity was observed in the presence of different agro-industrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused downregulation of the expression and activity of the ß-xylosidase. For example, the ß-xylosidase activity that was obtained in the presence of sugarcane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that ß-xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C. crescentus.