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How cuticular proteins (CPs) interact with chitin and with each other in the cuticle remains unresolved. We employed LC-MS/MS to identify CPs from 5-6 day-old adults of Anopheles gambiae released after serial extraction with PBS, EDTA, 2-8M urea, and SDS as well as those that remained unextracted. Results were compared to published data on time of transcript abundance, localization of proteins within structures and within the cuticle, as well as properties of individual proteins, length, pI, percent histidine, tyrosine, glutamine, and number of AAP[A/V/L] repeats. Thirteen proteins were solubilized completely, all were CPRs, most belonging to the RR-1 group. Eleven CPs were identified in both soluble fractions and the final pellet, including 5 from other CP families. Forty-three were only detected from the final pellet. These included CPRs and members of the CPAP1, CPF, CPFL, CPLCA, CPLCG, CPLCP, and TWDL families, as well as several low complexity CPs, not assigned to families and named CPLX. For a given protein, many histidines or tyrosines or glutamines appear to be potential participants in cross-linking since we could not identify any peptide bearing these residues that was consistently absent. We failed to recover peptides from the amino-terminus of any CP. Whether this implicates that location in sclerotization or some modification that prevents detection is not known. Soluble CPRs had lower isoelectric points than those that remained in the final pellet; most members of other CP families had isoelectric points of 8 or higher. Obviously, techniques beyond analysis of differential solubility will be needed to learn how CPs interact with each other and with chitin.

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum. The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Highly active antiretroviral therapy (HAART) significantly reduces HIV-1 replication and prevents progression to AIDS. However, residual low-level viremia (LLV) persists and long-lived viral reservoirs are maintained in anatomical sites. These reservoirs permit a recrudescence of viremia upon cessation of therapy and thus HAART must be maintained indefinitely. HIV-1 reservoirs include latently infected resting memory CD4⁺ T-cells and macrophages which may contribute to residual viremia. It has not been conclusively determined if a component of LLV may also be due to residual replication in cells with sub-therapeutic drug levels and/or long-lived chronically infected cells. In this study, RT-SHIV(mac239) diversity was characterized in five rhesus macaques that received a five-drug HAART regimen [tenofovir, emtricitabine, zidovudine, amdoxovir, (A, C, T, G nucleoside analogs) and the non-nucleoside reverse transcriptase (RT) inhibitor efavirenz]. Before maximal viral load suppression, longitudinal plasma viral RNA RT diversity was analyzed using a 454 sequencer. After suppression, LLV RT diversity (amino acids 65-210) was also assessed. LLV samples had viral levels less than our standard detection limit (50 viral RNA copies/mL) and few transient blips <200 RNA copies/mL. HAART was discontinued in three macaques after 42 weeks of therapy resulting in viral rebound. The level of viral divergence and the prevalence of specific alleles in LLV was similar to pre-suppression viremia. While some LLV sequences contained mutations not observed in the pre-suppression profile, LLV was not characterized by temporal viral evolution or apparent selection of drug resistance mutations. Similarly, resistance mutations were not detected in the viral rebound population. Interestingly, one macaque maintained a putative LLV predominant plasma clone sequence. Together, these results suggest that residual replication did not markedly contribute to LLV and that this model mimics the prevalence and phylogenetic characteristics of LLV during human HAART. Therefore, this model may be ideal for testing HIV-1 eradication strategies.

We performed an angle-resolved photoemission spectroscopy study of BaFe2As2, which is the parent compound of the so-called 122 phase of the iron-pnictide high-temperature superconductors. We reveal the existence of a Dirac cone in the electronic structure of this material below the spin-density-wave temperature, which is responsible for small spots of high photoemission intensity at the Fermi level. Our analysis suggests that the cone is slightly anisotropic and its apex is located very near the Fermi level, leading to tiny Fermi surface pockets. The bands forming the cone show an anisotropic leading edge gap away from the cone that suggests a nodal spin-density-wave description.

The discovery of high-temperature superconductivity in iron pnictides raised the possibility of an unconventional superconducting mechanism in multiband materials. The observation of Fermi-surface (FS)-dependent nodeless superconducting gaps suggested that inter-FS interactions may play a crucial role in superconducting pairing. In the optimally hole-doped Ba(0.6)K(0.4)Fe(2)As(2), the pairing strength is enhanced simultaneously (2Delta/T(c) approximately 7) on the nearly nested FS pockets, i.e., the inner hole-like (alpha) FS and the 2 hybridized electron-like FSs, whereas the pairing remains weak (2Delta/T(c) approximately 3.6) in the poorly nested outer hole-like (beta) FS. Here, we report that in the electron-doped BaFe(1.85)Co(0.15)As(2), the FS nesting condition switches from the alpha to the beta FS due to the opposite size changes for hole- and electron-like FSs upon electron doping. The strong pairing strength (2Delta/T(c) approximately 6) is also found to switch to the nested beta FS, indicating an intimate connection between FS nesting and superconducting pairing, and strongly supporting the inter-FS pairing mechanism in the iron-based superconductors.

Background: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs)., Results: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified., Conclusion: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Background: Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple., Results: Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes., Conclusion: Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of 'Royal Gala' apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.

The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5'-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.

Elastosis in human breast cancer has been compared with various prognostic indicators, but few studies have compared elastosis directly with prognosis. We examined 100 primary breast cancers for estrogen receptor (ER) and progesterone receptor (PR) content, tumor grade, and extent of elastosis and compared these data with clinical follow-up in 71 patients. The ER content was greater than 10 femtomoles (fmole) per milligram of protein in 68% of tumors, while PR content was greater than 10 fmole/mg of protein in 48%. Histologic study showed that 22% of the tumors were grade 1, 28% were grade 2, and 50% were grade 3. Grade 2 or grade 3 elastosis was seen in 36% of the tumors and grade 0 or grade 1 elastosis was seen in 64%. There was a negative correlation between tumor grade and elastosis grade and a positive correlation between ER content and elastosis grade. Elastosis grade and PR content did not correlate. A significant positive correlation existed between lymph node positivity and elastosis. No significant direct relationship between elastosis and tumor recurrence could be demonstrated once lymph node status was accounted for. Tumor elastosis correlates with histologic grade, receptor content, and lymph node involvement but does not appear to be a direct marker of prognosis.

An infant dying with pulmonary hypertension had a pulmonary vessel foreign body vasculitis as identified by light microscopy and characterized ultrastructurally by scanning electron microscopy and energy-dispersive x-ray analysis. The inclusions were of two distinct types: those containing silicon and titanium, and others consisting of talc. The possible sources of these inclusions and the importance of considering foreign body vasculitis in the pathogenesis of clinically idiopathic pulmonary hypertension are discussed.

1. The trans-epoxide of diethylstilboestrol and its pinacolone were synthesized chemically and the pinacolone shown to be formed from the epoxide by a non-enzymic process. 2. [14C]Diethylstiboestrol epoxide was converted by rat liver microsomal fraction into 4'-hydroxypropiophenone by a new type of cleavage reaction involving mono-oxygenase. Conditions for the formation of this metabolite and also water-soluble products were investigated together with the effect of inhibitors. A sex-difference in the conversion of diethylstilboestrol epoxide into 4'-hydroxypropiophenone and to polar and water-soluble products was observed. 3. Diethylstilboestrol epoxide was found to be a relatively stable compound that did not form a glutathione conjugate readily without further microsomal activation. A purified preparation of epoxide hydratase did not enhance its rate of conversion into the pinacolone. 4. Diethylstilboestrol epoxide was found to have about one-tenth the oestrogenic activity of diethylstilboestrol as measured by the increase in uterine weight or the induction of peroxidase in immature rat uteri. It was inactive as a mutagen when tested for its ability to inhibit bacteriophage phi X174 DNA viral replication. 5. The possible role of diethylstilboestrol epoxide as an intermediate in the metabolism of diethylstilboestrol and in mediating the harmful effects of this synthetic estrogen is discussed.

Since the recognition of the development of certain malignant neoplasms in association with thorium dioxide (Thorotrast), its presence has been documented by light microscopic appearance and time-consuming autoradiography. Energy dispersive x-ray microanalysis can be used in the rapid documentation of thorium in paraffin-embedded tissues and it is confirmed that thorium is the principal component of the granular deposits described by light microscopy.

In an episode of aflatoxicosis in feeder pigs, mortality was about 20%. Histopathologic findings characteristic of experimentally induced aflatoxicosis and the finding of aflatoxin B1 in the serum of pigs and in the cornbased feed confirmed the diagnosis. Aflatoxins B1 and B2 were found in the corn used to prepare the feed. Combine harvesting of the corn, which cracked a large percentage of the kernels, coupled with prolonged drying time of the corn probably contributed to the aflatoxin production. Although the corn was fed to adult swine without observable effect, 47 of the 250 feeder pigs developed typical signs of aflatoxicosis. Unseasonably cold weather apparently was a factor in initiating the onset of clinical signs and probably increased the severity of the disease.

A modified pectin agar medium was evaluated for the rapid isolation and presumptive identification of Yersinia enterocolitica. Of 118 isolates of Enterobacteriaceae tested, only the 13Y. enterocolitica and the three Klebsiella oxytoca strains produced colonies that depressed and sank into the agar. Yersinia enterocolitica was also easily identified in mixed cultures, even from inocula containing three times as many other Enterobacteriaceae organisms as Y. enterocolitica. The recovery of Y. enterocolitica was evaluated on Mueller-Hinton, pectin, Hektoen enteric, xylose lysine desoxycholate, Salmonella-Shigella, and MacConkey agars. Compared with Mueller-Hinton agar, the pectin agar showed a 100% recovery of Y. enterocolitica, with all strains having depressed colonies, while the other media showed lesser recoveries of only 5 to 25%, with no other discriminating colonial characteristic.

It has been shown that when purified polyoma (Py) virions are dissociated by incubation in 150 mM NaCl-50 mM Tris-HCl (pH 8.5) containing 1 mM EGTA and 3 mM DTT, two new polypeptides (MW 43.5K and 40K) are produced by proteolysis of virion polypeptide VP1. Proteolysis is blocked by diisopropyl fluorophosphonate (DFP) and phenylmethyl sulfonyl fluoride (PMSF), suggesting that the virion-associated enzyme is a serine protease. When Py virions were dissociated in the presence of radiolabeled DFP, only VP1 became labeled to any significant extent, which suggests that the protease activity is a property of this viral polypeptide and that the 43.5K and 40K species are produced by autodigestion.

The equilibria among spin-labeled amine local anesthetic species in dioleoylphosphatidylcholine liposomes at an anesthetic: lipid mole ratio of 1:100 are investigated. Electron spin resonance (ESR) spectra demonstrate that anesthetic mobility within the bilayer is charge-dependent, with the uncharged species the more mobile. Partition coefficient measurements confirm ESR evidence that changes in anesthetic mobility represent anesthetic-phospholipid interaction and not changes in bilayer fluidity. Spin-exchange attenuation experiments show that anesthetics within the bilayer are accessible to the aqueous medium. Dependence of tertiary-amine anesthetic pK on dielectric constant has been used to estimate the interfacial pK. We propose a model of equilibria among species of the tertiary amine anesthetic in the aqueous medium and those intercalated in the bilayer, including a species electrostatically bound to the lipid phosphate. Using experimentally determined equilibrium constants, the model provides the binding constant between the electrostatically bound and unbound cationic anesthetics within the bilayer. The model stimulates the pH dependence of the mobile fraction of total anesthetic population determined by subtraction techniques on experimental ESR spectra.

Two adults presented with frontal lobe masses. As visualized by computerized tomography, both lesions were large cysts with contrast-enhancing mural nodules and enhancing circumferential rims. En bloc resections of the mural nodules and cyst walls were performed. Pathological evaluation of each nodule disclosed a meningioma, and neoplastic cells were found in the distant cyst walls. Although the walls of large cysts associated with some meningiomas have been composed of reactive glia or collagen, the neoplastic character of the cysts in the present cases underscores the need for resection and careful pathological evaluation of the large cysts associated with meningiomas.

A 57-year-old man who died suddenly with severe bilateral mainstem bronchial collapse is described, and an alteration of the elastic tissue in the membranous portion of the bronchi is identified. The morphologic abnormalities, physiologic dynamics, and potential clinical consequences of such an alteration are discussed.