In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Dual inhibition of BRD4 and PI3K by SF2523 suppresses human prostate cancer cell growth in vitro and in vivo

Bromodomain-containing protein 4 (BRD4) and phosphatidylinositol 3-kinase (PI3K) are both key oncogenic proteins in human prostate cancer. In the current study, we examined the anti-prostate cancer cell activity by SF2523, a BRD4 and PI3K dual inhibitor. We showed that SF2523 potently inhibited survival and proliferation of the primary human prostate cancer cells. SF2523 induced profound apoptosis activation in prostate cancer cells. The dual inhibitor was yet non-cytotoxic to the prostate epithelial cells. At the molecular level, SF2523 downregulated BRD4-regulated genes (cyclin D1, c-Myc and androgen receptor) and almost blocked AKT-S6K1 activation in prostate cancer cells. In vivo, SF2523 intraperitoneal administration at the well-tolerated dose inhibited human prostate cancer xenograft growth in severe combined immunodeficient (SCID) mice. BRD4-regulated genes (cyclin D1, c-Myc and androgen receptor) and AKT-S6K1 activation were inhibited in SF2523-treated tumors. Together, dual inhibition of BRD4 and PI3K by SF2523 suppresses human prostate cancer cell growth in vitro and in vivo.     

MicroRNAs in Drosophila: The magic wand to enter the Chamber of Secrets?

MicroRNAs are small non-coding RNAs that are now recognised as key regulators of gene expression in eukaryotes. Over the past few years, hundreds of miRNAs have been identified from various organisms including vertebrates, nematodes, insects and plants. A high level of conservation of some miRNAs from animals to plants underlines their crucial role in eukaryotes. Although biogenesis and mode of action of miRNAs are now quite well understood, their numerous and specific regulatory functions remain to be unravelled. In this review, we summarise the current knowledge on miRNAs in insects, which was mainly acquired through the study of the fruit fly, Drosophila melanogaster.     

Drosophila E-cadherin and its binding partner Armadillo/ β-catenin are required for axonal pathway choices in the developing larval brain

The fly brain is formed by approximately hundred paired lineages of neurons, each lineage derived from one neuroblast. Embryonic neuroblasts undergo a small number of divisions and produce the primary neurons that form the functioning larval brain. In the larva, neuroblasts produce the secondary lineages that make up the bulk of the adult brain. Axons of a given secondary lineage fasciculate with each other and form a discrete bundle, the secondary axon tract (SAT). Secondary axon tracts prefigure the long axon connections of the adult brain, and therefore pathway choices of SATs made in the larva determine adult brain circuitry. Drosophila Shotgun/E-cadherin (DE-cad) and its binding partner Armadillo/β-catenin (β-cat) are expressed in newly born secondary neurons and their axons. The fact that the highly diverse, yet invariant pattern of secondary lineages and SATs has been recently mapped in the wild-type brain enabled us to investigate the role of DE-cad and β-cat with the help of MARCM clones. Clones were validated by their absence of DE-cad immuno-reactivity. The most significant phenotype consists in the defasciculation and an increased amount of branching of SATs at the neuropile-cortex boundary, as well as subtle changes in the trajectory of SATs within the neuropile. In general, only a fraction of mutant clones in a given lineage showed structural abnormalities. Furthermore, although they all globally express DE-cad and β-cat, lineages differ in their requirement for DE-cad function. Some lineages never showed morphological abnormalities in MARCM clones, whereas others reacted with abnormal branching and changes in SAT trajectory at a high frequency. We conclude that DE-cad/β-cat form part of the mechanism that control branching and trajectory of axon tracts in the larval brain.     

R-(-)-β-O-methylsynephrine, a natural product, inhibits VEGF-induced angiogenesis in vitro and in vivo

R-(-)-β-O-methylsynephrine (OMe-Syn) is an active compound isolated from a plant of the Rutaceae family. We conducted cell proliferation assays on various cell lines and found that OMe-Syn more strongly inhibited the growth of human umbilical vein endothelial cells (HUVECs) than that of other normal and cancer cell lines tested. In angiogenesis assays, it inhibited vascular endothelial growth factor (VEGF)-induced invasion and tube formation of HUVECs with no toxicity. The anti-angiogenic activity of OMe-Syn was also validated in vivo using the chorioallantonic membrane (CAM) assay in growing chick embryos. Expression of the growth factors VEGF, hepatocyte growth factor, and basic fibroblast growth factor was suppressed by OMe-Syn in a dose-dependent manner. Taken together, our results indicate that this compound could be a novel basis for a small molecule targeting angiogenesis.     

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.     

Complex interactions amongst N-cadherin, DLAR, and Liprin-α regulate Drosophila photoreceptor axon targeting

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-α play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-α function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-α in the developing growth cone.     

Are the diatoms Navicula sp. and Thalassiosira fluviatilis suitable to be fed to the benthic harpacticoid copepod Tisbe biminiensis?

The objective of this study was to compare the influence of the diets of two diatoms, Navicula sp. (benthic) and Thalassiosira fluviatilis (planctonic), on the development, fecundity and survival of the harpacticoid copepod Tisbe biminiensis. In order to determine the optimal concentration of food, 35 egg-bearing females were submitted to six algal concentrations and controls (without food). After 24 h, the content of the recipients was fixed with 4% formalin and then fecal pellets produced by each female were counted and measured. The larval development was studied by surveying 50 nauplii on each diet individually until the adult stage, at intervals of 6 h. The cast exoskeletons were removed to count the number of segments and for measurement. The fecundity was obtained counting the naupliar production every 48 h of groups containing 10 females in different algal concentrations in both diets. The diet influence on fecundity was tested by submitting four groups of 10 females fed on optimal algal concentrations based on the fecal pellet experiments. In the Navicula sp. concentration of 0.4 μg Chl-a/ml, considered to be optimal for fecal pellets production, the diatoms were shown toxic, resulting in a low survival rate and inhibiting the egg production of copepods. The optimal concentration considering fecundity was estimated to be 0.1 μg Chl-a/ml for both diets. Copepods fed on Navicula sp. presented a faster development rate and higher naupliar production compared to copepods fed on T. fluviatilis. Size and survival did not vary significantly among diets. The algal concentration interfered significantly in the reproductive success of females. Both very low and very high algal concentrations reduced reproductive success. Concluding, the benthic diatom Navicula sp. was more favorable to the copepod T. biminiensis than the planktonic diatom T. fluviatilis.     

Melphalan,alone or conjugated to an FSH-β peptide,kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of FSHβ. We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHβ consisting of amino acids 33 to 53 (FSHβ (33–53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33–53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33–53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33–53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33–53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33–53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.     

Hidden diversity uncovered in Hygrophorus sect. Aurei (Hygrophoraceae), including the Mediterranean H. meridionalis and the North American H. boyeri, spp. nov.

For many years, the binomial Hygrophorus hypothejus was widely applied to collections from various geographical regions in different continents, assuming a circum-boreal and circum-mediterranean distribution for this species. This hypothesis, however, had never been put to the test. To assess the diversity and species-limits within this complex of yellow-coloured waxcaps, a phylogenetic, morphological and taxonomical investigation into Hygrophorus sect. Aurei and similar species in sect. Olivaceoumbrini was carried out, including material of pan-European origin, as well as the east and west coasts of North America. Following sequencing of the ITS rDNA locus, nine lineages are confirmed in sect. Aurei, most of them highly continentalised. Of these, two are new to science, introduced here as Hygrophorus boyeri sp. nov., from Pinus banksiana and P. rigida forests in eastern North America and from P. muricata and P. contorta forests in western North America, and Hygrophorus meridionalis sp. nov., from Pinus brutia and Pinus halepensis forests in the island of Cyprus and mainland Greece. H. hypothejus is lectotypified and epitypified, and here resolved as a strictly European species, with the old forgotten taxon Hygrophorus siccipes revived as its North American vicariant. The placement of Hygrophorus fuligineus in sect. Aurei is phylogenetically confirmed and detailed comparisons between morphologically similar and phylogenetically affiliated taxa in sect. Aurei and sect. Olivaceoumbrini are provided. The chronic confusion associated with Hygrophorus fuscoalbus, a highly controversial taxon described from Germany nearly two centuries ago and variously interpreted since, is discussed, concluding that this name is too ambiguous to be applied to any currently recognized species.     

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1^−/− embryos. Inactivating β-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→β-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.     

Wnt5b stimulates adipogenesis by activating PPARγ, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a

Correct Wnt signaling is required for adipogenesis and alterations occur in Type 2 diabetes mellitus (T2DM). Gene expression studies showed that β-catenin independent Wnt5b was down-regulated in T2DM preadipocytes, while its paralog Wnt5a was unchanged. Our study aimed at defining the expression profile and function of Wnt5a and Wnt5b during adipogenesis by determining their effect on aP2 and PPARγ expression and assessing the level of β-catenin translocation in mouse 3T3-L1 preadipocytes. Additionally, we explored the effect on adipogenic capacity by Wnt5b overexpression in combination with stimulation of the β-catenin dependent or β-catenin independent Wnt signaling. Expression of Wnt5b was, like Wnt5a, down-regulated upon induction of differentiation and both inhibit β-catenin dependent Wnt signaling at the initiation of adipogenesis. Wnt5b additionally appears to be a potent enhancer of adipogenic capacity by stimulation of PPARγ and aP2. Down-regulation of Wnt5b could therefore contribute to decreased adipogenesis observed in T2DM diabetic subjects.     

In vitro susceptibilities of Neoscytalidium spp. sequence types to antifungal agents and antimicrobial photodynamic treatment with phenothiazinium photosensitizers

Neoscytalidium spp. are ascomycetous fungi consisting of pigmented and hyaline varieties both able to cause skin and nail infection. Their color-based identification is inaccurate and may compromise the outcome of the studies with these fungi. The aim of this study was to genotype 32 isolates morphologically identified as Neoscytalidiumdimidiatum or N. dimidiatum var. hyalinum by multilocus sequence typing (MLST), differentiate the two varieties by their sequence types, evaluate their susceptibility to seven commercial antifungal drugs [amphotericin B (AMB), voriconazole (VOR), terbinafine (TER), 5-flucytosine (5FC), ketoconazole (KET), fluconazole (FLU), and caspofungin (CAS)], and also to the antimicrobial photodynamic treatment (APDT) with the phenothiazinium photosensitizers (PS) methylene blue (MB), new methylene blue (NMBN), toluidine blue O (TBO) and the pentacyclic derivative S137. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, the APDT effects with each PS on the survival of ungerminated and germinated arthroconidia of both varieties were evaluated. Seven loci of Neoscytalidium spp. were sequenced on MLST revealing eight polymorphic sites and six sequence types (ST). All N. dimidiatum var. hyalinum isolates were clustered in a single ST. AMB, VOR and TER were the most effective antifungal agents against both varieties. The hyaline variety isolates were much less tolerant to the azoles than the isolates of the pigmented variety. APDT with S137 showed the lowest MIC for all the isolates of both varieties. APDT with all the PS killed both ungerminated and germinated arthroconidia of both varieties reducing the survival up to 5 logs. Isolates of the hyaline variety were also less tolerant to APDT. APDT with the four PS also increased the plasma membrane permeability of arthroconidia of both varieties but only NMBN and S137 caused peroxidation of the membrane lipids.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

Synthesis, in vitro and in vivo evaluation of 2-aryl-4H-chromene and 3-aryl-1H-benzo[f]chromene derivatives as novel α-glucosidase inhibitors

Herein we report a study of novel arylchromene derivatives as analogs of naturally occurring flavonoids with prominent α-glucosidase inhibitory properties. Novel inhibitors were identified via simple stepwise in silico screening, efficient synthesis, and biological evaluation. It is shown that 2-aryl-4H-chromene core retains pharmacophore properties while being readily available synthetically. A lead compound identified through screening inhibits yeast α-glucosidase with IC₅₀ of 62.26?µM and prevents postprandial hyperglycemia in rats at 2.2?mg/kg dose.     

The conversion of (24S)-24-ethylcholesta-5,22,25-trien-3β-ol into poriferasterol,both in vivo and with a cell-free homogenate of the alga Trebouxia sp.

[6-³H₁] (24S)-24-Ethylcholesta-5,22,25-trien-3β-ol added to the growth medium of a culture of Trebouxia sp. 213/3 was efficiently taken-up by the cells and converted into (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) which is one of the major sterols of this alga. A cell-free homogenate was obtained from Trebouxia which catalysed the NADPH-dependent reduction of [6-³H₁] (24S)-24-ethylcholesta-5,22,25-trien-3β-ol to yield poriferasterol. The δ²⁵-sterol reductase was found to be mainly localized in the microsomal fraction of the homogenate.     

Acute toxicity,critical body residues,Michaelis–Menten analysis of bioaccumulation,and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L^− 1 as CaCO₃ (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC50 values 3–3.5 × higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC50 values within and across species by ~ 2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis–Menten kinetics in all organisms, with greater hardness tending to decrease B_max with no consistent effect on K_d. Across species, acute Ni LC50 values tended to increase with both K_d and B_max values — i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K_NiBL values derived from Ni bioaccumulation correlated well with log K_NiBL values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.