Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Correlation of retinal alterations with vascular structure of macular neovascularisation in swept-source optical coherence tomography angiography in age-related macular degeneration

Purpose The aim of this study was to find out whether the vascular architecture of untreated macular neovascularisations (MNV) in neovascular age-related macular degeneration (nAMD) as visualised with optic coherence tomography angiography (OCTA) is associated with functional and known morphological alterations of the retina in optic coherence tomography (SD-OCT). Methods The study design was retrospective with consecutive patient inclusion. In 107 patients with newly diagnosed nAMD, MNV were detected by means of OCTA and automated quantitative vascular analysis was performed. The MNV characteristics measured were area, flow density, total vascular length (sumL), density of vascular nodes (numN), fractal dimension (FD) and average vascular width (avgW). These parameters were assessed for associations with vision (BCVA), central retinal thickness (CRT), fluid distribution, the elevation of any pigment epithelial detachment (PED), the occurrence of subretinal haemorrhage and atrophy. Results BCVA was significantly worse with greater MNV area and sumL. Fluid distribution differed significantly in relation to area (p < 0.005), sumL (p < 0.005) and FD (p = 0.001). Greater PED height was significantly associated with higher numN (p < 0.05) and lower avgW (p < 0.05). Atrophy was present significantly more often in MNV with larger area (p < 0.05), higher sumL (p < 0.05) and higher flow density (p = 0.002). None of the MNV parameters had a significant association with CRT or the occurrence of haemorrhage. Conclusion OCTA is not restricted to evaluation of secondary changes but offers the opportunity to analyse the vascular structure of MNV in detail. Differences in vascular morphology are associated with certain secondary changes in retinal morphology. There are thus grounds for optimism that further research may identify and classify OCTA-based markers to permit more individualised treatment of nAMD.

Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.

Dysregulation of GABAergic Signaling in Neurodevelomental Disorders: Targeting Cation-Chloride Co-transporters to Re-establish a Proper E/I Balance

The construction of the brain relies on a series of well-defined genetically and experience- or activity -dependent mechanisms which allow to adapt to the external environment. Disruption of these processes leads to neurological and psychiatric disorders, which in many cases are manifest already early in postnatal life. GABA, the main inhibitory neurotransmitter in the adult brain is one of the major players in the early assembly and formation of neuronal circuits. In the prenatal and immediate postnatal period GABA, acting on GABAA receptors, depolarizes and excites targeted cells via an outwardly directed flux of chloride. In this way it activates NMDA receptors and voltage-dependent calcium channels contributing, through intracellular calcium rise, to shape neuronal activity and to establish, through the formation of new synapses and elimination of others, adult neuronal circuits. The direction of GABAA-mediated neurotransmission (depolarizing or hyperpolarizing) depends on the intracellular levels of chloride [Cl−]i, which in turn are maintained by the activity of the cation-chloride importer and exporter KCC2 and NKCC1, respectively. Thus, the premature hyperpolarizing action of GABA or its persistent depolarizing effect beyond the postnatal period, leads to behavioral deficits associated with morphological alterations and an excitatory (E)/inhibitory (I) imbalance in selective brain areas. The aim of this review is to summarize recent data concerning the functional role of GABAergic transmission in building up and refining neuronal circuits early in development and its dysfunction in neurodevelopmental disorders such as Autism Spectrum Disorders (ASDs), schizophrenia and epilepsy. In particular, we focus on novel information concerning the mechanisms by which alterations in cation-chloride co-transporters (CCC) generate behavioral and cognitive impairment in these diseases. We discuss also the possibility to re-establish a proper GABAA-mediated neurotransmission and excitatory (E)/inhibitory (I) balance within selective brain areas acting on CCC.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

Treatment with Candida albicans biotherapic influences in vitro fungal adhesion to Ma-104 cells

Background: Oral candidiasis is an opportunist fungal infection in humans, mainly caused by Candida albicans. It occurs when the host presents an imbalance in the immune system and Candida spp., normally found in human flora, become able to develop the infection [1]. This disease is very common in HIV patients, and in all individuals that present immunossupression, such as patients treated with chemotherapy. Considering this scenario, the development of new medicines to treat oral candidiasis is mandatory. Aims: The aim of this study was to evaluate citotoxicity, morphology and quantify the adhesion rates of C. albicans to biotherapic-treated Ma104 cells. Methodology: The biotherapic was prepared following the Roberto Costa technique and Brazilian Homeopathic Pharmacopeia protocol [2]. Briefly, biotherapic 1X was prepared with 1 mL of aqueous solution containing 108 yeasts of living Candida albicans plus 9 ml of sterile distilled water. This solution was submmited to 100 mechanical succussions. Biotherapic 2X was obtained after addition of 1 ml of 1X solution in 9 ml of sterile distilled water and it was also submitted to 100 mechanical succussions. This procedure was repeated until biotherapic 30X was obtained. As a control, sterile dynamized water (30X) was used. The inhibition of fungal growth induced by biotherapic was evaluated by MTT method after 24 hours of treatment. The morphological aspects of Ma104-biotherapic-treated cells were analyzed by Giemsa staining after 5, 10 and 60 days, and compared with control groups (water 30X and untreated cells). Additionally, Ma104 cells were treated during 5 and 30 days with biotherapic in parallel with respective controls, and the index adhesion of yeast cells was quantified. Results: The biotherapic was not able to reduce the viability of treated C. albicans when compared with controls. On the other hand, Ma104 treated cells presented important morphological alterations after 60 days, such as: cytoplasmic vacuoles, halos around the nucleolus and elongation of the plasmatic membrane. These changes were not observed in ,untreated cells nor in ones treated with water 30X. The adhesion index to Ma104 cells was reduced around 27% after 5 and 30 days of treatment when compared to controls. Conclusion: These results showed that the biotherapic did not present any citotoxicity, but was able to modify the morphological aspects of Ma-104 cells. Additionally, the interaction between host cells and ethilogic agent is directly influenced by biotherapic treatment, suggesting a promising antifungal potential of this medicine.

Mitochondria-mediated Maternal-fetal Interactions and Consequences of Mitochondrial Dysregulation Indicate New Roles for Mitochondria in Hypertensive Pregnancies

Oxidative stress, placental mitochondrial morphological alterations, and impaired bioenergetics are associated with hypertensive disorders of pregnancy. Here we examined mitochondrial DNA mutational load in pregnant women with pregnancy-induced hypertension and reanalyzed publicly available high-throughput transcriptomic datasets from maternal and fetal tissues from normotensive and hypertensive pregnancies. Mitochondrial dysregulation was indicated by aberrant mitochondrial gene expression, and putative consequences were examined. Women with hypertensive pregnancy had elevated mitochondrial DNA mutational load. Maternal mitochondrial dysregulation in hypertensive pregnancies was associated with pathways involved in inflammation, cell death/survival, and placental development. In fetal tissues from hypertensive pregnancies, mitochondrial dysregulation was associated with increased extracellular vesicle production. Our study demonstrates mitochondria-mediated maternal-fetal interactions during healthy pregnancy and maternal mitochondrial dysregulation in hypertensive pregnancy development.

β-carbolines RCC and C5 induce death of Leishmania amazonensis intracellular amastigotes

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The β-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the β-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-β-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of β-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

Mesopore silica effect on chemical, thermal and tribological properties of polyimide composites

In this experimental study, the effects of mesoporous silica filler content on the chemical, thermal and tribological properties of polyimide composites were investigated. For that purpose, Pi/mesoporous silica composites were produced by in situ polymerization with various mesoporous silicas. After fabrication, thermal stability and chemical characterization were determined using TGA and FTIR. Morphological alterations were monitored with a scanning electron microscope (SEM). Texture structure (pore size and pore volume) were determined by the BJH method. Friction and wear properties were investigated by using a pin-on-disc arrangement. At the end of the study, minor shifts of Pi/mesoporous silica composites were observed. Thermal stability, as well as pore size and pore volume, was decreased with mesoporous silica. The coefficient of friction and specific wear rate decreased with the addition of mesoporous silica. Abrasive wear behaviors were seen for both neat Pi and Pi–Si composites. Hence, this study evidenced that the properties of Pi are influenced by mesoporous dimensions and content of Si employed.

Immunocompetent Mice Infected by Two Lineages of Dengue Virus Type 2: Observations on the Pathology of the Lung, Heart and Skeletal Muscle

Dengue virus (DENV) infection by one of the four serotypes (DENV-1 to 4) may result in a wide spectrum of clinical manifestations, with unpredictable evolution and organ involvement. Due to its association with severe epidemics and clinical manifestations, DENV-2 has been substantially investigated. In fact, the first emergence of a new lineage of the DENV-2 Asian/American genotype in Brazil (Lineage II) in 2008 was associated with severe cases and increased mortality related to organ involvement. A major challenge for dengue pathogenesis studies has been a suitable animal model, but the use of immune-competent mice, although sometimes controversial, has proven to be useful, as histological observations in infected animals reveal tissue alterations consistent to those observed in dengue human cases. Here, we aimed to investigate the outcomes caused by two distinct lineages of the DENV-2 Asian/American genotype in the lung, heart and skeletal muscle tissues of infected BALB/c mice. Tissues were submitted to histopathology, immunohistochemistry, histomorphometry and transmission electron microscopy (TEM) analysis. The viral genome was detected in heart and skeletal muscle samples. The viral antigen was detected in cardiomyocytes and endothelial cells of heart tissue. Heart and lung tissue samples presented morphological alterations comparable to those seen in dengue human cases. Creatine kinase serum levels were higher in mice infected with both lineages of DENV-2. Additionally, statistically significant differences, concerning alveolar septa thickening and heart weight, were observed between BALB/c mice infected with both DENV-2 lineages, which was demonstrated to be an appropriate experimental model for dengue pathogenesis studies on lung, heart and skeletal muscle tissues.