High-content phenotypic screening identifies novel chemistries that disrupt mosquito activity and development.

New classes of chemistries are needed to control insecticide resistant populations of mosquitoes and prevent transmission of vector-borne diseases (VBDs). Organismal screens of chemical collections have played an important role in the search for new vector insecticides and the identification of active ingredients (AIs) that cause rapid mortality of mosquitoes. Advances in image-based screening offer an opportunity to identify chemistries that operate via novel biochemical modes and investigate the range of phenotypes exhibited by mosquitoes following exposure to lethal and sub-lethal chemical dose. An automated, high throughput phenotypic screen (HTS) employing high-content imaging of first instar (L1) Aedes aegypti larvae was developed to identify chemistries associated with mortality and atypical morphological phenotypes. A pilot screen of the Library of Pharmacologically Active Compounds (LOPAC1280) identified 92 chemistries that disrupted larval activity and development, including conventional insecticides and chemistries known to modulate G protein-coupled receptors (GPCRs) and other molecular targets in mammalian systems. Secondary assay series were used to evaluate a selection of chemistries for impacts on mosquito activity, survival and development. Ritodrine hydrochloride reduced mobility of larvae but had no observable effect on survival and development of mosquitoes. High doses of metergoline suppressed larval activity and sub-lethal dose resulted in pupal mortality. Assay data support the utility of phenotypic screening and diverse entomological end-points for discovery of novel insecticidal chemical scaffolds. The insecticide discovery process must consider how multi-modal efficacy spectra contribute to vector and VBD control.

Inter- and intra-interviewer reliability of Italian version of Pediatric Evaluation of Disability Inventory (I-PEDI).

BACKGROUND: Childhood disabilities determine a range of immediate and long-term economic costs that have important implications for the well-being of the child, the family and the society. The Pediatric Evaluation of Disability Inventory (PEDI) measures capability and performance in children aged between 6 months and 7.5 years. It contains three scales: Functional Skills Scales (FSS), Caregiver Assistance Scale (CAS) and Modifications Scale (MS). The present study evaluated the measurement properties of the Italian version of the PEDI (PEDI-I) in patients with spastic cerebral palsy (CP). STUDY DESIGN: Reliability study. METHODS: The original PEDI was translated - including a cross-cultural adaptation - into Italian. Internal consistency and test-retest reliability were evaluated. RESULTS: Fifty-eight children with CP were recruited. According to inter-interviewer reproducibility, the FSS domain revealed intraclass correlation coefficient (ICC) values ranging between 0.94 and 1.00. CAS domain revealed ICC values ranging between 0.94 and 1.00. The SEM values ranged between 3.25 (SDD=8.98) for SF and 5.24 for SC (SDD=14.5). According to intra-interviewer reproducibility, the FSS domain revealed ICC values ranging between 0.99 and 1.00. CAS domain revealed ICC values ranging between 0.92 and 0.99. The SEM values ranged between 3.44 (SDD=9.5) for SF and 3.75 for SC (SDD=10.36). The inter-interviewer and intra-interviewer reproducibility results showed very high ICC values for both FFS and CAS domains. Cronbach's α ranged between 0.94 and 0.99, indicating excellent internal consistency within each domain of the PEDI-I. CONCLUSION: The inter-interviewer and intra-interviewer reproducibility results of PEDI-I showed very high ICC values for FFS and CAS domains. Therefore, we recommend its application to evaluate the effect of treatment in children with CP.

Mitochondrial specialization revealed by single muscle fiber proteomics: focus on the Krebs cycle.

We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

Prevalence of parvoviruses in commercial turkey flocks.

Turkey parvovirus belongs to the family Parvoviridae, subfamily Parvovirinae, Genus parvovirus. Since the initial report on turkey parvovirus in the United States appeared in 1983, there had been no further reports of parvovirus in turkeys until 2008. The aims of our study were to determine the prevalence of parvovirus in commercial turkey flocks using PCR; to determine their genetic relationship to previous strains identified in North America and Europe; and to test samples for enteric viruses by transmission electron microscopy (TEM). A total of 169 fecal samples collected from 42 turkey farms in four different states within the United States between 2000 and 2010 were examined. We found that the most frequently detected viruses by TEM were small round viruses, accounting for 52% of the examined samples; however, the PCR detected parvoviruses in 71% of the samples. The phylogenetic analysis of partial nonstructural gene sequences showed a certain degree of variability among the turkey samples tested in the study. Moreover, there was a clear dichotomy in the phylogenetic tree between chicken and turkey samples, with the exception of one turkey isolate from 2000, which clustered together with the chicken group.

Ras is involved in nerve-activity-dependent regulation of muscle genes.

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation-transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras-MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.

Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Examining Reliability and Validity of the Jebsen-Taylor Hand Function Test Among Children With Cerebral Palsy.

Manual dexterity has strongly predicted functional independence for daily life activities among children with cerebral palsy (CP). The Jebsen-Taylor Hand Function Test (JTHFT) is the most widely used assessment tool for exploring manual dexterity in the CP population, though no research has yet examined its psychometric properties for this use. This cross-sectional study explored the validity and internal consistency of the JTHFT in an Italian sample of inpatient and outpatient children with CP aged between 6-18 years (35 girls and 49 boys). We calculated internal consistency with Cronbach's alpha and tested validity against the Manual Ability Classification System (MACS) using Pearson's correlation coefficient. To better understand how the JTHFT compares with different levels of the MACS, we performed dominant hand timing variability for each test item. Results showed excellent internal consistency with a Cronbach's alpha of .944 and .911, respectively, for nondominant and dominant hands. There was also a statistically significant positive linear Pearson's correlation coefficient between the JTHFT and the MACS (p < .01). We observed high variability in writing performance (Item 1 of the JTHFT) within this sample for each level of the MACS. This study confirms that the JTHFT is a valid assessment tool when used in children with CP aged 6-18 years.

Efficient second harmonic generation from thin films of V-shaped benzo[b]thiophene based molecules.

We have designed an original approach for efficient Second Harmonic Generation of tailored V-shape benzo[b]thiophene molecular systems enabling versatile and flexible one-step, dry and technologically friendly thin film processing. The designed moieties show chi((2)) values at least as high as the reference LiNbO(3) single crystal, without poling processing and matching the constrains of integrated optical configuration for nonlinear optical devices. This may open the way to a new class of organic materials exploitable for photonic applications.

Mitochondrial Ca2+ homeostasis in intact cells.

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.

Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.

Microdomains of high intracellular calcium ion concentration, [Ca2+]i, have been hypothesized to occur in living cells exposed to stimuli that generate inositol 1,4,5-trisphosphate (IP3). Mitochondrially targeted recombinant aequorin was used to show that IP3-induced Ca2+ mobilization from intracellular stores caused increases of mitochondrial Ca2+ concentration, [Ca2+]m, the speed and amplitude of which are not accounted for by the relatively small increases in mean [Ca2+]i. A similar response was obtained by the addition of IP3 to permeabilized cells but not by perfusion of cells with Ca2+ at concentrations similar to those measured in intact cells. It is concluded that in vivo, domains of high [Ca2+]i are transiently generated close to IP3-gated channels and sensed by nearby mitochondria; this may provide an efficient mechanism for optimizing mitochondrial activity upon cell stimulation.

A radio ridge connecting two galaxy clusters in a filament of the cosmic web.

Galaxy clusters are the most massive gravitationally bound structures in the Universe. They grow by accreting smaller structures in a merging process that produces shocks and turbulence in the intracluster gas. We observed a ridge of radio emission connecting the merging galaxy clusters Abell 0399 and Abell 0401 with the Low-Frequency Array (LOFAR) telescope network at 140 megahertz. This emission requires a population of relativistic electrons and a magnetic field located in a filament between the two galaxy clusters. We performed simulations to show that a volume-filling distribution of weak shocks may reaccelerate a preexisting population of relativistic particles, producing emission at radio wavelengths that illuminates the magnetic ridge.

The treatment of femoral fractures in children with cerebral palsy.

OBJECTIVE: The purpose of this study is to retrospectively evaluate a group of children affected by cerebral palsy with a recent femoral fracture, and to analyse the results and complications in relation to the treatment used. MATERIALS AND METHODS: The analysis was performed on 36 children (21 M, 15 F, 8-14 years old) with cerebral palsy (7 diplegia, 28 tetraparesis, 1 hemiplegia) with a metaphyseal or a diaphyseal femoral fracture. The patients were subdivided into two groups according to their Gross Motor Function Classification System (GMFCS) level: level 2-3 (9 patients) and level 4-5 (27 patients), evaluating the presence of complications and malunions for each group at the end of each follow up. RESULTS: The fractures were displaced in 24 patients and nondisplaced in 12 patients. In 26 cases the treatment involved a closed reduction and immobilisation in a long leg hip spica cast for 7 weeks, while in 10 cases the treatment involved an open reduction-internal fixation (ORIF) followed by a 3-week period in a plaster coated fracture bandage. CONCLUSIONS: Taking into consideration the maximum possible recovery of function, an ORIF is preferable to prevent malunion, particularly in distal metaphysis and distal shaft fractures. In the GMFCS level 2-3 patients, surgery has allowed to recover, or at least maintain, the pre-fracture functional level, while in patients with GMFCS level 4-5, it has allowed to reduce the immobilisation times and prevent the development of decubitus lesions.

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.

BACKGROUND: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. RESULTS: In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator. CONCLUSION: GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.

Pathogenicity of turkey astroviruses in turkey embryos and poults.

The pathogenicity of turkey astrovirus 2001 (TAstV2001) and turkey astrovirus 1987 (TAstV1987) in specific-pathogen-free (SPF) turkey embryos and commercial poults was investigated. The virus shedding in poults was monitored using electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) during the 14-day experimental period. Both viruses caused enteritis and growth depression in SPF turkey embryos and poults. The TAstV2001 did not induce macroscopic or microscopic lesions in thymuses and bursas of embryos or poults. No macroscopic changes were observed in thymuses and bursas of embryos and poults inoculated with TAstV1987, and no statistically significant differences in bursa weight/ body weight ratios (P > 0.05) were detected. However, TAstV1987 infection resulted in microscopic lesions in bursas but not in thymuses of infected embryos and poults. Both TAstV2001 and TAstV1987 were shed during the whole 14-day experimental period as detected by EM and RT-PCR. These findings indicated that both TAstV1987 and TAstV2001 are etiologic agents of turkey enteritis. In addition, TAstV1987 might cause impairment of the immune system of infected poults. The pathogenicity of TAstV1987 is somewhat different from TAstV2001.

Spatial genetic structure in wild cardoon, the ancestor of cultivated globe artichoke: Limited gene flow, fragmentation and population history.

Nuclear and chloroplast markers and phenotypic characters were integrated to analyse the population genetic structure of wild cardoon, Cynara cardunculus var. sylvestris, the ancestor of cultivated globe artichoke, Cynara cardunculus var. scolymus on the island of Sardinia, Italy. The spatial scale ranged from a few metres to ∼200km. Wild cardoon appears to be genetically fragmented, with significant genetic divergence at various scales, indicating that gene flow is insufficient to counterbalance the effects of genetic drift or founder effects. Divergence between populations was higher for chloroplast (40%) than for nuclear markers (15%), suggesting that gene flow via seed was lower than via pollen. Two main genetic groups were detected; these correlated with differences in flowering time, capitula size, glossiness, and anthocyanin pigmentation. A complex population structure of wild cardoon emerged over small spatial scales, likely resulting from the interplay between gene dispersal, colonisation history and selective forces. Indeed, Sardinia appears to be a 'hybrid zone' of different gene pools. The island has unique diverse germplasm that has originated from hybridisation among different gene pools. The sampling of seeds from a few plants but from many sites is suggested as the best strategy to harvest the genetic diversity of wild cardoon.

Trichinella britovi from domestic to wild animals of Sardinia, Italy.

The zoonotic nematode Trichinella britovi has been documented in animals and/or humans of the Mediterranean islands of Corsica and Sardinia since 2004. From 2005 to 2007 in the Sardinia island, several surveys had shown that T. britovi was circulating among backyard and free-ranging pigs reared in the Orgosolo municipality but all attempts had failed to detect this parasite in wild susceptible animals. The aim of the present work was to investigate the circulation of T. britovi in pigs and wildlife of the Orgosolo municipality, and of surrounding municipalities and provinces in the 2010-2014 slaughtering/hunting seasons. The results show that the T. britovi circulation was still restricted to the Orgosolo municipality with a prevalence of 2.6% in free-ranging pigs and 0.2% in backyard pigs but, for the first time, this parasite was detected also in 0.4% of wild boar, and 27.6% of red foxes. No infection was detected in backyard pigs, wild boar, and red foxes of the other municipalities and provinces. Since 1978, African swine fever is endemic in Sardinia and foci of this virus are still active in the investigated areas favoring cannibalism and, consequently, the T. britovi transmission, due to the high mortality rate caused by this virus. This is the first documented report on the transmission of T. britovi between the domestic and the sylvatic cycle. The health authority of the island must provide a service to dispose animal carcasses and offal, stamping out illegal free-ranging pigs, and train hunters and pig owners to manage waste and by-products according to the EU regulations.

Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies.

The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca2+ concentration, [Ca2+]i, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2+]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca2+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2+]i; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2+]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca2+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca2+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca2+ in mitogenic stimulation of T cells.

Interactions of trialkyllead compounds with rat liver mitochondria.

The interactions of two trialkyllead (TAL) compounds, (trimethyl)Pb-Cl and (tributyl)Pb-Cl, with mitochodria from rat liver have been studied. A stimulation of the respiratory rate induced by the trialkyllead compounds added at low doses was observed which was not dependent on the presence of chloride in the medium. In contrast with the major current view, we propose that trialkyllead compounds behave as uncouplers of the oxidative phosphorylation and not (or not only) as Cl-/OH- exchangers. In fact the present results suggest that the TAL compounds enter the mitochondria as (alkyl)3Pb+ cations and are extruded as electroneutral (alkyl)3 Pb-OH compounds, the overall result being the transport of a proton through the membrane as in the case of classical uncouplers. The uncoupling effect could explain the toxicity of the compounds as a result of the decrease in the energy level of the cell. Furthermore, such a mechanism, in which the uptake of TAL compounds is supposed to be driven by a negative potential, could explain their preferential toxicity for neuronal cells, which maintain a higher negative-inside potential than most other cell types.