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The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pereira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Blood count is crucial for assessing bone marrow's cell production and differentiation during infections, gaging disease severity, and monitoring therapeutic responses. The profile of blood count in chronic forms of paracoccidioidomycosis (PCM) has been insufficiently explored. To better understand the changes in hematological cells in different stages of the PCM chronic form, we evaluated the blood count, including immature blood cells in automated equipment, before and during the treatment follow-up of 62 chronic PCM patients. Predominantly male (96.8%) with an average age of 54.3 (standard deviation SD 6.9) years, participants exhibited pre-treatment conditions such as anemia (45.2%), monocytosis (38.7%), and leukocytosis (17.7%), which became less frequent after clinical cure. Anemia was more prevalent in severe cases. Notably, hemoglobin and reticulocyte hemoglobin content increased, while leukocytes, monocytes, neutrophils, immature granulocytes, and platelets decreased. Chronic PCM induced manageable hematological abnormalities, mainly in the red blood series. Monocytosis, indicating monocytes' role in PCM's immune response, was frequent. Post-treatment, especially after achieving clinical cure, significant improvements were observed in various hematological indices, including immature granulocytes and reticulocyte hemoglobin content, underscoring the impact of infection on these parameters.

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MS E proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.

Paracoccidioidomycosis (PCM) is caused by Paracoccidioides spp.; during infection, some host mechanisms limit the availability of iron, thereby reducing its reproduction. However, Paracoccidioides spp. can evade the immune defense and, even under limited iron conditions, use this mineral for growth and dissemination. This study evaluated the iron metabolism of 39 patients who were diagnosed with chronic PCM from 2013 to 2021. The forms of iron before treatment and at the time of clinical cure were evaluated based on the following: serum ferritin levels (storage iron); total iron-binding capacity (TIBC) and transferrin saturation (TSAT) level (transport iron); red blood cell (RBC), hemoglobin (Hb), hematocrit (HCT), and soluble transferrin receptor (sTfR) levels; and sTfR/log ferritin ratio (functional iron). The mean age of the patients was 54.5 years (±6.7 years). Most patients were men (97.4%), rural workers (92.1%), and smokers (84.6%); furthermore, most had moderate disease severity (66.7%). After achieving clinical cure, we observed that serum ferritin levels decreased, and parameters of functional iron increased. The extent of alteration in these parameters were more pronounced in severe cases than in to mild or moderate cases. Furthermore, moderate correlations were observed between C-reactive protein and the Hb (r = -0.500; p = 0.002), RBC (r = -0.461; p = 0.005), HCT (r = -0.514; p = 0.001), and iron levels (r = -0.491; p = 0.002). However, it is possible to infer that PCM interferes with functional and storage iron because improvements in these parameters after treatment as well as associations with disease severity were observed. PCM can lead to anemia of inflammation, which can be differentiated from iron deficiency anemia by a careful investigation of the iron form parameters., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 de Brito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fungal infections represent a serious global health problem, causing damage to health and the economy on the scale of millions. Although vaccines are the most effective therapeutic approach used to combat infectious agents, at the moment, no fungal vaccine has been approved for use in humans. However, the scientific community has been working hard to overcome this challenge. In this sense, we aim to describe here an update on the development of fungal vaccines and the progress of methodological and experimental immunotherapies against fungal infections. In addition, advances in immunoinformatic tools are described as an important aid by which to overcome the difficulty of achieving success in fungal vaccine development. In silico approaches are great options for the most important and difficult questions regarding the attainment of an efficient fungal vaccine. Here, we suggest how bioinformatic tools could contribute, considering the main challenges, to an effective fungal vaccine.

Nitrogen is a crucial nutrient for microorganisms that compose essential biomolecules. However, hosts limit this nutrient as a strategy to counter infections, therefore, pathogens use adaptive mechanisms to uptake nitrogen from alternative sources. In fungi, nitrogen catabolite repression (NCR) activates transcription factors to acquire nitrogen from alternative sources when preferential sources are absent. Formamidase has been related to nitrogen depletion in Aspergillus nidulans through formamide degradation to use the released ammonia as a nitrogen source. In Paracoccidioides spp., formamidase is highly expressed in transcriptomic and proteomic analyses. Here, we aim to investigate the importance of formamidase to Paracoccidioides lutzii . Thereby, we developed a P. lutzii silenced strain of fmd gene (AsFmd) by antisense RNA technology using Agrobacterium tumefaciens -mediated transformation (ATMT). The AsFmd strain led to increased urease expression, an enzyme related to nitrogen assimilation in other fungi, suggesting that P. lutzii might explore urease as an alternative route for ammonia metabolism as a nitrogen source. Moreover, formamidase was important for fungal survival inside macrophages, as fungal recovery after macrophage infection was lower in AsFmd compared to wild-type (WT) strain. Our findings suggest potential alternatives of nitrogen acquisition regulation in P. lutzii , evidencing formamidase influence in fungal virulence.

Paracoccidioidomycosis (PCM) is a systemic mycosis with high incidence in Latin America, caused by species of the genus Paracoccidioides spp. Brazil is considered to be the endemic center of this disease, which is identified as the eighth cause of mortality from chronic infectious disease in the country. There are several specific diagnostic methods in PCM, such as microbiological, immunological, histopathological, and molecular. However, the standard laboratory diagnosis depends mostly on fungus direct observation - the gold standard of PCM diagnosis. The implementation of new technologies, such as Fourier Transform Infrared (FTIR), can contribute to the clinical diagnosis trial of this disease. Here, we evaluated a new strategy for the diagnosis of PCM by using blood serum FTIR spectra from 20 patients with PCM and 20 healthy individuals. Machine learning algorithms were able to provide an overall accuracy of 91.67% by using Cubic SVM in the PCA data from FTIR results., (Copyright © 2022. Published by Elsevier B.V.)

Systemic mycoses have been viewed as neglected diseases and they are responsible for deaths and disabilities around the world. Rapid, low-cost, simple, highly-specific and sensitive diagnostic tests are critical components of patient care, disease control and active surveillance. However, the diagnosis of fungal infections represents a great challenge because of the decline in the expertise needed for identifying fungi, and a reduced number of instruments and assays specific to fungal identification. Unfortunately, time of diagnosis is one of the most important risk factors for mortality rates from many of the systemic mycoses. In addition, phenotypic and biochemical identification methods are often time-consuming, which has created an increasing demand for new methods of fungal identification. In this review, we discuss the current context of the diagnosis of the main systemic mycoses and propose alternative approaches for the identification of new targets for fungal pathogens, which can help in the development of new diagnostic tests.

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37-100 °C) and pH (2-10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5-10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii., Methodology and Main Findings: This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome., Conclusions: This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented., Competing Interests: The authors have declared that no competing interests exist.

Flavonoids-compounds abundant in balanced daily diets-have been extensively investigated for biological activity. The pronounced antiproliferative effects of flavonoids have prompted studies to elucidate their mode of action against tumor cells. The anticancer properties of myricetin, a 3',4',5'-tri-hydroxylated flavonol, have been confirmed for a number of neoplasms, but myricitrin, its 3- O -rhamnoside derivative found in fruits and other parts of edible plants, has been scarcely investigated as a chemopreventive agent. This study evaluated the antiproliferative potential of myricitrin obtained from Combretum lanceolatum (Combretaceae) against MCF7 (breast), PC-3 (prostate), HT-29 (colon), 786-0 (kidney), and HL-60 (acute promyelocytic leukemia) cancer cell lines, using the sulforhodamine B and tetrazolium salt assays. Myricitrin proved most effective in inhibiting growth of HL-60 cells (GI 50  = 53.4  μ mol·L -1 ), yet showed weak antiproliferative activity against other cell lines. Possible cytotoxic mechanisms involving inhibition of topoisomerases I and II α by myricitrin were also evaluated, revealing inhibitory activity only against topoisomerase II α . The results suggested that topoisomerase II α inhibition is the probable mechanism responsible for the antiproliferative activity of myricitrin. In vivo mutagenicity by myricitrin and its possible antimutagenic effect on doxorubicin-induced DNA damage were also investigated by performing the somatic mutation and recombination test (SMART) on Drosophila melanogaster . Myricitrin proved nonmutagenic to the offspring of standard (ST) and high-bioactivation (HB) crosses, while cotreatments with doxorubicin revealed the antimutagenic properties of myricitrin, even under conditions of high metabolic activation.

Background: Thrombocytopenia (TP) is the major event associated with linezolid (LZD) therapy. We investigated the incidence and risk factors for thrombocytopenia in hospitalized adults who received LZD (1200mg/day) between 2015 and 2017. HIV-positive, death during follow-up and those with a baseline platelet count ≤100×10 3 /mm 3 were excluded., Method: TP was defined as a decrease in platelet count of ≥20% from the baseline level at the initiation of linezolid therapy and a final count of <100×10 3 /mm 3 . The odds ratios (OR) for thrombocytopenia were obtained using multivariate stepwise logistic regression analysis., Main Results: A total of 66 patients were included (mean age [SD] 62 [18], male gender [%], 37 [56]). LZD-associated TP was identified in 12 patients (18.2%). For TP, the adjusted OR [95% CI] of the platelet count ≤200×10 3 /mm 3 , serum creatinine and renal impairment at baseline were 5.66 [1.15-27.9], 4.57 [1.26-16.5] and 9.41 [1.09-80.54], respectively. Male gender and dosage per weight per day (DPWD) >20mg/kg/day were not risk factors., Conclusion: The results showed that the incidence of linezolid-induced thrombocytopenia was lower in patients with normal renal function and higher in those with platelet counts ≤200×10 3 /mm 3 or serum creatinine >1.5mg/dL at the start of the treatment., (Copyright © 2019 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Fungi of the Paracoccidioides genus are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis restricted to the countries of Latin America. Currently, the Paracoccidioides complex is represented by Paracoccidioides lutzii , Paracoccidioides americana , Paracoccidioides brasiliensis , Paracoccidioides restrepiensis , and Paracoccidioides venezuelensis . Even with advances in techniques used for diagnosing fungal diseases, high rates of false-positive results for PCM are still presented. Additionally, there is no efficient antigen that can be used to follow up the efficiency of patient treatment. The immunoproteomic is considered a powerful tool for the identification of antigens. In addition, antigens are molecules recognized by the immune system, which make them excellent targets for diagnostic testing of diseases caused by microorganisms. In this vein, we investigated which antigens are secreted by species representing Paracoccidioides complex to increase the spectrum of molecules that could be used for future diagnostic tests, patient follow-up, or PCM therapy. To identify the profile of antigens secreted by Paracoccidioides spp., immunoproteomic approaches were used combining immunoprecipitation, followed by antigen identification by nanoUPLC-MS E -based proteomics. Consequently, it was possible to verify differences in the exoantigen profiles present among the studied species. Through a mass spectrometry approach, it was possible to identify 79 exoantigens in Paracoccidioides species. Using bioinformatics tools, two unique exoantigens in P. lutzii species were identified, as well as 44 epitopes exclusive to the Paracoccidioides complex and 12 unique antigenic sequences that can differentiate between Paracoccidioides species. Therefore, these results demonstrate that Paracoccidioides species have a range of B-cell epitopes exclusive to the complex as well as specific to each Paracoccidioides species. In addition, these analyses allowed us the identification of excellent biomarker candidates for epidemiology screening, diagnosis, patient follow-up, as well as new candidates for PCM therapy., (Copyright © 2020 Moreira, Oliveira, Silva, Inácio, Bailão, Parente-Rocha, Cruz-Leite, Paccez, de Almeida Soares, Weber and Borges.)

Background/aim: Hodgkin's lymphoma (HL) accounts for 1% of cancers and is of largely unknown pathogenesis. This study intended to find clues about potential causes (etiology) of HL through observations on patients and their families., Patients and Methods: In 37 years of a medical oncology practice in Basel, Switzerland, 36 out of 2201 patients (1.6%) had HL. Etiological information has been obtained from the patients with the help of a German translation of the NCI Medical History Questionnaire for Cancer Etiology, and through the study of their medical charts., Results: Findings of etiological interest were observed in the medical history of 26 out of the 36 patients with HL; these findings were grouped as follows: benign tumors (25%), carcinogenic exposures (19%), immunologic disorders (19%), secondary malignancies (14%), infections (11%), congenital disorders (8%) and tonsillectomies (6%). Twenty-two out of 36 patients had one or more relatives with cancer., Conclusion: Personal and family history of patients with HL is a readily available useful tool for etiology research. The fact that HL is frequently associated with other cancers in patients and their relatives points to causal genetic factors., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective. To compare performance outcomes of students completing a longitudinal pharmacy practice experience at a single site with those of students completing advanced pharmacy practice experiences (APPEs) at a variety of practice sites. Methods. Performance data and other meaningful outcomes from a cohort of students completing a six-month experience within a single health system (longitudinal group) were compared to data from a group of students completing six months of similar APPEs at multiple health systems (control group). Results. Motivators for participating in the longitudinal program varied, with students in the program identifying practice interest alignment, schedule simplification, and desire to do a residency; and students choosing to not participate in identifying schedule flexibility and experience at a variety of practice sites. The 16 longitudinal students differed from the 20 control students at baseline in grade point average and the number of scheduled community pharmacy APPEs. Performance measures for longitudinal students and control students did not differ significantly, but the difference between students' self-perceived effort and preceptor perception of student effort was significantly different for control students compared to longitudinal students. Residency match rates and number of students receiving recognition for high academic performance were similar in the two groups. Conclusion. Students participating in the longitudinal program had different motivators but comparable outcomes to students who completed similar APPEs at a variety of practice sites., (© 2019 American Association of Colleges of Pharmacy.)

Protective immunity against intracellular pathogens, including bacteria, usually relies on cellular immunity. However, antibodies are also implicated in mediating protection against intracellular bacteria. In case of airway infection with Legionella pneumophila (Lpn), the causative agent of Legionnaires' disease, pre-existing Lpn-specific antibodies were shown to afford protection within two days of infection. Here we dissected the early kinetics of Ab-mediated protection against airway Lpn infection and observed two kinetically and mechanistically distinct phases of protection by passively administered antibodies. Within the first hour of infection, Lpn-opsonizing antibodies provided almost 10-fold protection in an antibody Fc-dependent, but FcR-independent manner. Later on, by two days post infection, Lpn-specific Ab-mediated protection strictly involved FcγR, Syk kinase activity in alveolar macrophages and induction of reactive oxygen species (ROS). The findings presented here contribute to the understanding of the mechanisms of Ab-mediated control of Lpn infection in actively or passively immunized individuals., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic disease caused by RET gene germline mutations that is characterized by medullary thyroid carcinoma (MTC) associated with other endocrine tumors. Several reports have demonstrated that the RET mutation profile may vary according to the geographical area. In this study, we collected clinical and molecular data from 554 patients with surgically confirmed MTC from 176 families with MEN2 in 18 different Brazilian centers to compare the type and prevalence of RET mutations with those from other countries. The most frequent mutations, classified by the number of families affected, occur in codon 634, exon 11 (76 families), followed by codon 918, exon 16 (34 families: 26 with M918T and 8 with M918V) and codon 804, exon 14 (22 families: 15 with V804M and 7 with V804L). When compared with other major published series from Europe, there are several similarities and some differences. While the mutations in codons C618, C620, C630, E768 and S891 present a similar prevalence, some mutations have a lower prevalence in Brazil, and others are found mainly in Brazil (G533C and M918V). These results reflect the singular proportion of European, Amerindian and African ancestries in the Brazilian mosaic genome.

Introduction: Studies have demonstrated that pathogens react to the harsh conditions in human tissues by inducing mechanisms that promote survival., Methods: Persistence and biofilm-forming ability were evaluated during stress conditions that mimic those in the host., Results: Carbon-source availability had a positive effect on Staphylococcus epidermidis RP62A adhesion during hypoxia, accompanied by a decrease in pH. In contrast, iron limitation led to decreased surface-adherent biomass, accompanied by an increase medium acidification and lactate levels. Interestingly, iron starvation and hypoxia induced persister cells in planktonic culture., Conclusions: These findings highlight the role of host stress in the virulence of S. epidermidis.

Background: The elimination of malaria depends on the blocking of transmission and of an effective treatment. In Brazil, artemisinin therapy was introduced in 1991, and here we present a performance overview during implementation outset years., Methods: It is a retrospective cohort (1991 to 2002) of patients treated in a tertiary centre of Manaus, with positive microscopic diagnosis of Plasmodium falciparum malaria, under treatment with using injectable or rectal artemisinin derivatives, and followed over 35-days to evaluate parasite clearance, death and recurrence., Findings: This cohort outcome resulted 97.6% (1554/1593) of patients who completed the 35-day follow-up, 0.6% (10/1593) of death and 1.8% (29/1593) of follow-up loss. All patients that died and those that presented parasitaemia recurrence had pure P. falciparum infections and received monotherapy. Considering patients who completed 35-day treatment, 98.2% (1527/1554) presented asexual parasitaemia clearance until D4 and 1.8% (27/1554) between D5-D10. It is important to highlight that had no correlation between the five treatment schemes and the sexual parasite clearance. Finally, it is noteworthy that we were able to observe also gametocytes carriage during all follow-up (D0-D35)., Main Conclusions: Artemisinin derivatives remained effective in the treatment of falciparum malaria during first 12-years of use in north area of Brazil.

Paracoccidioidomycosis (PCM) is a systemic disease caused by thermodymorphic fungi of the Paracoccidioides brasiliensis complex, (Paracoccidioides spp.). Patients with PCM reveal specific cellular immune impairment. Despite the effective treatment, quiescent fungi can lead to relapse, usually late, the serological diagnosis of which has been deficient. The present study was carried out with the objective of investigating a biomarker for the identification of PCM relapse and another molecule behaving as an immunological recovery biomarker; therefore, it may be used as a cure criterion. In the evolutionary analysis of the proteins identified in PCM patients, comparing those that presented with those that did not reveal relapse, 29 proteins were identified. The interactions observed between the proteins, using transferrin and haptoglobin, as the main binding protein, were strong with all the others. Patient follow-up suggests that cerulosplamin may be a marker of relapse and that transferrin and apolipoprotein A-II may contribute to the evaluation of the treatment efficacy and avoiding a premature decision., Competing Interests: The authors have declared that no competing interests exist.

Objective: To evaluate a longitudinal experiential training model for advanced pharmacy practice experiences (APPEs)., Innovation: A six-month longitudinal pilot program named the Focused Institutional Longitudinal Experience (FILE) program was developed at two academic medical centers to maximize active participation of the student and minimize the time spent orienting and onboarding students to each APPE experience. A unique component of the FILE program is the longitudinal service project, which involved a medication use evaluation, including a review of published literature and drug policy recommendations to medical center quality committees., Analysis: Student ratings regarding the quality and value of the FILE student experience was compared to the traditional APPE model. Nine quality measures were compared (e.g. amount of opportunity for direct patient care experience, learning, integration into healthcare team, and accountability for patient outcomes) between students from the FILE program to peers completing similar APPEs outside the FILE program. FILE students and APPE preceptors also completed surveys regarding the value of several program aspects., Key Findings: There was no difference between FILE and non-FILE student self-rated measures of APPE quality, and thus the decision to participate in a longitudinal APPE program should be based on the personal preference of the student. Students in the FILE program agreed or strongly agreed (mean score 4.3) that they felt prepared for post-graduate training at the completion of the program. The potential value that students in a longitudinal program might bring to the site is reinforced by the general agreement by preceptors in the end of year survey that FILE students take less of their time to orient to their service and the trend toward perception that FILE students are more likely to independently participate in patient care activities., Next Steps: To address feedback on preceptor-mentor role and the desire for more interaction with pharmacy residents, students are now paired with a pharmacy resident, and the student and resident work together on the service project with a clinical pharmacist as an advisor. Updated standards of practice clearly delineate the roles and responsibilities of students, residents, and the clinical pharmacist preceptor. Annual surveys of FILE students and preceptors provide necessary feedback to continuously improve the quality of the program., (© University of Minnesota Libraries Publishing.)

The sensitivity of the double agar gel immunodiffusion test is about 90% in patients with untreated paracoccidioidomycosis (PCM), but it is much lower in cases of relapse. In addition, serum from patients with PCM caused by Paracoccidioides lutzii, frequent in the Midwest region of Brazil, do not react with the classical antigen obtained from Pb B-339. These findings showed the need for alternative diagnostic methods, such as biological markers through proteomics. The aim of this study was to identify biomarkers for the safe identification of PCM relapse and specific proteins that could distinguish infections caused by Paracoccidioides brasiliensis from those produced by Paracoccidioides lutzii. Proteomic analysis was performed in serum from 9 patients with PCM caused by P. brasiliensis, with and without relapse, from 4 patients with PCM produced by P. lutzii, and from 3 healthy controls. The comparative evaluation of the 29 identified plasma proteins suggested that the presence of the immunoglobulin (Ig) alpha-2 chain C region and the absence of Ig heavy chain V-III TIL indicate infection by P. lutzii. In addition, the absence of complement factor B protein might be a predictor of relapse. The evaluation of these proteins in a higher number of patients should be carried out in order to validate these findings., Competing Interests: The authors have declared that no competing interests exist.

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) - an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MS E ) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii , strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization⁻time-of-flight (MALDI⁻TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI.

The anti-inflammatory, antiproliferative and cytoprotective activity of the Attalea phalerata Mart. ex Spreng pulp oil was evaluated by in vitro and in vivo methods. As for the chemical profile, the antioxidant activity was performed by spectrophotometry, and the profile of carotenoids and amino acids by chromatography. Our data demonstrated that A. phalerata oil has high carotenoid content, antioxidant activity and the presence of 5 essential amino acids. In the in vitro models of inflammation, the oil demonstrated the capacity to inhibit COX1 and COX2 enzymes, the production of nitric oxide and also induces macrophages to spreading. In the in vivo models of inflammation, the oil inhibited edema and leukocyte migration in the Wistar rats. In the in vitro model of antiproliferative and cytoprotective activity, the oil was shown inactive against the kidney carcinoma and prostate carcinoma lineage cells and with cytoprotective capacity in murine fibroblast cells, inhibiting the cytotoxic action of doxorubicin. Therefore, it is concluded that A. phalerata pulp oil has anti-inflammatory effects with nutraceutical properties potential due to the rich composition. Moreover, the oil also has cytoprotective activity probably because of its ability to inhibit the action of free radicals.

Objective. To characterize and determine the quality of the student experience in an attending pharmacist model (APM). Methods. In-depth interviews were conducted with students completing an advanced pharmacy practice experience (APPE) at two general medicine services using the APM over a 2-year time period. Quantitative information about student learning and interprofessional interactions were extracted from student evaluations of the site. Data from the mixed model were analyzed to identify strengths of the APM and areas needing improvement. Results. Strengths of the APM included positive student interaction with the pharmacy resident and more students reporting full integration in and accountability to the interprofessional team for patient outcomes compared to students in non-teaching models. A few students at one site reported a need for greater delineation of expectations, more initial support from preceptors, and initial responsibility for fewer patients. These factors were modified before the second APM year and subsequent reports from students at this site were uniformly positive. Students at the second site did not note areas needing improvement. The APM increased student capacity at both sites. Conclusion. The attending pharmacist model provided a high quality learning experience for students, particularly with regard to integration into and accountability for patient outcomes to the interprofessional team. Qualitative research methods enabled precise detection of areas needing improvement at one site and confirmed that changes made at that site improved the student experience.

Background: Immune thrombocytopenia is an immune disease characterized by thrombocytopenia and bleeding due to platelet antibodies against platelet membrane glycoproteins. Human platelet antigens are derived from polymorphisms of these glycoproteins. The aim of this study was to investigate human platelet antigen frequencies in immune thrombocytopenia patients from the state of Amazonas, Brazil and investigate the potential association between specific antigens and risk for immune thrombocytopenia., Method: Human platelet antigen typing was performed by BeadChip technology to determine allelic variants of 11 systems (HPA-1 to HPA-9, HPA-11 and HPA-15). Thirty-six patients (8 male and 28 female) with a median age of 34 years (range: 9-69 years) were evaluated and compared with data from Amazonas blood donors., Results: Platelet counts varied from 3 to 98×10 9 /L. The allele frequencies were 0.944 for HPA-1a, 0.056 for HPA-1b, 0.847 for HPA-2a, 0.153 for HPA-2b, 0.555 for HPA-3a, 0.444 for HPA-3b, 0.805 for HPA-5a, 0.222 for HPA-5b, 0.9975 for HPA-9a, 0.025 for HPA-9b, 0.486 for HPA-15a and 0.513 for HPA-15b. Among immune thrombocytopenia individuals, no b allele of the HPA-4, -6, -7, -8 and -11 were found., Conclusions: The results suggest HPA-1a, HPA-3b and HPA-5b are immune thrombocytopenia-specific autoepitopes., (Copyright © 2017 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Background: Human platelet antigens (HPAs) are alloantigens derived from polymorphisms in platelet-surface glycoproteins. The occurrence of alloantibodies against HPAs can lead to platelet destruction and subsequent thrombocytopenia. Brazilians have a high rate of racial admixture, and the knowledge of HPA polymorphisms in particular donors from north Brazil, who have a large Amerindian influence, is a relevant strategy to prevent alloimmunisation., Objective: Our aim was investigate the HPA allele's frequencies in the Amazonas blood donors., Methods: We performed HPA genotyping among 200 Amazonas blood donors by microarray for 11 HPA biallelic systems, including six of the most clinically significant systems (HPA-1 to -5 and -15) and five others (HPA-6 to -9 and -11) that have been also associated with alloimmunisation, amounting to 22 HPA alleles., Results: The obtained allele frequencies were compared with data of 38 populations worldwide to determine the hierarchical relationship and estimated the probability of mismatch platelets. The allele frequencies were 0·862 for HPA-1a, 0·137 for HPA-1b, 0·852 for HPA-2a, 0·147 for HPA-2b, 0·665 for HPA-3a, 0·335 for HPA-3b, 0·995 for HPA-4a, 0·005 for HPA-4b, 0·892 for HPA-5a, 0·107 for HPA-5b, 0·997 for HPA-9a, 0·005 for HPA-9b, 0·502 for HPA-15a and 0·497 for HPA-15b. The incompatibility risks are higher for HPA-15 and HPA-3, followed by HPA-1, -2 and -5., Conclusion: We found differences among populations worldwide, and it is interesting to note the indigenous and European influences in this region, reinforcing the heterogeneity in the ancestry of Brazilians. The results will be helpful in providing information for platelet transfusion to avoid alloimmunisation., (© 2016 British Blood Transfusion Society.)

The ability to respond to stressful conditions is essential for most living organisms. In pathogenic organisms, this response is required for effective transition from a saprophytic lifestyle to the establishment of pathogenic interactions within a susceptible host. Hyperosmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, including the alteration of protein expression and cellular volume as well as size regulation. In this work, we evaluated the proteomic profile of Paracoccidioides lutzii Pb01 yeast cells during osmotic stress induced by potassium chloride. We performed a high accuracy proteomic technique (NanoUPLC-MS(E)) to identify differentially expressed proteins during osmotic shock. The data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of cell wall components were modulated, which suggested cell wall remodeling. In addition, alterations in the energy metabolism were observed. Furthermore, proteins involved in amino acid metabolism and hydrogen peroxide detoxification were modulated during osmotic stress. Our study suggests that P. lutzii Pb01. presents a vast osmoadaptative response that is composed of different proteins that act together to minimize the effects caused by osmotic stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections.

Objective: To identify specific preceptor teaching-coaching, role modeling, and facilitating behaviors valued by pharmacy students and to develop measures of those behaviors that can be used for an experiential education quality assurance program., Methods: Using a qualitative research approach, we conducted a thematic analysis of student comments about excellent preceptors to identify behaviors exhibited by those preceptors. Identified behaviors were sorted according to the preceptor's role as role model, teacher/coach, or learning facilitator; measurable descriptors for each behavior were then developed., Results: Data analysis resulted in identification of 15 measurable behavior themes, the most frequent being: having an interest in student learning and success, making time for students, and displaying a positive preceptor attitude. Measureable descriptors were developed for 5 role-modeling behaviors, 6 teaching-coaching behaviors, and 4 facilitating behaviors., Conclusion: Preceptors may need to be evaluated in their separate roles as teacher-coach, role model, and learning facilitator. The developed measures in this report could be used in site quality evaluation.

Background: The pathological hallmarks of Parkinson's disease are intracellular inclusions composed mainly of misfolded α-synuclein (αSYN). Under physiological conditions αSYN is mostly localized in synapses. In addition, a portion of αSYN is secreted to the extracellular space, where it may be sequestered by neighboring cells and could induce inflammatory responses. The mechanisms of αSYN internalization and signal transduction are not unequivocally clarified. In this work we investigated in primary mouse astrocytes the involvement of toll-like receptor 4 (TLR4) in the induction of inflammatory responses upon exposure to purified human αSYN produced in bacteria., Results: The mRNA induction of pro-inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase-2 was significantly reduced in TLR4 knockout astrocytes. The αSYN-mediated activation of c-Jun N-terminal kinases and p38 mitogen-activated protein kinase tended to be diminished, and nuclear translocation of the p65 subunit of nuclear factor κB was abolished in TLR4 knockout astrocytes. In contrast, the uptake of exogenous αSYN was unaffected by TLR4 knockout., Conclusions: Extracellular αSYN can activate pro-inflammatory TLR4 pathways in astrocytes, whereas αSYN uptake is independent of TLR4.

Background: Despite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen., Results: In the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides., Conclusions: These data indicate that Paracoccidioides is a pathogen that has several plasminogen-binding proteins that likely play important roles in pathogen-host interaction. In this context, FBA is a protein that might be involved somehow in the processes of invasion and spread of the fungus during infection.

Abs are able to mediate local protection from pulmonary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. L. pneumophila is able to infect alveolar macrophages in the lung and replicates intracellularly in a vacuolar compartment with endoplasmic reticulum-like characteristics. However, Abs opsonize the bacteria and confer an FcR-mediated signal to phagocytic host cells that vetoes the bacterial evasion strategies, thereby efficiently targeting the bacteria to intracellular lysosomal degradation. In this study we analyzed the prevalence of pathogen-specific IgG subclasses present in immunized mice and found that the presence of IgG2c and IgG3 correlated with reduced bacterial titers after intranasal infection. We then isolated different IgG subclasses and compared their differential prophylactic potential in restricting airway L. pneumophila replication. We found that all IgG subclasses were effective in restricting pulmonary airway infection in mice when administered at high and equivalent doses. However, at limiting Ab concentrations we found a superior role of IgG2c in restricting L. pneumophila replication in a prophylactic setting. Furthermore, we assessed the therapeutic efficacy of administering an mAb during an established infection and found that bacterial titers could be reduced very efficiently with such a treatment. Thus, we propose the therapeutic use of Abs for the treatment of intracellular bacterial infections in situations where antibiotics might be ineffective., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Objective: To determine how colleges and schools of pharmacy interpreted the Accreditation Council for Pharmacy Education's (ACPE's) Standards 2007 definitions for core advanced pharmacy practice experiences (APPEs), and how they differentiated community and institutional practice activities for introductory pharmacy practice experiences (IPPEs) and APPEs., Methods: A cross-sectional, qualitative, thematic analysis was done of survey data obtained from experiential education directors in US colleges and schools of pharmacy. Open-ended responses to invited descriptions of the 4 core APPEs were analyzed using grounded theory to determine common themes. Type of college or school of pharmacy (private vs public) and size of program were compared., Results: Seventy-one schools (72%) with active APPE programs at the time of the survey responded. Lack of strong frequent themes describing specific activities for the acute care/general medicine core APPE indicated that most respondents agreed on the setting (hospital or inpatient) but the student experience remained highly variable. Themes were relatively consistent between public and private institutions, but there were differences across programs of varying size., Conclusion: Inconsistencies existed in how colleges and schools of pharmacy defined the core APPEs as required by ACPE. More specific descriptions of core APPEs would help to standardize the core practice experiences across institutions and provide an opportunity for quality benchmarking.

A newly-approved carotid patch, derived from porcine small intestinal submucosa (SIS), is thought to allow functional tissue regeneration by acting as a biologic scaffold of extracellular matrix. We report three cases of asymptomatic pseudoaneurysm after SIS patch closure. At exploration there were intact suture lines, no growth from cultures, and central patch herniation. Histopathologic examination showed postendarterectomy neointima in the artery and disorganized collagen in the pseudoaneurysm. SIS patch remnants adjacent to macrophage infiltration and neovascularization indicated ongoing processes of degradation and synthesis. Imbalances between degradation and host tissue synthesis are problems that may unpredictably affect SIS patch integrity., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

DJ-1 is a ubiquitous protein regulating cellular viability. Recessive mutations in the PARK7/DJ-1 gene are linked to Parkinson's disease (PD). Although the most dramatic L166P point mutation practically eliminates DJ-1 protein and function, the effects of other PD-linked mutations are subtler. Here, we investigated two recently described PD-associated DJ-1 point mutations, the A179T substitution and the P158Δ in-frame deletion. [A179T]DJ-1 protein was as stable as wild-type [wt]DJ-1, but the P158Δ mutant protein was less stable. In accord with the notion that dimer formation is essential for DJ-1 protein stability, [P158Δ]DJ-1 was impaired in dimer formation. Similar to our previous findings for [M26I]DJ-1, [P158Δ]DJ-1 bound aberrantly to apoptosis signal-regulating kinase 1. Thus, the PD-associated P158Δ mutation destabilizes DJ-1 protein and function. As there is also evidence for an involvement of DJ-1 in multiple system atrophy, a PD-related α-synucleinopathy characterized by oligodendroglial cytoplasmic inclusions, we studied an oligodendroglial cell line stably expressing α-synuclein. α-Synuclein aggregate dependent microtubule retraction upon co-transfection with tubulin polymerization-promoting protein p25α was ameliorated by [wt]DJ-1. In contrast, DJ-1 mutants including P158Δ failed to protect in this system, where we found evidence of apoptosis signal-regulating kinase 1 (ASK1) involvement. In conclusion, the P158Δ point mutation may contribute to neurodegeneration by protein destabilization and hence loss of DJ-1 function., (© 2012 International Society for Neurochemistry.)

Legionella pneumophila is the causative agent of the potentially fatal Legionnaires' disease in humans. Mice have proved to be valuable model organisms to study the pathogenesis of this intracellular bacterium, as well as immune responses against it. In this chapter we describe a selection of mouse infection protocols to study the innate and adaptive immune responses raised after an infection with Legionella. Included are protocols for systemic and pulmonary infections, surgical collection of organs as well as determination of cell composition, cytokines, and antibody titers therein. Furthermore, we describe an immunohistology protocol to analyze lung tissue sections by fluorescence microscopy.

Upon uptake into a host cell, the intracellular bacterium Legionella pneumophila is not degraded on the lysosomal pathway but efficiently establishes a highly specialized replicative vacuole in which it readily multiplies. As many Icm/Dot type 4 secretion translocated bacterial effectors contribute to the establishment of this subcellular compartment in close interaction with host cell trafficking pathways, the analysis of the intracellular localization of this bacterium during infection is of pivotal importance to dissect the cellular and bacterial components of this process. In this chapter we describe a protocol for immunofluorescence microscopy in fixed mammalian and amoebal cells as well as transfection protocols to produce host cells expressing fluorescently labeled proteins as intracellular trafficking markers.

The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires' disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5'-GA-3' and 5'-UG-3' repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.

Industrial production of β-lactam antibiotics by the filamentous fungus Penicillium chrysogenum is based on successive classical strain improvement cycles. This review summarizes our current knowledge on the results of this classical strain improvement process, and discusses avenues to improve β-lactam biosynthesis and to exploit P. chrysogenum as an industrial host for the production of other antibiotics and peptide products. Genomic and transcriptional analysis of strain lineages has led to the identification of several important alterations in high-yielding strains, including the amplification of the penicillin biosynthetic gene cluster, elevated transcription of genes involved in biosynthesis of penicillin and amino acid precursors, and genes encoding microbody proliferation factors. In recent years, successful metabolic engineering and synthetic biology approaches have resulted in the redirection of the penicillin pathway towards the production of cephalosporins. This sets a new direction in industrial antibiotics productions towards more sustainable methods for the fermentative production of unnatural antibiotics and related compounds., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chrysogenum. It includes construction of expression plasmids, their transformation into fungal strains, cultivation of transformants, microscopy analysis, as well as additional protocols on staining of fungal cells with organelle-specific dyes like Hoechst 33342, MitoTracker DeepRed, and FM4-64.

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.

Background: Trans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is causally related to the neurodegenerative diseases frontotemporal dementia and amyotrophic lateral sclerosis being the hallmark protein in the disease-characteristic neuropathological lesions and via genetic linkage. Histone deacetylase 6 (HDAC6) is an established target of the RNA-binding protein TDP-43. HDAC6 is an unusual cytosolic deacetylase enzyme, central for a variety of pivotal cellular functions including aggregating protein turnover, microtubular dynamics and filopodia formation. All these functions are important in the context of neurodegenerative proteinopathies involving TDP-43. We have previously shown in a human embryonic kidney cell line that TDP-43 knockdown significantly impairs the removal of a toxic, aggregating polyQ ataxin-3 fusion protein in an HDAC6-dependent manner. Here we investigated the influence of TDP-43 and its target HDAC6 on neurite outgrowth., Results: Human neuroblastoma SH-SY5Y cells with stably silenced TDP-43 showed a significant reduction of neurite outgrowth induced by retinoic acid and brain-derived neurotrophic factor. Re-transfection with TDP-43 as well as HDAC6 rescued retinoic acid-induced neurite outgrowth. In addition, we show that silencing of HDAC6 alone is sufficient to reduce neurite outgrowth of in vitro differentiated SH-SY5Y cells., Conclusions: TDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases.

Background: Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species., Results: Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest--glnK and glnB, encoding PII regulatory proteins--were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium., Conclusion: In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

Intracellular pathogen-specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of target cells either via neutralization of the critical surface epitopes required for host cell entry, complement-mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)-bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen-specific T-cell responses., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)