A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Nannizzia species causing dermatophytosis in cats and dogs: First report of Nannizzia incurvata as an etiological agent in Brazil.

Dermatophytosis is a superficial cutaneous infection, most commonly caused by fungal species such as Microsporum canis, Nannizzia gypsea (Microsporum gypseum), and Trichophyton mentagrophytes in dogs and cats. The zoonotic potential of these species is concerning, as companion animals are increasingly close to their owners. Therefore, the objectives of the study were to evaluate the current prevalence of Nannizzia-causing canine and feline dermatophytosis in Curitiba and Metropolitan Region, as well as perform phenotypic and phylogenetic characterizations of these isolates. Thus, 241 skin and fur samples from 163 dogs and 78 cats were analyzed from 2020 to 2021. The samples were obtained from animals of three sources: Veterinary Hospital of the Federal University of Paraná, animal shelters, and private clinics. The diagnosis was performed through phenotypic characterization and sequencing ITS rDNA region. Among 97 positive samples for dermatophytes, Nannizzia was identified in 14 (14.4%) samples, while other dermatophyte genera were found in the remaining 83 (85.6%) samples. Among the canine samples, nine (90%) were N. gypsea, and one (10%) was N. incurvata. Whereas in feline samples, three (75%) were N. gypsea, and one (25%) was N. incurvata. It was concluded that among 97 animals infected with dermatophytes, dogs (24.4%; 10/41) were significantly more affected by Nannizzia than cats (7.1%; 4/56) (P < .05). According to molecular analyses, the ITS rDNA region provided satisfactory results for species-level identification of Nannizzia, confirming the first report of N. incurvata as an etiological agent of canine and feline dermatophytosis in Brazil.

Nannizzia genus affected significantly more dogs (24.4%) than cats (7.1%) (P < .05). The ITS rDNA exhibited higher accuracy for identifying dermatophytes compared to phenotypic diagnosis, allowing the confirmation of the first reports of N. incurvata as an etiological agent of dermatophytosis in dogs and cats in Brazil.

Sugarcane: an unexpected habitat for black yeasts in Chaetothyriales.

Sugarcane (Saccharum officinarum, Poaceae) is cultivated on a large scale in (sub)tropical regions such as Brazil and has considerable economic value for sugar and biofuel production. The plant is a rich substrate for endo- and epiphytic fungi. Black yeasts in the family Herpotrichiellaceae (Chaetothyriales) are colonizers of human-dominated habitats, particularly those rich in toxins and hydrocarbon pollutants, and may cause severe infections in susceptible human hosts. The present study assessed the diversity of Herpotrichiellaceae associated with sugarcane, using in silico identification and selective isolation. Using metagenomics, we identified 5833 fungal sequences, while 639 black yeast-like isolates were recovered in vitro. In both strategies, the latter fungi were identified as members of the genera Cladophialophora, Exophiala, and Rhinocladiella (Herpotrichiellaceae), Cyphellophora (Cyphellophoraceae), and Knufia (Trichomeriaceae). In addition, we discovered new species of Cladophialophora and Exophiala from sugarcane and its rhizosphere. The first environmental isolation of Cladophialophora bantiana is particularly noteworthy, because this species up to now is exclusively known from the human host where it mostly causes fatal brain disease in otherwise healthy patients.

Plasma Thermogram Parameters Differentiate Status and Overall Survival of Melanoma Patients.

Melanoma is the fifth most common cancer in the United States and the deadliest of all skin cancers. Even with recent advancements in treatment, there is still a 13% two-year recurrence rate, with approximately 30% of recurrences being distant metastases. Identifying patients at high risk for recurrence or advanced disease is critical for optimal clinical decision-making. Currently, there is substantial variability in the selection of screening tests and imaging, with most modalities characterized by relatively low accuracy. In the current study, we built upon a preliminary examination of differential scanning calorimetry (DSC) in the melanoma setting to examine its utility for diagnostic and prognostic assessment. Using regression analysis, we found that selected DSC profile (thermogram) parameters were useful for differentiation between melanoma patients and healthy controls, with more complex models distinguishing melanoma patients with no evidence of disease from patients with active disease. Thermogram features contributing to the third principal component (PC3) were useful for differentiation between controls and melanoma patients, and Cox proportional hazards regression analysis indicated that PC3 was useful for predicting the overall survival of active melanoma patients. With the further development and optimization of the classification method, DSC could complement current diagnostic strategies to improve screening, diagnosis, and prognosis of melanoma patients.

Sample Processing Considerations for Protein Stability Studies of Low Concentration Biofluid Samples using Differential Scanning Calorimetry.

BACKGROUND: The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To circumvent this problem, sample processing methods are employed to increase the protein concentration in analyzed samples. However, for some techniques, like differential scanning calorimetry (DSC) that characterizes thermally-induced unfolding of biomolecules, sample processing must not affect native protein structure and stability. METHODS: We evaluated centrifugal concentration and stirred cell ultrafiltration, two common methods of sample concentration characterized by a low risk of protein denaturation, with the goal of establishing a protocol for DSC analysis of low concentration biospecimens. RESULTS: Our studies indicate that both methods can affect protein stability assessed by DSC and, even after optimization of several parameters, the obtained DSC profile (thermogram) suggested that sample processing affects the structure or intermolecular interactions of component proteins contributing to altered thermal stability detectable by DSC. We also found a relationship between changes in thermograms and low protein concentration, indicating that diluting biospecimens to concentrations below 0.1 mg/mL can perturb the intermolecular environment and affect the structure of proteins present in the solution. CONCLUSION: Dilution of samples below 0.1 mg/mL, as well as concentration of samples with low protein content, resulted in affected thermogram shapes suggesting changes in protein stability. This should be taken into account when concentrating dilute samples or employing techniques that lower the protein concentration (e.g., fractionation), when downstream applications include techniques, such as DSC, that require the preservation of native protein forms.

Colostrum-Induced Temporary Changes in the Expression of Proteins Regulating the Epithelial Barrier Function in the Intestine.

The intestinal wall and epithelial cells are interconnected by numerous intercellular junctions. Colostrum (Col), in its natural form, is a secretion of the mammary gland of mammals at the end of pregnancy and up to 72 h after birth. Recently, it has been used as a biologically active dietary supplement with a high content of lactoferrin (Lf). Lf, a glycoprotein with a broad spectrum of activity, is becoming more popular in health-promoting supplements. This study aims to investigate whether Col supplementation can affect small and large intestine morphology by modulating the expression of selected proteins involved in tissue integrity. We examined the thickness of the epithelium, and the length of the microvilli, and assessed the expression of CDH1, CDH2, CTNNB, CX43, VCL, OCLN, HP, MYH9, and ACTG2 gene levels using qRT-PCR and at the protein level using IHC. Additionally, to evaluate whether the effect of Col supplementation is temporary or persistent, we performed all analyses on tissues collected from animals receiving Col for 1, 3, or 6 months. We noticed a decrease in CDH1 and CDH2 expression, especially after 3 months of supplementation in the large intestine and in CTNNB in the small intestine as well as increased levels of CX43 and CTNNB1 in the small intestine. The present data indicate that Col can temporarily alter some components of the cell adhesion molecules involved in the formation of the cellular barrier.

The Utility of Differential Scanning Calorimetry Curves of Blood Plasma for Diagnosis, Subtype Differentiation and Predicted Survival in Lung Cancer.

Early detection of lung cancer (LC) significantly increases the likelihood of successful treatment and improves LC survival rates. Currently, screening (mainly low-dose CT scans) is recommended for individuals at high risk. However, the recent increase in the number of LC cases unrelated to the well-known risk factors, and the high false-positive rate of low-dose CT, indicate a need to develop new, non-invasive methods for LC detection. Therefore, we evaluated the use of differential scanning calorimetry (DSC) for LC patients' diagnosis and predicted survival. Additionally, by applying mass spectrometry, we investigated whether changes in O- and N-glycosylation of plasma proteins could be an underlying mechanism responsible for observed differences in DSC curves of LC and control subjects. Our results indicate selected DSC curve features could be useful for differentiation of LC patients from controls with some capable of distinction between subtypes and stages of LC. DSC curve features also correlate with LC patients' overall/progression free survival. Moreover, the development of classification models combining patients' DSC curves with selected plasma protein glycosylation levels that changed in the presence of LC could improve the sensitivity and specificity of the detection of LC. With further optimization and development of the classification method, DSC could provide an accurate, non-invasive, radiation-free strategy for LC screening and diagnosis.

Molecular Identification and Antimicrobial Activity of Foliar Endophytic Fungi on the Brazilian Pepper Tree (Schinus terebinthifolius) Reveal New Species of Diaporthe.

The presence of endophytes promotes the biosynthesis of secondary plant metabolites. In this study, endophytic fungi were isolated from Schinus terebinthifolius to investigate their diversity and antimicrobial activity. A total of 272 endophytic fungi was obtained. These belonged to nine different genera: Alternaria, Colletotrichum, Diaporthe, Epicoccum, Fusarium, Pestalotiopsis, Phyllosticta, Xylaria, and Cryptococcus. Notably, Diaporthe foliorum was introduced as a new species, with accompanying morphological descriptions, illustrations, and a multigene phylogenetic analysis (using ITS, TEF1, TUB, HIS, and CAL). Among the 26 fungal morphotypes evaluated for antimicrobial activity, five strains had inhibitory effects against pathogenic microorganisms. Xylaria allantoidea CMRP1424 extracts showed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Diaporthe terebinthifolii CMRP1430 and CMRP1436 showed antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Meanwhile, D. foliorum CMRP1321 and D. malorum CMRP1438 extracts inhibited C. albicans alone. Three classes of chemical compounds were identified in D. foliorum CMRP1438 extracts: ferric chloride, potassium hydroxide, and vanillin-sulfuric acid. In conclusion, the endophytic isolates were able to produce bioactive agents with pharmaceutical potential as antibacterial and antifungal agents. As such, they may provide fresh leads in the search for new, biological sources of drug therapies.

Genomics and Virulence of Fonsecaea pugnacius, Agent of Disseminated Chromoblastomycosis.

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.

Environmental prospecting of black yeast-like agents of human disease using culture-independent methodology.

Melanized fungi and black yeasts in the family Herpotrichiellaceae (order Chaetothyriales) are important agents of human and animal infectious diseases such as chromoblastomycosis and phaeohyphomycosis. The oligotrophic nature of these fungi enables them to survive in adverse environments where common saprobes are absent. Due to their slow growth, they lose competition with common saprobes, and therefore isolation studies yielded low frequencies of clinically relevant species in environmental habitats from which humans are thought to be infected. This problem can be solved with metagenomic techniques which allow recognition of microorganisms independent from culture. The present study aimed to identify species of the family Herpotrichiellaceae that are known to occur in Brazil by the use of molecular markers to screen public environmental metagenomic datasets from Brazil available in the Sequence Read Archive (SRA). Species characterization was performed with the BLAST comparison of previously described barcodes and padlock probe sequences. A total of 18,329 sequences was collected comprising the genera Cladophialophora, Exophiala, Fonsecaea, Rhinocladiella and Veronaea, with a focus on species related to the chromoblastomycosis. The data obtained in this study demonstrated presence of these opportunists in the investigated datasets. The used techniques contribute to our understanding of environmental occurrence and epidemiology of black fungi.

Selective isolation of agents of chromoblastomycosis from insect-associated environmental sources.

Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation.

S1P Signaling in the Tumor Microenvironment.

Sphingosine-1-phosphate (S1P), together with other phosphosphingolipids, has been found to regulate complex cellular function in the tumor microenvironment (TME) where it acts as a signaling molecule that participates in cell-cell communication. S1P, through intracellular and extracellular signaling, was found to promote tumor growth, angiogenesis, chemoresistance, and metastasis; it also regulates anticancer immune response, modulates inflammation, and promotes angiogenesis. Interestingly, cancer cells are capable of releasing S1P and thus modifying the behavior of the TME components in a way that contributes to tumor growth and progression. Therefore, S1P is considered an important therapeutic target, and several anticancer therapies targeting S1P signaling are being developed and tested in clinics.

Primary Central Nervous System Infection by Histoplasma in an Immunocompetent Adult.

Central nervous system (CNS) infection by Histoplasma capsulatum is a rare disease in immunocompromised individuals in endemic areas. About one quarter of cases result from hematogenous dissemination. A 23-year-old upholsterer with chronic occipital headache had developed intracranial hypertension and dizziness, incoordination with ataxic gait, and acute confusion 5 months prior to admission. Laboratory examinations and chest roentgenogram were normal. Postcontrast T1-weighted MRI of the brain revealed a multiple ring-enhancing cerebellar, brain stem and parietal lobe lesions, and meningeal contrast enhancement. Cerebrospinal fluid culture was positive for H. capsulatum species complex, which was confirmed by phylogenetic analysis. Thirteen years after the diagnosis and treatment, there was no H. capsulatum recurrence; sequels related to complications due to the ventriculoperitoneal shunt. This case shows a primary neurological presentation of cerebral histoplasmosis, without meningitis or disseminated disease in nonimmune-compromised patient. The authors propose a categorization of the diagnosis of CNS histoplasmosis. Routine diagnostics of sibling species within the H. capsulatum complex proved to be difficult.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis.

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.

Increased methylation upstream of the MEG3 promotor is observed in acute myeloid leukemia patients with better overall survival.

BACKGROUND: The delta-like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3(MEG3) locus (DLK1-MEG3 locus) plays a critical role in the maintenance and differentiation of hematopoietic stem cells. Accumulating evidence implicates the imprinted genes from this locus, DLK1 and MEG3, in the development and progression of acute myeloid leukemia (AML). However, the contribution of this locus to the treatment response of patients and their survival is unknown. METHODS: DNA methylation of select CG dinucleotide-containing amplicons (CpG sites) within the DLK1-MEG3 locus and within differentially methylated regions of other imprinted loci was assessed in the mononuclear cells of 45 AML patients by combined bisulfite restriction analysis. Methylation results were compared with patient response to first-round induction therapy and overall survival. Multivariable analysis was employed to identify independent prognostic factors for patient overall survival in AML. RESULTS: Increased methylation at CpG sites within the MEG3 promotor region was observed in AML patients having longer overall survival. In addition, patients with shorter overall survival had increased expression of DLK1 and MEG3, and methylation at the MEG3-DMR CpG site inversely correlated with MEG3 expression. Multivariable analysis revealed that methylation at CG9, a non-imprinted CpG site within the MEG3 promotor region which contains a CCCTC-binding factor (CTCF)-binding DNA sequence, is an independent prognostic factor for the overall survival of AML patients. CONCLUSIONS: The results of our pilot study underscore the importance of the DLK1-MEG3 locus in AML development and progression. We identify CG9 methylation as an independent prognostic factor for AML patient survival, which suggests that distinct miRNA signatures from the DLK1-MEG3 locus could reflect varying degrees of cell stemness and present novel opportunities for personalized therapies in the future. These data provide a foundation for future studies into the role of higher-order chromatin structure at DLK1-MEG3 in AML.

[Corrigendum] Pituitary sex hormones enhance the pro­metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase­1.

After the publication of the article, the authors realize that they overlooked stating that the author Magda Kucia was the recipient of an OPUS grant (grant no. UMO­2016/21/B/NZ4/00201). Therefore, the Acknowledgements section of the paper should have read as follows (the added text is highlighted in bold): "This study was supported by NIH grants 2R01 DK074720 and R01HL112788, the Stella and Henry Endowment, and NCN Harmonia grant UMO­2014/14/M/NZ3/00475 to M.Z.R., and OPUS grant UMO­2016/21/B/NZ4/00201 to M.K." The authors regret their oversight in failing to include this information in the Acknowledgements section of their paper. [the original article was published in International Journal of Oncology 50: 317-328, 2017; DOI: 10.3892/ijo.2016.3787].

Analysis of the Paternally-Imprinted DLK1-MEG3 and IGF2-H19 Tandem Gene Loci in NT2 Embryonal Carcinoma Cells Identifies DLK1 as a Potential Therapeutic Target.

The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Estressores ocupacionais e estratégias de enfrentamento / Occupational stressors and coping strategies / Factores de estrés ocupacionales y estrategias de enfrentamiento / Stresseurs professionnels et les stratégies de coping

O presente estudo teve como objetivo conhecer a vivência dos estressores ocupacionais e estratégias de enfrentamento utilizadas na perspectiva dos professores. Participaram do estudo oito professoras provenientes de duas escolas públicas municipais de uma cidade da região metropolitana de Porto Alegre, RS. Como instrumento de coleta de dados foi utilizado o método do diário em sua modalidade qualitativa, estruturado em dois eixos temáticos estabelecidos a priori: estressores e estratégias de enfrentamento utilizadas. Os registros foram analisados por meio da técnica de análise de conteúdo categorial-temática. Como resultados, foram identificados cinco estressores de natureza psicossocial: relação professor-alunos; falta de apoio de familiares de alunos; sobrecarga de papel; conciliar trabalho-família e conciliar trabalho-vida pessoal. As estratégias de enfrentamento referidas foram as com foco na emoção e evitação.

The present study had as objective to know the experience of the occupational stressors and coping strategies used from the perspective of the teachers. Eight female teachers from two municipal public schools of a city in the metropolitan region of Porto Alegre, RS, participated in the study. As a data collection instrument, the journal method was used in its qualitative modality, structured in two thematic axes established a priori: stressors and coping strategies used. The records were analyzed using the categorical-thematic content analysis technique. As results, five stressors of a psychosocial nature were identified: teacher-student relationship; lack of support from student relatives; paper overload; reconciling work-family and reconciling work-life. The coping strategies referred to were those focused on emotion and avoidance.

Este estudio tuvo como objetivo conocer a la vivencia de los factores de estrés ocupacionales y estrategias de enfrentamiento utilizadas en la perspectiva de los profesores. Participaron del estudio ocho profesoras de escuelas públicas municipales de una ciudad de la región central de Porto Alegre, RS- Brasil. Como instrumento de colecta de datos fue utilizado el método del diario en su modalidad cualitativa, estructurado en dos ejes temáticos establecidos a priori: factores de estrés y estrategias de enfrentamiento utilizadas. Los registros fueron analizados por medio de la técnica de análisis de contenido categorial-temática. Como resultado, fueron identificados cinco factores de estrés de naturaleza psicosocial: relación profesor-alumno; falta de apoyo de familiares de alumnos; sobrecarga de papel; conciliar trabajo-familia y trabajo-vida personal. Las estrategias de enfrentamiento referidas fueron las con enfoque en la emoción y prevención.

La présente étude a eu l'objectif de connaître l'expérience des stresseurs professionnels et aussi des stratégies de coping utilisées sur le point de vue des enseignants. Huit enseignantes, de deux écoles publiques municipales d'une ville de la région métropolitaine de Porto Alegre, RS, ont participé à l'étude. Le journal intime - dans la modalité qualitative - a été utilisé comme outil pour collecter les donnés. Ils ont été structurés sur deux axes thématiques prévues à priori: des stresseurs et des stratégies de coping utilisées. Les enregistrements ont été analysés à l'aide de la technique d'analyse de contenu catégorielle thématique. On a pu identifier cinq stresseurs de nature psychosocial : de la relation enseignant-élèves; de l'absence de soutien par les familles des élèves; surcharge de rôles; difficulté de conciliation entre le travail et la famille et entre le travail et la vie personnelle. Les stratégies de coping mentionnées étaient celles axées sur l'émotion et l'évitement.

Novel evidence that pituitary sex hormones regulate migration, adhesion, and proliferation of embryonic stem cells and teratocarcinoma cells.

The pituitary sex hormones (SexHs): follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) regulate several functions crucial for reproduction, including oogenesis, spermatogenesis, and lactation. An important source of prolactin-like hormones, known as lactogens, is the placenta, and lactogens bind to the PRL receptor (PRLR) with high affinity and thereby mimic the actions of PRL. Recently, it has been demonstrated that pituitary SexHs were involved in metastatic lung cancer, certain sarcomas, and leukemia. In the present study we aimed to investigate whether FSH, LH, and PRL were able to stimulate stem cells involved in early development. To address this issue we employed a murine embryonic stem cell line (ES-D3) as well as two teratocarcinoma cell lines, P19 (murine) and NTera2 (human). We determined that all these cells expressed SexH receptors at the mRNA and protein levels and that stimulation of these receptors induced phosphorylation of p42/44 MAPK, p38 MAPK, and AKT. Moreover, ES-D3, P19, and NTera2 cells responded with increased migration and adhesion to physiological concentrations of pituitary SexHs. In view of these findings we proposed that maternal-derived pituitary SexHs regulate the biology of stem cells involved in early development.