Functional reconstruction of a eukaryotic-like E1/E2/(RING) E3 ubiquitylation cascade from an uncultured archaeon.

The covalent modification of protein substrates by ubiquitin regulates a diverse range of critical biological functions. Although it has been established that ubiquitin-like modifiers evolved from prokaryotic sulphur transfer proteins it is less clear how complex eukaryotic ubiquitylation system arose and diversified from these prokaryotic antecedents. The discovery of ubiquitin, E1-like, E2-like and small-RING finger (srfp) protein components in the Aigarchaeota and the Asgard archaea superphyla has provided a substantive step toward addressing this evolutionary question. Encoded in operons, these components are likely representative of the progenitor apparatus that founded the modern eukaryotic ubiquitin modification systems. Here we report that these proteins from the archaeon Candidatus 'Caldiarchaeum subterraneum' operate together as a bona fide ubiquitin modification system, mediating a sequential ubiquitylation cascade reminiscent of the eukaryotic process. Our observations support the hypothesis that complex eukaryotic ubiquitylation signalling pathways have developed from compact systems originally inherited from an archaeal ancestor

The quality assessment of commercial Lycium berries using LC-ESI-MS/MS and chemometrics

Lycium (also known as Goji berry) is used in traditional Chinese medicine (TCM) with claimed benefits, including eye and liver protection, immune system fortification and blood glucose control. The commercially available product comes from either the L. barbarum or L. chinense species, with the former dominating the marketplace due to its better taste profile. The main objective of this study was to develop a validated LC-ESI-MS/MS method to quantify multiple key bio-active analytes in commercially available Lycium berries and to qualitatively assess these samples using a principal component analysis (PCA). A LC-ESI-MS/MS method for the quantitation of seven analytes selected using the Herbal Chemical Marker Ranking System (Herb MaRS) was developed. The Herb MaRS ranking system considered bioavailability, bioactivity and physiological action of each target analyte, its intended use and the commercial availability of an analytical standard. After method optimization combining high resolving power with selective detection, seven analytes were quantified and the Lycium samples were quantitatively profiled. Chromatographic spectra were also obtained using longer run-time LC-UV and GC-MS methods in order to qualitatively assess the samples using a principal component analysis (PCA). The result of the method validation procedure was a 15.5 min LC-ESI-MS/MS method developed for the quantification of seven analytes in commercial Lycium samples. Wide variation in analyte concentration was observed with the following results (analyte range in mg/g): rutin, 16.1–49.2; narcissin, 0.37–1.65; nictoflorin, 0.26–0.78; coumaric acid, 6.84–12.2; scopoletin, 0.33–2.61; caffeic acid, 0.08–0.32; chlorogenic acid, 1.1–9.12. The quantitative results for the L. barbarum and L. chinense species samples indicate that they cannot be di_erentiated based on the bio-actives tested. A qualitative assessment using PCA generated from un-targeted LC-UV and GC-MS phytochemical spectra led to the same conclusion. The un-targeted quantitative and qualitative phytochemical profiling indicates that commercial L. barbarum and L. chinense cannot be distinguished using chemical analytical methods. Genetic fingerprinting and pharmacological testing may be needed to ensure the efficacy of commercial Lycium in order to validate label claims

Looking through the 'window of opportunity': is there a new paradigm of podiatry care on the horizon in early rheumatoid arthritis?

Over the past decade there have been significant advances in the clinical understanding and care of rheumatoid arthritis (RA). Major paradigm changes include earlier disease detection and introduction of therapy, and 'tight control' of follow-up driven by regular measurement of disease activity parameters. The advent of tumour necrosis factor (TNF) inhibitors and other biologic therapies have further revolutionised care. Low disease state and remission with prevention of joint damage and irreversible disability are achievable therapeutic goals. Consequently new opportunities exist for all health professionals to contribute towards these advances. For podiatrists relevant issues range from greater awareness of current concepts including early referral guidelines through to the application of specialist skills to manage localised, residual disease activity and associated functional impairments. Here we describe a new paradigm of podiatry care in early RA. This is driven by current evidence that indicates that even in low disease activity states destruction of foot joints may be progressive and associated with accumulating disability. The paradigm parallels the medical model comprising early detection, targeted therapy, a new concept of tight control of foot arthritis, and disease monitoring

Quality control methods for herbal medicine : a multifaceted approach

The past decade has seen an unprecedented growth in the popularity of complementary medicines in Western countries. As the popularity of complementary medicines continues to grow, serious concerns have been raised about their quality and safety. Herbal medicine quality control (QC) and assurance (QA) poses a great challenge, as the large assortment of complex chemicals and the compositional variation found within herbal mixtures makes analysis especially difficult. Here, the two commonly used paradigms for herbal quality assessment; the compound- and pattern-based approaches are examined using model systems. The compound-based paradigm focuses on the quantitative analysis of one or more chemical constituents of an herb or formulation in order to ensure quality and hence product consistency. The complex nature of the 13-herb Endoherb formulation made it an exemplar model for assessing this approach. The methodology used in this study demonstrates how the systematic QC and QA of a complex herbal mixture can be carried out using a logical and systematic method for the selection of the analytes, and then developing a method for their sensitive, specific and accurate analysis. Several methods of analysis including high-performance liquid chromatography (LC) with photodiode array (PDA) and mass spectrometric (MS) detection, as well as gas chromatography (GC) with MS and flame ionisation detection (FID) were employed for the specific analysis of the selected analytes based on factors such as their polarity, volatility and presence of a chromophore. Sample preparation is rapid and simple, utilising sonication as the extraction method. The results show that increasing the organic modifier in the extraction solvent, even for predominantly polar analytes can increase the analyte extraction efficiency. The pattern-based method compares the chromatographic profile of the extract of a new batch with that of a target or reference batch. Combined with new genetic and pharmacological methods of analysis, the pattern-based approach can be an important addition to the characterisation of herbal authenticity and quality, especially in regards to the quality of the raw materials. E. arvense was used as a model to observe the effect of worldwide cultivation on phytochemical profile, genomic profile and pharmacological activity. LC-MS was shown to provide excellent sensitivity, resolution, and reproducibility for the chemical characterisation of the E. arvense extracts. Chromatographic pre-processing of LC-MS profile data using the statistical software 'R' was necessary for subsequent statistical analysis as the instrumental contribution to the profiles made the detection of peaks cumbersome and the statistical inferences inaccurate. The novel use of k-nearest neighbour analysis combined with principal component analysis allowed for an objective classification of sample grouping. Chemical measurement of the antioxidant capacity of the E. arvense extracts provided a basic and preliminary way to establish quality standards of pharmacological equivalence. Importantly, the results indicate that the implied biological effect of the extracts is not reflected in their chemical profile. Finally, DNA barcoding using the loci rbcL and matK was successfully used to authenticate the raw materials

Structural studies of the Type IX secretion system inner membrane complex

The Type IX Secretion System (T9SS) is a protein transport system found exclusively in the Bacteroidetes phylum of Gram-negative bacteria. The T9SS transports folded substrates across the outer membrane, including virulence factors of human pathogens, polysaccharide utilisation enzymes of environmental bacteria, and cell-surface adhesin molecules involved in Bacteroidetes gliding motility The inner membrane proteins GldL and GldM use the proton-motive force to energise both the T9SS and the gliding motility apparatus. I present structures from five different species of the transmembrane core of the GldLM complex, solved using electron cryo-microscopy. The GldLM complex has two copies of GldM with one transmembrane helix each, surrounded by five copies of GldL with two transmembrane helices each. Most of the cytoplasmic domain of GldL was unresolved. I also present a low-resolution structure of the full length orthologous PorLM complex from Porphyromonas gingivalis. Several polar residues are present in the transmembrane helices of GldL and GldM, suggesting that they may have a role in conducting protons across the inner membrane. I generated mutant strains where these polar residues were substituted with non-polar alternatives and found that some of these strains were deficient in T9SS and gliding motility. A universally conserved glutamate residue in GldL was essential to T9SS and gliding motility activity. In the structures of GldLM, one copy of this glutamate residue appears to form a salt bridge with a conserved arginine residue in one copy of GldM. I propose that a proton flow-dependent alternation of which copy of GldM is involved in salt bridge formation drives rotation of the GldM dimer, which spans the periplasm to connect the inner membrane and outer membrane components of the T9SS. These results reveal the GldLM complex to be a third class of ion-driven rotary motor after the ATP synthase and the MotAB flagellar stator family.</p

From classical taxonomy to genome and metabolome : towards comprehensive quality standards for medicinal herb raw materials and extracts

Fundamental to herbal medicine quality is the use of 'authentic' medicinal herb species. Species, however, 'represent more or less arbitrary and subjective man-made units'. Against this background, we discuss, with illustrative examples, the importance of defining species boundaries by accommodating both the fixed (shared) diagnostic and varying (within-species) traits in medicinal herb populations. We emphasize the role of taxonomy, floristic information and genomic profiling in authenticating medicinal herb species, in addition to the need to include within species phytochemical profile variations while developing herbal extract identification protocols. We outline the application of species-specific genomic and phytochemical markers, chemoprofiling and chemometrics as additional tools to develop qualifying herbal extract references. We list the diagnostic traits available subsequent to each step during the medicinal herb extract manufacturing process and delineate limits to qualification of extract references

Structures of the Type IX Secretion/gliding motility motor from across the phylum Bacteroidetes

Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221–233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases

PickYOLO: Fast deep learning particle detector for annotation of cryo electron tomograms

Particle localization (picking) in digital tomograms is a laborious and time-intensive step in cryogenic electron tomography (cryoET) analysis often requiring considerable user involvement, thus becoming a bottleneck for automated cryoET subtomogram averaging (STA) pipelines. In this paper, we introduce a deep learning framework called PickYOLO to tackle this problem. PickYOLO is a super-fast, universal particle detector based on the deep-learning real-time object recognition system YOLO (You Only Look Once), and tested on single particles, filamentous structures, and membrane-embedded particles. After training with the centre coordinates of a few hundred representative particles, the network automatically detects additional particles with high yield and reliability at a rate of 0.24–3.75 s per tomogram. PickYOLO can automatically detect number of particles comparable to those manually selected by experienced microscopists. This makes PickYOLO a valuable tool to substantially reduce the time and manual effort needed to analyse cryoET data for STA, greatly aiding in high-resolution cryoET structure determination

Validation of a method for the simultaneous determination of four schisandra lignans in raw herb and commercial dried aqueous extracts of Schizandra chinensis (Wu wei zi) by RP-LC with DAD

A rapid and specific reversed-phase high performance liquid chromatography (RP-LC) method with photodiode array detection (DAD) was developed and validated for the determination of four common schisandra lignans, schisandrin (1), schisandrol B (2), deoxyshisandrin (3) and γ-schisandrin (4), in raw herb materials and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi). The extraction solvent and extraction method were optimised where it was found that a 4 h Soxhlet extraction using methanol was successful at extracting >99.5% of each of the schisandra lignans analysed from the raw herb material. The sample preparation process for the dried aqueous extract samples involved sonication using methanol for 2 Ã 30 min. The herb and extract solutions were separated on a Varian Microsorb-MV 100-5 C18 column using a gradient mixture of 0.1% aqueous phosphoric acid and acetonitrile. Subsequent detection and quantitation of the schisandra lignans was performed at 210 nm. The correlation coefficients of the linear regression analysis performed on these calibration curves were >0.9996 for all four schisandra lignans assayed. The detection limits and quantification limits ranged from 0.12 to 0.57 and 0.41 to 1.89 mg g−1, respectively. The mean recoveries of the various analytes ranged from 92.20 to 107.01%. The method was used to investigate the levels of the four mentioned components in herb samples and dried aqueous extracts. The identities of the chromatographic peaks were confirmed by (+) electrospray ionisation LC–MS/MS

Genomic DNA extraction and barcoding of endophytic fungi

Endophytes live inter-and/or intracellularly inside healthy aboveground tissues of plants without causing disease. Endophytic fungi are found in virtually every vascular plant species examined. The origins of this symbiotic relationship between endophytes go back to the emergence of vascular plants. Endophytic fungi receive nutrition and protection from their hosts while the plants benefit from the production of fungal secondary metabolites, which enhance the host plants' resistance to herbivores, pathogens, and various abiotic stresses. Endophytic fungi have attracted increased interest as potential sources of secondary metabolites with agricultural, industrial, and medicinal use. This chapter provides detailed protocols for isolation of genomic DNA from fungal endophytes and its use in polymerase chain reaction-based amplification of the internal transcribed spacer region between the conserved flanking regions of the small and large subunit of ribosomal RNA for barcoding purposes