Understanding the dynamics of rhizosphere microbial communities is essential for predicting future ecosystem function, yet most research focuses on either spatial or temporal processes, ignoring ...combined spatio-temporal effects. Using pyrosequencing, we examined the spatio-temporal dynamics of a functionally important community of rhizosphere microbes, the arbuscular mycorrhizal (AM) fungi. We sampled AM fungi from plant roots growing in a temperate grassland in a spatially explicit manner throughout a year. Ordination analysis of the AM fungal assemblages revealed significant temporal changes in composition and structure. Alpha and beta diversity tended to be negatively correlated with the climate variables temperature and sunshine hours. Higher alpha diversity during colder periods probably reflects more even competitive interactions among AM fungal species under limited carbon availability, a conclusion supported by analysis of beta diversity which highlights how resource limitation may change localized spatial dynamics. Results reveal distinct AM fungal assemblages in winter and summer at this grassland site. A seasonally changing supply of host-plant carbon, reflecting changes in temperature and sunshine hours, may be the driving force in regulating the temporal dynamics of AM fungal communities. Climate change effects on seasonal temperatures may therefore substantially alter future AM fungal community dynamics and ecosystem functioning.
No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first ...collected their remains, they included perhaps the 'strangest animals ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The advent of next-generation sequencing has allowed huge amounts of DNA sequence data to be produced, advancing the capabilities of microbial ecosystem studies. The current challenge is to identify ...from which microorganisms and genes the DNA originated. Several tools and databases are available for annotating DNA sequences. The tools, databases and parameters used can have a significant impact on the results: naïve choice of these factors can result in a false representation of community composition and function. We use a simulated metagenome to show how different parameters affect annotation accuracy by evaluating the sequence annotation performances of MEGAN, MG-RAST, One Codex and Megablast. This simulated metagenome allowed the recovery of known organism and function abundances to be quantitatively evaluated, which is not possible for environmental metagenomes. The performance of each program and database varied, e.g. One Codex correctly annotated many sequences at the genus level, whereas MG-RAST RefSeq produced many false positive annotations. This effect decreased as the taxonomic level investigated increased. Selecting more stringent parameters decreases the annotation sensitivity, but increases precision. Ultimately, there is a trade-off between taxonomic resolution and annotation accuracy. These results should be considered when annotating metagenomes and interpreting results from previous studies.
The sequence annotation accuracies of metagenomic analysis tools were evaluated using a simulated metagenome.
Graphical Abstract Figure.
The sequence annotation accuracies of metagenomic analysis tools were evaluated using a simulated metagenome.
The blood fluke Schistosoma mansoni can live for years in the hepatic portal system of its human host and so must possess very effective mechanisms of immune evasion. The key to understanding how ...these operate lies in defining the molecular organisation of the exposed parasite surface. The adult worm is covered by a syncytial tegument, bounded externally by a plasma membrane and overlain by a laminate secretion, the membranocalyx. In order to determine the protein composition of this surface, the membranes were detached using a freeze/thaw technique and enriched by sucrose density gradient centrifugation. The resulting preparation was sequentially extracted with three reagents of increasing solubilising power. The extracts were separated by 2‐DE and their protein constituents were identified by MS/MS, yielding predominantly cytosolic, cytoskeletal and membrane‐associated proteins, respectively. After extraction, the final pellet containing membrane‐spanning proteins was processed by liquid chromatographic techniques before MS. Transporters for sugars, amino acids, ions and other solutes were found together with membrane enzymes and proteins concerned with membrane structure. The proteins identified were categorised by their function and putative location on the basis of their homology with annotated proteins in other organisms.
The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the ...lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes.
We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands.
A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class ...of photosensitiser, exemplified by
DC324
, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of
in vitro
cellular assays confirmed ROS formation and
in vivo
cytotoxic activity was exemplified
via
irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.
Drug-like, donor-acceptor diphenylacetylenes cause efficient cell death upon photoactivation and hence have potential phototherapeutic applications.
Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for ...conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.
The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood.
The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Schistosomiasis, caused by parasitic helminths, remains a serious human disease in the tropics. Cercariae of Schistosoma mansoni infect their hosts by direct skin penetration, aided by secretions ...from acetabular and head glands. Both proteolytic and immunomodulatory properties have been ascribed to the released material, but to date only five isoforms of elastase and one putative anti-inflammatory protein (Sm16) have been cloned. We analyzed secretions from mechanically transformed cercariae by two-dimensional electrophoresis. An average gel image was created and compared with a separation of soluble larval extract, revealing a less complex spot pattern in the secretions with 60% of the spots matched to the larval extract. Subsequent tandem mass spectrometric analysis identified 48 spots from the released material, representing approximately 80% of its normalized volume. Twenty-nine of these are likely to originate in the vesicles, and 18 are likely to originate in the cytosol of the glands (the latter class being present due to holocrine secretion); one is unknown. The vesicular proteins were significantly more enriched than the cytosolic proteins in the released material when compared with the larval extract. A novel metalloproteinase (termed SmPepM8) was the second most abundant constituent after three isoforms of cercarial elastase. In addition, a dipeptidyl peptidase IV (SmDPP IV) was discovered but in much smaller quantity. A new serine protease inhibitor (SmSerp_c) was also prominent. Along with Sm16, four potential immunomodulators were identified, three with similarity to venom allergens (SmSCP_a, _b, and _c) and one with homology to the potassium channel blockers in scorpion venom (SmKK7). Interrogation of the expressed sequence tag database found transcripts encoding the majority of vesicular proteins present solely in the intramolluscan stages of the life cycle. Distinct patterns of radiolabel incorporation suggested three separate origins for the vesicular proteins. All the novel constituents merit investigation as vaccine candidates, and the potential immunomodulators merit investigation as therapeutic agents.
The intestinal helminth parasite,
Heligmosomoides polygyrus bakeri offers a tractable experimental model for human hookworm infections such as
Ancylostoma duodenale and veterinary parasites such as
...Haemonchus contortus. Parasite excretory–secretory (ES) products represent the major focus for immunological and biochemical analyses, and contain immunomodulatory molecules responsible for nematode immune evasion. In a proteomic analysis of adult
H. polygyrus secretions (termed HES) matched to an extensive transcriptomic dataset, we identified 374 HES proteins by LC–MS/MS, which were distinct from those in somatic extract HEx, comprising 446 identified proteins, confirming selective export of ES proteins. The predominant secreted protein families were proteases (astacins and other metalloproteases, aspartic, cysteine and serine-type proteases), lysozymes, apyrases and acetylcholinesterases. The most abundant products were members of the highly divergent venom allergen-like (VAL) family, related to
Ancylostoma secreted protein (ASP); 25 homologues were identified, with VAL-1 and -2 also shown to be associated with the parasite surface. The dominance of VAL proteins is similar to profiles reported for
Ancylostoma and
Haemonchus ES products. Overall, this study shows that the secretions of
H. polygyrus closely parallel those of clinically important GI nematodes, confirming the value of this parasite as a model of helminth infection.
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► We identify 374 proteins secreted by adult worms of the model nematode parasite Heligmosomoides polygyrus. ► Multiple proteases, lysozymes, apyrases and acetylcholinesterase were identified. ► The dominant gene family in the secretome are members of the venom allergen-like (VAL) family of no known function. ► The secretion of VAL proteins and enzymes is also found in secretomes of major human and veterinary nematodes. ► Up to 90 novel gene products were found to be secreted, all bearing signal peptides, by the adult parasites.
Low‑q Bicelles Are Mixed Micelles Caldwell, Tracy A; Baoukina, Svetlana; Brock, Ashton T ...
The journal of physical chemistry letters,
08/2018, Letnik:
9, Številka:
15
Journal Article
Recenzirano
Odprti dostop
Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of “isotropic” bicelles by small-angle neutron ...scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.
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