Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

Background

Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples.

Methods

Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview''s automated scanning system.

Results

All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively.

Conclusions

The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.     

Knowledge,Attitudes and Practices Related to Visceral Leishmaniasis in Rural Communities of Amhara State: A Longitudinal Study in Northwest Ethiopia

Background

In the northwest of Ethiopia, at the South Gondar region, there was a visceral leishmaniasis (VL) outbreak in 2005, making the disease a public health concern for the regional health authorities ever since. The knowledge on how the population perceives the disease is essential in order to propose successful control strategies.

Methodology/Principal findings

Two surveys on VL knowledge, attitudes and practices were conducted at the beginning (May 2009) and at the end (February 2011) of a VL longitudinal study carried out in rural communities of Libo Kemkem and Fogera, two districts of the Amhara Regional State. Results showed that VL global knowledge was very low in the area, and that it improved substantially in the period studied. Specifically, from 2009 to 2011, the frequency of proper knowledge regarding VL signs and symptoms increased from 47% to 71% (p<0.0001), knowledge of VL causes increased from 8% to 25% (p<0.0001), and knowledge on VL protection measures from 16% to 55% (p<0.0001). Moreover, the improvement observed in VL knowledge was more marked among the families with no previous history of VL case. Finally, in 2011 more than 90% of the households owned at least an impregnated bed net and had been sprayed, and attitudes towards these and other protective measures were very positive (over 94% acceptance for all of them).

Conclusions/Significance

In 2009 the level of knowledge regarding VL was very low among the rural population of this area, although it improved substantially in the study period, probably due to the contribution of many actors in the area. VL patients and relatives should be appropriately informed and trained as they may act as successful health community agents. VL risk behavioural patterns are subject to change as attitudes towards protective measures were very positive overall.     

Vaccination with Recombinant Adenoviruses Expressing the Peste des Petits Ruminants Virus F or H Proteins Overcomes Viral Immunosuppression and Induces Protective Immunity against PPRV Challenge in Sheep

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.     

The ecosystem engineering role of the Neotropical crab <Emphasis Type="Italic">Cardisoma guanhumi</Emphasis> on mangrove soil properties

The land crab Cardisoma guanhumi is one of the most common species in mangroves of the American Atlantic coast and Caribbean islands however, studies of its effects on the physical and chemical soil properties are scarce. This study compares specific physicochemical properties of soil between C. guanhumi burrows (B) and adjacent zones (AZ), and provides the first insights on their role as an ecosystem engineer in mangroves. The study was conducted in an estuarine system dominated by Rhizophora mangle, located at the Río Chico estuary, Miranda state of Venezuela. Random soil samples were taken digging each burrow until reaching the bottom and at the same depth for AZ. Data analysis was carried out using Bayesian inference. Credible mean differences between B and AZ, were found for sand (B?=?26.53?±?10.76, AZ?=?17.25?±?5.7%), silt (B?=?73.16?±?10.77, AZ?=?82.42?±?5.69%), pH (B?=?8.71?±?0.36, AZ?=?9.12?±?0.30), soil organic matter (SOM, B?=?0.43?±?0.21, AZ?=?0.17?±?0.06%), total N (TN, B?=?786?±?232, AZ?=?529?±?107 µg g^?1), Mg (B?=?4.42?±?0.60, AZ?=?3.48?±?0.71 cmol_c kg^?1) and K (B?=?0.12?±?0.05 AZ?=?0.06?±?0.02 cmol_c kg^?1). Chemical variables as SOM, K, Mg and TN showed the highest values of effect size (>?1.4). With the exception of the pH, all chemicals variables—which were different between B and AZ—showed strong and decisive evidences of correlations with SOM. When SOM variable was controlled, the relationships between pH–TN, TN–K and Mg–K decreased, even though the correlation evidence between each pair remained. Differences in chemical contents found in B respect to AZ suggest that the activities of C. guanhumi (feeding, moulting, excretion and defecation) within their burrows promote the spatial heterogeneity of mangrove soils.     

Decreasing the Expression of GABA<Subscript>A</Subscript> α5 Subunit-Containing Receptors Partially Improves Cognitive,Electrophysiological, and Morphological Hippocampal Defects in the Ts65Dn Model of Down Syndrome

Trisomy 21 or Down syndrome (DS) is the most common cause of intellectual disability of a genetic origin. The Ts65Dn (TS) mouse, which is the most commonly used and best-characterized mouse model of DS, displays many of the cognitive, neuromorphological, and biochemical anomalies that are found in the human condition. One of the mechanisms that have been proposed to be responsible for the cognitive deficits in this mouse model is impaired GABA-mediated inhibition. Because of the well-known modulatory role of GABA_A α5 subunit-containing receptors in cognitive processes, these receptors are considered to be potential targets for improving the intellectual disability in DS. The chronic administration of GABA_A α5-negative allosteric modulators has been shown to be procognitive without anxiogenic or proconvulsant side effects. In the present study, we use a genetic approach to evaluate the contribution of GABA_A α5 subunit-containing receptors to the cognitive, electrophysiological, and neuromorphological deficits in TS mice. We show that reducing the expression of GABA_A α5 receptors by deleting one or two copies of the Gabra5 gene in TS mice partially ameliorated the cognitive impairments, improved long-term potentiation, enhanced neural differentiation and maturation, and normalized the density of the GABAergic synapse markers. Reducing the gene dosage of Gabra5 in TS mice did not induce motor alterations and anxiety or affect the viability of the mice. Our results provide further evidence of the role of GABA_A α5 receptor-mediated inhibition in cognitive impairment in the TS mouse model of DS.     

Analysis of differentially upregulated proteins in ptsHIcrr− and rppH− mutants in Escherichia coli during an adaptive laboratory evolution experiment

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS⁻ derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH⁻) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH⁻ mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS⁻ and rppH⁻ mutations in the deep of a proteomic approach to understanding the relevance of rppH⁻ phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH⁻, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH⁻ were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS⁻ strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS⁻ derivatives during the ALE experiment. This result suggests that the evolving PTS⁻ population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH⁻ mutation in the PTS⁻ background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

     

Echoes of the whispering land: interacting roles of vicariance and selection in shaping the evolutionary divergence of two <Emphasis Type="Italic">Calceolaria</Emphasis> (Calceolariaceae) species from Patagonia and Malvinas/Falkland Islands

A key to understanding the origin and identity of young species lays on the knowledge of the Quaternary climatic oscillations’ effect on gene flow and vicariance. Even though the effect of climatic fluctuations is relatively well understood for southern hemisphere plant species, little is known about their effect on the evolutionary histories of species from mainland and islands. Thus, we investigated whether Quaternary climate-driven fluctuations translated into lineage divergence and speciation, followed or not by climatic niche differentiation, in two allopatric plant species, Calceolaria uniflora and C. fothergillii from Patagonia and Malvinas/Falkland islands, respectively. We sampled the range of both species, and sequenced two chloroplastic (cpDNA; trnS–trnG and trnH–psbA), and one single copy “anonymous” non-coding nuclear region (nDNA). We performed phylogeographic and dating analyses, and adjusted spatio-temporal diffusion models. We complemented molecular evidence with climatic niche differentiation analyses and species paleo-distribution projections. A species coalescent reconstruction based on multi-locus data retrieved both species as monophyletic. Estimates from cpDNA indicated the species diverged during the Great Patagonian Glaciation. Chloroplast and nuclear DNA showed east–west distribution of the main genetic groups but with contrasting spatial genetic diversity. The spatio-temporal diffusion analyses showed that between 1–0.8 Mya and 570 Kya the lineage leading to C. fothergillii diverged from C. uniflora and arrived to the islands. Climatic niche projections hindcasted range expansions during glaciations, and contractions during the interglacial periods. Comparisons of climatic niches between the two study species indicated that temperature variables show evidence of niche conservatism while precipitation regimes supported niche divergence, even when considering the background environmental divergence. Our study indicates that glacial fluctuations affected the mainland/islands connections favouring speciation mediated not only by isolation, but also by climatic niche differentiation.     

Photoacclimation and photoregulation strategies of Corallina (Corallinales,Rhodophyta) across the NE Atlantic

This study characterizes the photoacclimation and photoregulation mechanisms that allow calcified macroalgae of the genus Corallina (Corallinales, Rhodophyta) to dominate rock pool habitats across the NE Atlantic despite the highly variable irradiance regimes experienced. Rapid light curves (RLCs) were performed with pulse amplitude modulation (PAM) fluorometry in situ across a full seasonal cycle in the UK intertidal with C. officinalis and C. caespitosa. Latitudinal comparisons were performed across the full extent of C. officinalis’ range in the NE Atlantic (Iceland–northern Spain), and for C. caespitosa in northern Spain. Ex situ RLCs with dark recovery were further employed to assess the optimal, as compared with actual, photophysiology across seasons and latitudes. Corallina species were shown to photoacclimate at seasonal timescales to changing irradiance, increasing light-harvesting during low-light autumn/winter periods and protecting photosystems during high-light summer conditions. Seasonal photoacclimation was achieved through alteration in the number of photosystem (PS) units (PSII and light harvesting antennae) over time. Non-photochemical quenching (NPQ) served as an important photoregulation mechanism utilized by Corallina to prevent or minimize photoinhibition over shorter time scales (seconds–hours), though the efficiency of NPQ was dependent on the seasonal-acclimated state. With increasing latitude the efficiency of photoregulation decreased, representing potential differential photoadaptation of Corallina across species ranges in the NE Atlantic. In contrast, highly conserved inter-specific patterns in photophysiological responses to irradiance were apparent. This study demonstrates the photophysiological mechanisms allowing Corallina to optimize use of the variable irradiance conditions apparent in rock pool environments, when and how they are employed, and their limitations.     

Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis

Tau is a microtubule‐associated neuronal protein found mainly in axons. However, its presence in dendrites and dendritic spines is particularly relevant due to its involvement in synaptic plasticity and neurodegeneration. Here, we show that Tau plays a novel in vivo role in the morphological and synaptic maturation of newborn hippocampal granule neurons under basal conditions. Furthermore, we reveal that Tau is involved in the selective cell death of immature granule neurons caused by acute stress. Also, Tau deficiency protects newborn neurons from the stress‐induced dendritic atrophy and loss of postsynaptic densities (PSDs). Strikingly, we also demonstrate that Tau regulates the increase in newborn neuron survival triggered by environmental enrichment (EE). Moreover, newborn granule neurons from Tau^?/? mice did not show any stimulatory effect of EE on dendritic development or on PSD generation. Thus, our data demonstrate that Tau^?/? mice show impairments in the maturation of newborn granule neurons under basal conditions and that they are insensitive to the modulation of adult hippocampal neurogenesis exerted by both stimulatory and detrimental stimuli.     

Molecular systematics of teioid lizards (Teioidea/Gymnophthalmoidea: Squamata) based on the analysis of 48 loci under tree‐alignment and similarity‐alignment

We infer phylogenetic relationships within Teioidea, a superfamily of Nearctic and Neotropical lizards, using nucleotide sequences. Phylogenetic analyses relied on parsimony under tree‐alignment and similarity‐alignment, with length variation (i.e. gaps) treated as evidence and as absence of evidence, and maximum‐likelihood under similarity‐alignment with gaps as absence of evidence. All analyses produced almost completely resolved trees despite 86% of missing data. Tree‐alignment produced the shortest trees, the strict consensus of which is more similar to the maximum‐likelihood tree than to any of the other parsimony trees, in terms of both number of clades shared, parsimony cost and likelihood scores. Comparisons of tree costs suggest that the pattern of indels inferred by similarity‐alignment drove parsimony analyses on similarity‐aligned sequences away from more optimal solutions. All analyses agree in a majority of clades, although they differ from each other in unique ways, suggesting that neither the criterion of optimality, alignment nor treatment of indels alone can explain all differences. Parsimony rejects the monophyly of Gymnophthalmidae due to the position of Alopoglossinae relative to Teiidae, whereas support of Gymnophthalmidae by maximum‐likelihood was low. We address various nomenclatural issues, including Gymnophthalmidae Fitzinger, 1826 being an older name than Teiidae Gray, 1827. We recognize three families in the arrangement Alopoglossidae + (Teiidae + Gymnophthalmidae). Within Gymnophthalmidae we recognize Cercosaurinae, Gymnophthalminae, Rhachisaurinae and Riolaminae in the relationship Cercosaurinae + (Rhachisaurinae + (Riolaminae + Gymnophthalminae)). Cercosaurinae is composed of three tribes—Bachiini, Cercosaurini and Ecpleopodini—and Gymnophthalminae is composed of three—Gymnophthalmini, Heterodactylini and Iphisini. Within Teiidae we retain the currently recognized three subfamilies in the arrangement: Callopistinae + (Tupinambinae + Teiinae). We also propose several genus‐level changes to restore the monophyly of taxa.