Mixed colonies of two species of congeneric stingless bees (Hymenoptera: Apinae,Meliponini) display environmentally‐acquired and endogenously‐produced recognition signals

Nestmate recognition is fundamental for the maintenance of social organization in insect nests. It is becoming well recognized that cuticle hydrocarbons mediate the recognition process, although the origin of recognition cues in stingless bees remains poorly explored. The present study investigates the effects of endogenously‐produced and environmentally‐acquired components in cuticular hydrocarbons in stingless bees. The tests are conducted using colonies of Plebeia droryana Friese and Plebeia remota Holmberg. Recognition tests are performed with four different groups: conspecific nestmates, conspecific non‐nestmates, heterospecifics and conspecific, genetically‐related individuals that emerge in a heterospecific nest. This last group is produced by introducing brood cells of P. droryana into a P. remota colony, and the resulting adult bees are tested for acceptance 10 days after emergence. For all groups, 15 individuals are sampled for chemical analysis. The results show the acceptance of all conspecific nestmates, and the rejection of almost every conspecific non‐nestmate and every heterospecific bee. Genetically‐related individuals emerging from heterospecific nests present intermediate rejection (66.7% rejection). Chemical analysis shows that P. droryana individuals emerging in a P. remota nest have small amounts of alkene and diene isomers found in P. remota cuticle that are not found in workers from the natal nest. The data clearly show that the majority of the compounds present in P. droryana cuticle are endogenously produced, although a few unsaturated compounds are acquired from the environment, increasing the chemical differences and, consequently, the rejection percentages.     

Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.     

A systematic study of the development of the airway (bronchial) system of the avian lung from days 3 to 26 of embryogenesis: a transmission electron microscopic study on the domestic fowl, Gallus gallus variant domesticus

In the embryo of the domestic fowl, Gallus gallus variant domesticus, the lung buds become evident on day 3 of development. After fusing on the ventral midline, the single entity divides into left and right primordial lungs that elongate caudally while diverging and shifting towards the dorsolateral aspects of the coelomic cavity. On reaching their definitive topographical locations, the lungs rotate along a longitudinal axis, attach, and begin to slide into the ribs. First appearing as a solid cord of epithelial cells that runs in the proximal-distal axis of the developing lung, progressively, the intrapulmonary primary bronchus begins to canalize. In quick succession, secondary bronchi sprout from it in a craniocaudal sequence and radiate outwards. On reaching the periphery of the lung, parabronchi (tertiary bronchi) bud from the secondary bronchi and project into the surrounding mesenchymal cell mass. The parabronchi canalize, lengthen, increase in diameter, anastomose, and ultimately connect the secondary bronchi. The luminal aspect of the formative parabronchi is initially lined by a composite epithelium of which the peripheral cells attach onto the basement membrane while the apical ones project prominently into the lumen. The epithelium transforms to a simple columnar type in which the cells connect through arm-like extensions and prominently large intercellular spaces form. The atria are conspicuous on day 15, the infundibulae on day 16, and air capillaries on day 18. At hatching (day 21), the air and blood capillaries have anastomosed profusely and the blood-gas barrier become remarkably thin. The lung is well developed and potentially functionally competent at the end of the embryonic life. Thereafter, at least upto day 26, no further consequential structures form. The mechanisms by which the airways in the avian lung develop fundamentally differ from those that occur in the mammalian one. Compared with the blind-ended bronchial system that inaugurates in the mammalian lung, an elaborate, continuous system of air conduits develops in the avian one. Further studies are necessary to underpin the specific molecular factors and genetic processes that direct the morphogenesis of an exceptionally complex and efficient respiratory organ.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

A novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers for the enrichment and determination of polychlorinated biphenyls in fish samples

The novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers (rGO@m‐MIPs) were successfully synthesized as adsorbents for six kinds of polychlorinated biphenyls (PCBs) in fish samples. rGO@m‐MIPs was prepared by surface molecular imprinting technique. Besides, Fe₃O₄ nanoparticles (NPs) were employed as magnetic supporters, and rGO@Fe₃O₄ was in situ synthesis. Different from functional monomer and cross‐linker in traditional molecularly imprinted polymer, here, 3,4‐dichlorobenzidine was employed as dummy molecular and poly(ethylene‐co‐vinyl alcohol) was adopted as the imprinted polymers. After morphology and inner structure of the magnetic adsorbent were characterized, the adsorbent was employed for disperse solid phase extraction toward PCBs and exhibited great selectivity and high adsorption efficiency. This material was verified by determination of PCBs in fish samples combined with gas chromatography‐mass spectrometry (GC‐MS) method. According to the detection, the low detection limits (LODs) of PCBs were 0.0035–0.0070 µg l⁻¹ and spiked recoveries ranged between 79.90 and 94.23%. The prepared adsorbent can be renewable for at least 16 times and expected to be a new material for the enrichment and determination of PCBs from contaminated fish samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of two components of the female sex pheromone of the sugarcane‐borer Diatraea flavipennella (Lepidoptera: Crambidae)

Analysis by gas chromatography with electroantennographic detection of extracts of pheromone glands derived from calling females of the sugarcane‐borer Diatraea flavipennella revealed two antennally active compounds. These components were identified as (Z)‐9‐hexadecenal (Z9–16:Ald) and (Z)‐11‐hexadecenal (Z11–16:Ald) by comparison of the retention times of the natural compounds and the synthetic compounds supported by two‐dimensional gas chromatography – time‐of‐flight mass spectrometric analysis and the positions of the double bounds in the chains were confirmed from the mass spectral fragmentation patterns of their dimethyldisulphide adducts. The analysis indicated that Z9–16:Ald and Z11–16:Ald were present in the sex pheromone in the proportions 25 : 75. Trace amounts of tetradecanal, hexadecanal, (Z)‐7‐hexadecenal (Z7–16:Ald), (Z)‐9‐hexadecen‐1‐ol and (Z)‐11‐hexadecen‐1‐ol were also found in the extract, but of these only Z9–16:Ald and Z11–16:Ald appeared to be antennally active. Behavioural bioassays demonstrated that a binary blend composed of Z9–16:Ald and Z11–16:Ald in the ratio of 25 : 75 induced a response in D. flavipennella virgin males similar to that elicited by live virgin females or by an hexane extract of the pheromone glands of calling females. Z9–16:Ald and Z11–16:Ald are, therefore, considered to be the major constituents of the female sex pheromone of D. flavipennella.     

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.     

IMPROVED DETECTION AND CHARACTERIZATION OF FUCOXANTHIN‐TYPE CAROTENOIDS: NOVEL PIGMENTS IN EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

A new atmospheric pressure chemical ionization mass spectrometry (APCI‐LC/MS) method improved detection and aided characterization of fucoxanthin related carotenoids, revealing the coccolithophorid Emiliania huxleyi (Lohm.) Hay et Mohler (strain MBA 92, Plymouth) to contain a wider range of acyloxyfucoxanthins than reported previously. The diversity is confirmed as arising from differences in the length of the alkanoic acid substituent esterified at position C‐19′. Acyloxyfucoxanthins with substituents of between four and eight carbons at the C‐19′ position have been detected in a culture of Emiliania huxleyi, where previously only 19′‐butanoyloxyfucoxanthin and 19′‐hexanoyloxyfucoxanthin have been reported in the literature. Novel fucoxanthinol derivatives were also found. The detection of these novel carotenoids in Emiliania huxleyi permits detailed studies of the impact of environmental factors on individual components of the complex pool of fucoxanthin‐type carotenoids in this organism.     

Toxicological characterization of Fukuyoa paulensis (Dinophyceae) from temperate Australia

Dinoflagellates of the genus Gambierdiscus are known to produce neurotoxins that cause the human illness ciguatera, a tropical and sub‐tropical fish poisoning. Some species from the Gambierdiscus genus were recently re‐classified into a new genus, Fukuyoa based on their phylogenetic and morphological divergence, however, little is known about their distribution, ecology and toxicology. Here we report the first occurrence of F. paulensis in the temperate coastal waters of eastern Australia and characterize its toxicology. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) did not detect the presence of ciguatoxins, however, a putative maitotoxin congener (MTX‐3) was present. Similarly, high maitotoxin‐like activity was detected in High Performance Liquid Chromatography (HPLC) fractionated cell extracts using a Ca²⁺ influx bioassay on a Fluorescent Imaging Plate Reader (FLIPR), but no ciguatoxin‐like activity was detected.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

Chemical Variation in Essential Oils from the Oleo‐gum Resin of Boswellia carteri: A Preliminary Investigation

Frankincense, the oleo‐gum resin of Boswellia species, has been an important element of traditional medicine for thousands of years. Frankincense is still used for oral hygiene, to treat wounds, and for its calming effects. Different Boswellia species show different chemical profiles, and B. carteri, in particular, has shown wide variation in essential oil composition. In order to provide insight into the chemical variability in authentic B. carteri oleoresin samples, a hierarchical cluster analysis of 42 chemical compositions of B. carteri oleo‐gum resin essential oils has revealed at least three different chemotypes, i) an α‐pinene‐rich chemotype, ii) an α‐thujene‐rich chemotype, and iii) a methoxydecane‐rich chemotype.     

Morphological,Secondary Metabolite and ITS (rDNA) Variability within Usnic Acid‐Containing Lichen Thalli of Xanthoparmelia Explored at the Local Scale of Rock Outcrop in W‐Alps

Lichen secondary metabolites (LSMs) are regarded with interest for valuable biological properties, but chemical variability among/within lichen taxa has been only fragmentarily characterized by advanced analytical techniques. Knowledge of variability at a local geographic scale has been particularly neglected, while it should address the collection of chemically homogeneous materials to test and exploit LSMs. Here we evaluated the chemical variability of 48 Xanthoparmelia specimens from two rock outcrops in Western Italian Alps, representative of nine morphotypes and sixteen rDNA ITS haplotypes. Qualitative and quantitative analyses were performed by HPLC‐DAD‐ESI‐MS₂ and UPLC‐HDR‐DAD, respectively, and revealed the occurrence of 18 LSMs. Chemical partition allowed distinguishing six chemical groups, only partially overlapping with distinct morphotypes and three divergent haplotype groups, which, overall, accounted for the co‐occurrence of different taxa only in part identifiable with species described for Europe. Some morphotypes were variable in presence and concentration of LSMs, and chemical divergences also characterized single ITS haplotypes. Accordingly, the collection of chemically homogeneous materials, even at a local scale, may be not properly addressed by morphological features and ITS barcoding, and should be confirmed by a specimen‐level chemical characterization.     

Headspace Solid-Phase Micro-Extraction Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS)-Based Untargeted Metabolomics Analysis for Comparing the Volatile Components from 12 <i>Panax</i> Herbal Medicines

Quality control of ginseng currently is mainly based on ginsenoside analysis, but rarely focuses on the volatile organic components. In the current work, an untargeted metabolomics approach, by headspace solid-phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS), was elaborated and further employed to holistically compare the compositional difference of the volatile components simultaneously from 12 Panax herbal medicines, which included P. ginseng (PG), P. quinquefolius (PQ), P. notoginseng (PN), red ginseng (PGR), P. ginseng leaf (PGL), P. quinquefolius leaf (PQL), P. notoginseng leaf (PNL), P. ginseng flower (PGF), P. quinquefolius flower (PQF), P. notoginseng flower (PNF), P. japonicus (PJ), and P. japonicus var. major (PJvm). Chromatographic separation was performed on an HP-5MS elastic quartz capillary column using helium as the carrier gas, enabling good resolution within 1 h. We were able to characterize totally 259 volatile compounds, including 82 terpenes (T), 46 alcohols (Alc), 29 ketones (K), 25 aldehydes (Ald), 21 esters (E), and the others. By analyzing 90 batches of ginseng samples based on the untargeted metabolomics workflows, 236 differential ions were unveiled, and accordingly 36 differential volatile components were discovered. It is the first report that simultaneously compares the compositional difference of volatile components among 12 Panax herbal medicines, and useful information is provided for the quality control of ginseng aside from the well-known ginsenosides.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.