Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Systems biology meets stress ecology: linking molecular and organismal stress responses in Daphnia magna

Background: Ibuprofen and other nonsteroidal anti-inflammatory drugs have been designed to interrupt eicosanoid metabolism in mammals, but little is known of how they affect nontarget organisms. Here we report a systems biology study that simultaneously describes the transcriptomic and phenotypic stress responses of the model crustacean Daphnia magna after exposure to ibuprofen. Results: Our findings reveal intriguing similarities in the mode of action of ibuprofen between vertebrates and invertebrates, and they suggest that ibuprofen has a targeted impact on reproduction at the molecular, organismal, and population level in daphnids. Microarray expression and temporal real-time quantitative PCR profiles of key genes suggest early ibuprofen interruption of crustacean eicosanoid metabolism, which appears to disrupt signal transduction affecting juvenile hormone metabolism and oogenesis. Conclusion: Combining molecular and organismal stress responses provides a guide to possible chronic consequences of environmental stress for population health. This could improve current environmental risk assessment by providing an early indication of the need for higher tier testing. Our study demonstrates the advantages of a systems approach to stress ecology, in which Daphnia will probably play a major role.

Morpho-physiological characterization of spring wheat genotypes under drought stress

Water-deficit is a severe abiotic stress and major constraint to wheat productivity with effect on plant growth and development. The objective of this study was to characterize drought tolerant and susceptible spring wheat cultivars on the basis of physiological and yield attributes. The experiment was comprised of two irrigation regimes i.e. irrigated and 65% drought stress and ten wheat cultivars viz. Anmol, Moomal, Sarsabz, Bhittai, Pavon, SKD-1, TD-1, Kiran, Marvi and Mehran. Results indicated significant effect of water stress on stomatal dimension, stomatal conductance, relative leaf water content and grain yield with no effect on stomatal density. The irrigation × cultivars interaction was non-significant for grain yield only. Cultivars like Anmol, Moomal, Bhittai, Sarsabz proved to be drought tolerant with smaller stomatal dimensions, less stomatal conductance and more relative water content under water stress and produced higher grain yield. While decrease in relative water contents and grain yield, and increase in stomatal attributes was observed in drought susceptible cultivars such as Marvi, TD-1 and SKD-1 hence proved to be drought susceptible.

Awareness of number in children with severe and profound learning difficulties: three exploratory case studies

This paper reports on exploratory work investigating how children with severe and profound learning difficulties register an awareness of small quantities and how they might use this information to inform their understanding. It draws on studies of typically developing children and investigates their application to pupils whose response to conventional mathematical tasks are often limited because they lack relevance and interest. The responses of the three pupils to individualized learning contexts mirror the progression suggested in the literature, namely from awareness of number to simple actions using number cues to problem-solving behaviour

Evaluation of tangerine hybrid resistance to Alternaria alternata

The Alternaria Brown Spot, caused by Alternaria alternata, is a major fungal disease in some kinds of tangerines, tangor, mandarins and pomelos. In Brazil as well as worldwide, A. alternata can cause necrosis in fruits, branches and leaves, causing substantial profit loss. In the present research, in laboratory conditions and in the field, we evaluated the resistance to the fungus, in leaves and fruits, for 22 varieties and hybrids of tangerines. To this end, we evaluated genotypes belonging to the Germplasm Bank of the Estacao Experimental de Citricultura de Bebedouro. The resistant genetic materials (found in leaves and fruits) represented four varieties of clementines (Citrus clementina); six varieties of mandarins (two belonging to C. reticulata, two to C. tangerina, one to C. deliciosa and one to C. nobilis); one tangelo (C. tangerina x C. paradisi); two mandarin hybrids (one resulting from crossing C nobilis x C. deliciosa and the other from crossing C. clementina x C. reticulata); one tangor hybrid (C. clementina) and two satsuma hybrids (C. unshiu x C. deliciosa). We also determined a relation between the inoculation of leaves and fruits. The resistance and susceptibility following inoculation in leaves and fruits supports a relationship between these organs and the physiological responses observed for the evaluated genotypes. (C) 2009 Elsevier B.V. All rights reserved.

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of the administration of a catalase inhibitor into the fourth cerebral ventricle on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke

OBJECTIVE: ,,,,,Previous studies have demonstrated a relationship between brain oxidative stress and cardiovascular regulation. We evaluated the effects of central catalase inhibition on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke. ,,,, ,,,, ,,,,,METHODS: ,,,,,Male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SH) (16 weeks old) were implanted with a stainless steel guide cannula leading into the fourth cerebral ventricle (4th V). The femoral artery and vein were cannulated for arterial pressure and heart rate measurement and drug infusion, respectively. The rats were exposed to sidestream cigarette smoke for 180 minutes/day, 5 days/week for 3 weeks (CO: 100-300 ppm). The baroreflex was tested using a pressor dose of phenylephrine (8 μg/kg, bolus) and a depressor dose of sodium nitroprusside (50 μg/kg, bolus). Cardiovascular responses were evaluated before and 5, 15, 30 and 60 minutes after injection of a catalase inhibitor (3-amino-1,2,4-triazole, 0.001 g/100 μL) into the 4th V. ,,,, ,,,, ,,,,,RESULTS: ,,,,,Vehicle administration into the 4th V did not affect the cardiovascular response, whereas administration of the central catalase inhibitor increased the basal HR and attenuated the bradycardic peak (p<0.05) to a greater extent in WKY rats exposed to sidestream cigarette smoke than in WKY rats exposed to fresh air. However, in spontaneously hypertensive rats, the effect of the catalase inhibitor treatment was stronger in the fresh air condition (p<0.05). ,,,, ,,,, ,,,,,CONCLUSION: ,,,,,Administration of a catalase inhibitor into the 4th V combined with exposure to sidestream cigarette smoke has a stronger effect in WKY rats than in SH rats.

Molecular characterization of Cryptosporidium spp. in dairy calves from the state of So Paulo, Brazil

Cryptosporidiosis is a common protozoan disease observed in a wide range of vertebrate hosts, including ruminants. Cattle can be a potential reservoir of Cryptosporidium spp., leading to environmental contamination with oocysts of zoonotic species. The molecular characterization of Cryptosporidium spp. isolated from cattle from the state of So Paulo, Brazil, was accomplished using nested polymerase chain reaction for amplification of fragments of the 18S rRNA gene and the glycoprotein GP60 gene, following sequencing of amplified fragments. Positivity for Cryptosporidium was found in 10.7% (21/196) of the samples. Four species of Cryptosporidium were identified: C. andersoni, C. bovis, C. parvum subtype IIaA15G2R1, and C. ryanae. To the best of our knowledge, this is the first report of infection by C. ryanae and C. parvum IIaA15G2R1 in cattle from Brazil.

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Histopathology of the tegument of rabbits infested by Rhipicephalus sanguineus (ACARI: IXODIDAE) ticks and exposed to selamectin (active principle of acaricide Revolution®, Pfizer)

Ticks are hematophagous ectoparasites which can transmit several diseases to the host during their feeding process. When ticks mechanically damage the tissue, they eventually induce inflammatory responses on the skin spot where they are fixed. One of the alternatives to control these ectoparasites is the use of chemical substances like selamectin - the active principle of Pfizer's antiparasitic Revolution® - a macrocyclic lactone capable of doing neurotoxic damage to the tick and eventually eliminating infestation in dogs and cats. The purpose of this study was to analyze, using histological and histochemical techniques, the occurrence of morphophysiological alterations in the skin of the host rabbits exposed to selamectin and infested with Rhipicephalus sanguineus (Acari: Ixodidae). Histologically, the exposed and infested rabbits showed a partial and/or total decrease in the stratum corneum and the epithelium decreased in the number of cell layers, consequently reducing the stratification (thinning) and quite pronounced formations of sub-epidermal edemas with consequent disorganization of collagen fibers in the dermal layer's connective tissue. Histochemical tests showed strong periodic acid-Schiff-positive reaction in the hair follicle and some regions of the dermis, besides resynthesis of collagen fibers detected by Mallory's trichrome technique. The obtained results showed that selamectin acts like a toxicant agent when in contact with the skin of the rabbit infested with ticks, inducing morphophysiological alterations in the acute inflammatory process in the animal's tegument. Selamectin is a chemical substance which has a dose-dependent action since higher concentrations cause greater morphophysiological damage in the skin of rabbits. © 2013 Springer-Verlag Berlin Heidelberg.

A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B

Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2μg/ml for the 11 isolates studied. The same results were found by FC. The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-alpha, whereas S. flexneri induced only the production of TNF-alpha. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL) 20 and TNF-alpha. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Leptin as a link between the immune system and kidney-related diseases: leading actor or just a coadjuvant?

Food intake and nutritional status modify the physiological responses of the immune system to illness and infection and regulate the development of chronic inflammatory processes, such as kidney disease. Adipose tissue secretes immune-related proteins called adipokines that have pleiotropic effects on both the immune and neuroendocrine systems, linking metabolism and immune physiology. Leptin, an adipose tissue-derived adipokine, displays a variety of immune and physiological functions, and participates in several immune responses. Here, we review the current literature on the role of leptin in kidney diseases, linking adipose tissue and the immune system with kidney-related disorders. The modulation of this adipose hormone may have a major impact on the treatment of several immune- and metabolic-related kidney diseases.

In vivo and in vitro effects of antioxidant compounds and PPAR γ modulators on rainbow trout oxidative stress and lipid metabolism.

The aquafeed use of raw plant materials, as protein and lipid sources, has been considered and approved as a sustainable alternative to fish products (fish meal and oils) because the current trend to use high-lipid diets has been shown to induce undesirable increase in fat depots or further physiological alterations, such as induction of oxidative stress. In the aquaculture perspective, the addition of natural substances with antioxidant properties is an emerging strategy for protecting biological systems and foodstuffs from oxidative damage. Among natural substances, hydroxytyrosol (HT) and caffeic acid (CA) have attracted considerable attention as food antioxidant additives and modulators of physiological and molecular pathways involved in energy metabolism and adiposity. The aim of this study was to evaluate the effects of CA and HT on lipid metabolism and oxidative stress of rainbow trout (Oncorhynchus mykiss). In vitro results showed the potential anti-obesogenic effects of the compounds CA and HT on the adipose tissue of the rainbow trout. To support these data, in vitro assays performed (MTT, ORO, immunofluorescence) resulted in accordance among them; only results from proliferating cell nuclear antigen (PCNA) assay were not significant. In vivo results showed a possible anti-obesogenic effect of CA in liver and HT in adipose tissue. Regarding oxidative stress, we could hypothesize a possible anti-oxidant role of CA in liver.

The effect of African Americans' ethnographic paradigms and cultural explanatory model of breast cancer in framing the male partner's attitudes and behaviors about the woman's breast cancer diagnosis

The disparate burden of breast cancer-related morbidity and mortality experienced by African American women compared with women of other races is a topic of intense debate in the medical and public health arenas. The anomaly is consistently attributed to the fact that at diagnosis, a large proportion of African American women have advanced-stage disease. Extensive research has documented the impacts of cultural factors and of socioeconomic factors in shaping African American women's breast-health practices; however, there is another factor of a more subtle influence that might have some role in establishing these women's vulnerability to this disease: the lack of or perceived lack of partner support. Themes expressed in the research literature reflect that many African American breast cancer patients and survivors consider their male partners as being apathetic and nonsupportive. ^ The purpose of this study was to learn how African American couples' ethnographic paradigms and cultural explanatory model of breast cancer frame the male partners' responses to the women's diagnosis and to assess his ability to cope and willingness to adapt to the subsequent challenges. The goal of the study was to determine whether these men's coping and adaptation skills positively or negatively affect the women's self-care attitudes and behaviors. ^ This study involved 4 African American couples in which the woman was a breast cancer survivor. Participants were recruited through a community-based cancer support group and a church-based cancer support group. Recruitment sessions were held at regular meetings of these organizations. Accrual took 2 months. In separate sessions, each male partner and each survivor completed a demographic survey and a questionnaire and were interviewed. Additionally, the couples were asked to participate in a communications activity (Adinkra). This activity was not done to fulfill any part of the study purpose and was not included in the data analysis; rather, it was done to assess its potential use as an intervention to promote dialogue between African American partners about the experience of breast cancer. ^ The questionnaire was analyzed on the basis of a coding schema and the interview responses were analyzed on the principles of hermeneutic phenomenology. In both cases, the instruments were used to determine whether the partner's coping skills reflected a compassionate attitude (positive response) versus an apathetic attitude (negative response) and whether his adaptation skills reflected supportive behaviors (the positive response) versus nonsupportive behaviors (the negative response). Overall, the women's responses showed that they perceived of their partners as being compassionate, yet nonsupportive, and the partner's perceived of themselves likewise. Only half of the women said that their partners' coping and adaptation abilities enabled them to relinquish traditional concepts of control and focus on their own well-being. ^ The themes that emerged indicate that African American men's attitudes and behaviors regarding his female partner's diagnosis of breast cancer and his ability to cope and willingness to adapt are influenced by their ritualistic mantras, folk beliefs, religious teachings/spiritual values, existential ideologies, socioeconomic status, and environmental factors and by their established perceptions of what causes breast cancer, what the treatments and outcomes are, and how the disease affects the entire family, particularly him. These findings imply that a culturally specific intervention might be useful in educating African American men about breast cancer and their roles in supporting their female partners, physically and psychologically, during diagnosis, treatment, and recovery. ^