Molecular Verification of the UK National Collection of Cultivated Liriope and Ophiopogon Plants

open access articleA collection of cultivated Liriope and Ophiopogon plants was established in 1996–1998 and subsequently hosted at a horticultural college. Uncertainties about the identification of the accessions, compounded by potential errors in propagation and labelling have led to waning confidence in the identities of the plants in the collection. The potential for using DNA barcoding to determine the species identities of the accessions was investigated. The DNA barcode regions of the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) were amplified. DNA sequence analysis allowed the sequences of the accessions to be compared to reference sequences in public databases. A simple haplotype map of the characteristic polymorphic positions in the rbcL regions was used to clearly distinguish between the two genera and assign Ophiopogon accessions to individual species or sub-groups of species. The ITS sequence data confirmed these genus and species assignations and provided greater resolution to distinguish between closely related species. The combination of two DNA barcodes allowed most of the accessions to be assigned to individual species. This molecular verification confirmed the identity of about 70% of the accessions, with the remaining 30% demonstrating a range of mistaken identities at the species and genus level

DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region

open access articleThere is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 103 ITS copies µL−1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples

open access articleLiriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5′ end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLa) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs

Sequence-Specific Detection of Aristolochia DNA – A Simple Test for Contamination of Herbal Products

open access articleHerbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies

JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy

Decreasing JunB expression causes muscle atrophy, whereas overexpression induces hypertrophy and blocks atrophy via myostatin inhibition and regulation of atrogin-1 and MuRF expression via FoxO3

The Near-Earth Object Surveyor Mission

The Near-Earth Object (NEO) Surveyor mission is a NASA observatory designed to discover and characterize near-Earth asteroids and comets. The mission's primary objective is to find the majority of objects large enough to cause severe regional impact damage ($>$140 m in effective spherical diameter) within its five-year baseline survey. Operating at the Sun-Earth L1 Lagrange point, the mission will survey to within 45 degrees of the Sun in an effort to find the objects in the most Earth-like orbits. The survey cadence is optimized to provide observational arcs long enough to reliably distinguish near-Earth objects from more distant small bodies that cannot pose an impact hazard. Over the course of its survey, NEO Surveyor will discover $\sim$200,000 - 300,000 new NEOs down to sizes as small as $\sim$10 m and thousands of comets, significantly improving our understanding of the probability of an Earth impact over the next century.Comment: accepted to PS

The First Post-Kepler Brightness Dips of KIC 8462852

We present a photometric detection of the first brightness dips of the unique variable star KIC 8462852 since the end of the Kepler space mission in 2013 May. Our regular photometric surveillance started in October 2015, and a sequence of dipping began in 2017 May continuing on through the end of 2017, when the star was no longer visible from Earth. We distinguish four main 1-2.5% dips, named "Elsie," "Celeste," "Skara Brae," and "Angkor", which persist on timescales from several days to weeks. Our main results so far are: (i) there are no apparent changes of the stellar spectrum or polarization during the dips; (ii) the multiband photometry of the dips shows differential reddening favoring non-grey extinction. Therefore, our data are inconsistent with dip models that invoke optically thick material, but rather they are in-line with predictions for an occulter consisting primarily of ordinary dust, where much of the material must be optically thin with a size scale <<1um, and may also be consistent with models invoking variations intrinsic to the stellar photosphere. Notably, our data do not place constraints on the color of the longer-term "secular" dimming, which may be caused by independent processes, or probe different regimes of a single process

Role of Autophagy in the control of muscle mass

Protein degradation in skeletal muscle cells is essentially mediated by the activity of two highly conserved pathways, the ubiquitin-proteasome and the autophagy-lysosome pathway. In the ubiquitin-proteasome pathway, target proteins are conjugated to multiple ubiquitin moieties and ubiquitin-tagged proteins are degraded within the proteasome complex (Lecker et al., 2006; Mammucari et al., 2007). The ubiquitin-proteasome system is constitutively active in normal skeletal muscle and is responsible for the turnover of most soluble and myofibrillar muscle proteins. In the autophagy-lysosome system, portions of cytoplasm and cell organelles are sequestered into vacuoles, called autophagosomes, that are delivered to the lysosomes for the degradation of their content by acidic hydrolases (Lum et al., 2005). Also the autophagy system is constitutively active in skeletal muscle. The ubiquitin-proteasome system is constitutively active in muscle but its activity increases significantly during muscle atrophy due to activation of two ubiquitin-ligases: Atrogin-1/Mafbx and Murf1 (Gomes et al., 2001). The activation of these two genes is regulated by the transcription factor FoxO3. This factor is normally phosphorylated and inactivated by AKT / PKB. Conversely when this pathway is suppressed (eg during muscle atrophy), FoxO3 translocates into the nucleus where it can transactivate its target genes (Sandri et al., 2004; Stitt et al., 2004). Alteration of autophagy has been observed in various myopathies caused by genetic defects of lysosomal components, e.g. Pompe's and Danon's disease, or by drugs that inhibit lysosomal function, such as chloroquine (Shintani and Klionsky, 2004). During muscle atrophy induced by various debilitating conditions (such as fasting and diabetes), there is activation of several genes, named "Atrophy-Related-Genes" or “Atrogenes”. Among the atrogenes, two most-induced are two ubiquitin-ligases, Atrogin-1 and Murf1. Several autophagy genes belong to the “Atrogenes”. These genes are: LC3, GABARAP and BNIP3. During the first part of my PhD we focused on the transcriptional regulation of the autophagy genes. Our hypothesis was that FoxO3 can coordinate the ubiquitin-proteasome and the autophagy-lysosome system. To characterize the mechanisms that control the autophagic/lysosomal pathway during muscle atrophy in vivo, we first determined whether the Akt/mTOR pathway is involved in the regulation of some of autophagy-related genes. During starvation and denervation, two different models of muscle wasting, the Autophagy-Related-Genes are induced. Moreover these autophagy-related genes are suppressed by Akt, and acute activation of Akt in transgenic mice inhibits autophagy in atrophying muscle. Importantly mTOR pathway did not appear to play a significant role in the activation of the autophagic/lysosomal pathway during muscle atrophy. Indeed the regulation of autophagy-related genes and the formation of autophagic vesicles are not induce either by rapamycin, an inhibitor of mTOR, or by knocking down of mTOR. These findings are in agreement with previous studies (Kochl et al., 2006; Mordier et al., 2000; Sarkar et al., 2007; Yamamoto et al., 2006). We used gain- and loss-of-function experiments to determine the role of FoxO3 in the autophagic/lysosomal pathway. These experiments found two novel FoxO3 targets that regulate autophagy. LC3 and Bnip3 promoters contain several potential FoxO binding sites and ChIP (Chromatin-ImmunoPrecipitation) experiments on atrophying muscles showed that FoxO3 binds chromatin of their promoters in specific sites. The regions of FoxO3 interaction were cloned upstream luciferase gene and functional studies confirmed that FoxO3 transactivates LC3 and BNIP3 genes. Moreover, loss-function experiments showed that BNIP3 upregulation is necessary for autophagy induction in adult muscle. Finally, we asked whether the induction of autophagy is secondary to the activation of the ubiquitin-proteasome system. Inhibition of ubiquitin-proteasome system by pharmacological or genetic approach, did not affect autophagy, suggesting that the two degradation pathways are independently controlled by FoxO3 (Mammucari et al., 2007). Thus, FoxO3 coordinates the two major proteolytic systems of the cell. In the second part of my PhD I focused my studies on the role of basal autophagy in skeletal muscle homeostasis. It is known that excessive activation of autophagy aggravates muscle wasting by removing portion of cytoplasm, proteins, and organelles (Dobrowolny et al., 2008; Mammucari et al., 2007; Wang et al., 2005; Zhao et al., 2007). Conversely, inhibition of lysosome-dependent degradation causes myopathies like Pompe and Danon diseases, and autophagy inhibition is thought to play a role in many myopathies with inclusions or with abnormal mitochondria (Levine and Kroemer, 2008; Temiz et al., 2009). To understand the exact role of autophagy in physiology of skeletal muscle we have generated conditional knockout for Atg7 gene to block autophagy specifically in skeletal muscle. The Atg7 protein is crucial for the formation of the autophagy vesicles by the activations of different Atg proteins and for the formation of the autophagosome. To understand the role of the autophagy in adult skeletal muscle, Atg7 floxed mice were crossed with mice that express the Cre-recombinase under the muscle-specific promoter Myosin light chain 1f. Muscle-specific deletion of Atg7, resulted in profound muscle atrophy, accumulation of protein aggregates that are positive for p62/SQSTM1 and age-dependent decrease in force. Moreover Atg7 null muscles showed accumulation of abnormal mitochondria, distension of sarcoplasmic reticulum, sarcomere disorganization, and formation of aberrant concentric membranous structures. Moreover, muscle loss is more exacerbated in autophagy knockout mice during denervation and fasting. These results suggest that the autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Moreover Atg7 null muscles showed activation of endoplasmic reticulum chaperones, such as BiP, as well as the phosphorylation of eIF2α, suggesting an ongoing unfolded protein response. The failure of protein-folding quality control in Atg7 null mice induces endoplasmic reticulum stress which can generate ROS, and suppression of protein synthesis which can contribute to muscle atrophy (Masiero et al., 2009). To further confirm our findings in adulthood, we generated a tamoxifen-inducible muscle-specific Atg7 knockout mice. In this case, the floxed Atg7 mice were crossed with mice expressing the Cre-recombinase fused with a modified estrogen receptor, under the control of a muscle-specific promoter (Human Skletal Muscle). When animals are treated with tamoxifen (an estrogen analogue that has a high affinity for the modified estrogen receptor), the Cre-recombinase is stabilized and can recombinate the loxP site. Identical results were obtained in inducible Atg7 null muscles. These mice showed p62/SQSTM1 accumulation, muscle atrophy and decrease in force generation. Morphological analyses revealed accumulation of abnormal mitochondria in small atrophic fibers and the number of centrally nucleated fibers were more abundant after acute Atg7 deletion than in non-inducible autophagy-deficient muscles (Masiero et al., 2009). Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.Nel muscolo scheletrico, la degradazione proteica è principalmente mediata da due sistemi altamente conservati: il sistema ubiquitina-proteasoma e il sistema autofagico-lisosomiale. Nel sistema ubiquitina-proteasoma, le proteine destinate alla degradazione vengono poli-ubiquitinate e successivamente veicolate e degradate nel proteasoma. Tale sistema è costitutivamente attivo nel normale muscolo scheletrico ed è responsabile per il riciclo di proteine muscolari solubili e proteine miofibrillari (Lecker et al., 2006; Mammucari et al., 2007). Nel sistema autofagico-lisosomiale, porzioni citoplasmatiche e organelli vengono sequestrati all’interno di vescicole (autofagosomi), i quali successivamente si fondono con i lisosomi (Lum et al., 2005). Anche tale sistema è costitutivamente attivo nel muscolo scheletrico. Il sistema ubiquitina-proteosoma è costitutivamente attivo nel muscolo, però la sua attività aumenta in maniera significativa durante l’atrofia muscolare, dovuto all’attivazione di due ubiquitine-ligasi: Atrogin-1/Mafbx e Murf1 (Gomes et al., 2001). L’attivazione di questi due geni è regolata dal fattore di trascrizione FoxO3. Tale fattore è normalmente fosforilato e inattivo quando la via di segnale AKT/PKB è attiva; di contro quando tale via è repressa (ad esempio durante l'atrofia muscolare) il fattore di trascrizione può traslocare nel nucleo dove può attivare la trascrizione dei suoi geni target (Sandri et al., 2004; Stitt et al., 2004). Durante l’atrofia muscolare indotta da diverse condizioni debilitanti (ad esempio: digiuno e diabete), vi è l’attivazione di diversi geni, comunemente chiamati “Atrogenes” e i più indotti sono le due ubiquitine-ligasi Atrogin-1 e Murf-1. Tra questi “Atrogenes” fanno parte anche geni correlati all’autofagia. Questi geni sono: LC3, GABARAP e BNIP3. Durante la prima parte del mio dottorato di ricerca, ci siamo concentrati sulla regolazione trascrizionale dei geni dell’autofagia. La nostra ipotesi era che FoxO3 potesse regolare sia il sistema ubiquitina-proteasoma sia il sistama autofagico-lisosomiale a livello del muscolo scheletrico. Per caratterizzare i meccanismi che regolano il sistema autofagico durante l'atrofia muscolare in vivo, abbiamo analizzato se la via di segnale Akt/mTOR fosse coinvolta nella regolazione di alcuni geni autofagici. Durante l’atrofia muscolare indotta dal digiuno e dalla denervazione, abbiamo osservato che tali geni sono indotti (Mammucari et al., 2007). Comunque l’induzione di questi geni sono inibiti quando la via di segnale Akt è attiva e inoltre l’attivazione acuta di tale sistema, mediante l’utilizzo dei topi transgenici per Akt, inibisce il sistema autofagico durante l’atrofia muscolare. Inoltre, abbiamo osservato che la via di segnale mTOR non sembra svolgere un ruolo significativo nella attivazione della via autofagica-lisosomiale durante l'atrofia muscolare. Infatti la regolazione di geni autofagici e la formazione delle vescicole autofagiche non erano indotte sia in seguito al trattamento degli animali con il farmaco rapamicina (inibitore di mTOR), sia abbattendo mTOR. Questi risultati sono in accordo con studi precedenti (Kochl et al., 2006; Mordier et al., 2000; Sarkar et al., 2007; Yamamoto et al., 2006). Per capire il ruolo di FoxO3 nella regolazione del sistema autofagico-lisosomiale, ci siamo avvalsi di diverse metodiche sperimentali che consistevano nella gain/loss function. Tali esperimenti ci hanno permesso di identificare due nuovi geni bersaglio per Foxo3, i quali sono coinvolti nella regolazione dell’autofagia. Questi geni sono LC3 e Bnip3. L’analisi dei promotori di LC3 e Bnip3 ha evidenziato alcuni potenziali siti per l’interazione con il fattore di trascrizione FoxO3. Mediante l’utilizzo della metodica ChIP (Chromatin–ImmunoPrecipitation) abbiamo dimostrato che FoxO3, durante condizioni di atrofia, si lega in siti specifici dei promotori. Per validare queste osservazioni abbiamo condotto degli studi funzionali e quindi le regioni di interazione FoxO3 sono state clonate a monte del gene della luciferasi. Questi studi funzionali hanno confermato che FoxO3 è in grado di indurre l’espressione dei geni LC3 e BNIP3. Ulteriori esperimenti di loss-function hanno inoltre documentato che l’induzione di BNIP3 è necessaria per l’attivazione dell’ autofagia nel muscolo scheletrico adulto. Infine ci siamo chiesti se l'induzione del sistema autofagico fosse un evento secondario o no rispetto all'attivazione del sistema ubiquitina-proteasoma. L'inibizione del sistema ubiquitina-proteasoma, mediante approccio farmacologico o genetico, non ha influenzato l'autofagia, suggerendo che le due vie di degradazione proteica siano controllate da FoxO3 in modo indipendente (Mammucari et al., 2007). Questo ha dimostrato che il fattore di trascrizione FoxO3 è in grado di regolare due diversi sistemi proteolitici nel muscolo scheletrico. Nella seconda parte del mio dottorato ci siamo concentrati sulla comprensione del ruolo del sistema autofagico basale nell'omeostasi del muscolo scheletrico. E’ noto che una eccessiva attivazione dell’autofagia induce una esacerbata atrofia muscolare, dovuta ad una sproporzionata eliminazione di porzioni citoplasmatiche, proteine ed organelli (Dobrowolny et al., 2008; Mammucari et al., 2007; Wang et al., 2005; Zhao et al., 2007). Di contro, l'inibizione del sistema, dovuto a difetti genetici degli enzimi lisosomiali o a farmaci che inibiscono la funzione lisosomiale, come la clorochina (Shintani e Klionsky, 2004), causa diverse miopatie come le malattie di Pompe e di Danon. Si pensa che l'inibizione del sistema autofagico giochi un ruolo in molte miopatie caratterizzate da inclusioni, o che presentano mitocondri anormali (Levine e Kroemer, 2008; Temiz et al., 2009). In ogni caso il ruolo specifico del sistema autofagico nel muscolo scheletrico non è stato determinato. Per comprendere il ruolo esatto del sistema autofagico nella fisiologia del muscolo scheletrico, abbiamo generato dei topi transgenici-condizionali, in cui è stato deleto il gene Atg7 specificatamente a livello del muscolo scheletrico. Quindi per tale scopo, topi transgenici Atg7flox sono stati incrociati con dei topi esprimenti l’enzima Cre-recombinasi, regolata da un promotore muscolo-specifico (Myosin Light Chain 1f). La proteina Atg7 è fondamentale per la formazione delle vescicole autofagiche, mediante l’attivazione di diverse proteine Atg, e per la formazione degli autofagosomi. La delezione del gene Atg7 induce una profonda atrofia muscolare, formazione di aggregati proteici che risultano essere positivi per la proteina p62/SQSTM1 e una diminuzione della forza muscolare che è correlata con l’età dell’animale. Inoltre mediante microscopia elettronica, abbiamo rilevato che tali animali presentano dei depositi di mitocondri anormali, distensione reticolo sarcoplasmatico, disorganizzazione del sarcomero, e la formazione di strutture membranose aberranti e concentriche. Per di più, la perdita muscolare è più accentuata nei topi durante la denervazione ed il digiuno. Questi risultati suggeriscono che il sistema autofagico nel muscolo scheletrico è importante per evitare la perdita di massa muscolare e per mantenere l'integrità delle miofibre. Inoltre l’inibizione di Atg7 ha mostrato l'attivazione di proteine chaperonine associate al reticolo endoplasmatico, in particolare la proteina BIP, così come la fosforilazione di eIF2α, fattore d’inizio della traduzione, suggerendo una continua attivazione delle vie implicate nella regolazione delle proteine mal formate. La presenza di proteine alterate nei topi transgenici induce stress del reticolo endoplasmatico, che può generare ROS, e la soppressione della sintesi proteica, che possono contribuire ad atrofia muscolare (Masiero et al., 2009). Per confermare i dati ottenuti nei topi transgenici-condizionali, abbiamo generato un altro tipo di topo transgenico tamoxifen-inducibile per Atg7 muscolo specifico. In questo caso i topi Atg7flox sono stati incrociati con dei topi esprimenti la Cre-recombinasi fusa con un recettore degli estrogeni modificato, sotto il controllo di un promotore muscolo-specifico (Human Skletal Muscle). In condizioni normali il gene Atg7 sarà espresso in tutti i tessuti perché, in assenza del ligando per gli estrogeni, la proteina di fusione è bloccata e inattivata a livello citoplasmatico da un complesso di Heat Shock Protein. Quando trattiamo gli animali con il Tamoxifen, un analogo degli estrogeni che presenta un’alta affinità per il recettore degli estrogeni modificato, il legame del composto determina il distacco del complesso delle Heat Shock Protein e la traslocazione della proteina di fusione nel nucleo, dove può esplicare la sua attività enzimatica. In questo modo, in seguito al trattamento con il Tamoxifen, si ottiene la delezione del gene Atg7 solo a livello muscolare. Anche in questo tipo di transgenico si sono avuti gli stessi risultati ottenuti con i topi transgenici-condizionali per Atg7. Infatti si sono osservati aggregati proteici positivi per la proteina p62/SQSTM1, atrofia muscolare e riduzione della forza muscolare. Inoltre l’analisi morfologica, ha rilevato degli accumuli di mitocondri alterati nelle fibre atrofiche, ed un più abbondante numero di fibre con nuclei centrali dopo la delezione del gene Atg7 in maniera acuta rispetto agli animali non-inducibili (Masiero et al., 2009)