Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity.

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Inference of time-ordered multibody interactions.

We introduce time-ordered multibody interactions to describe complex systems manifesting temporal as well as multibody dependencies. First, we show how the dynamics of multivariate Markov chains can be decomposed in ensembles of time-ordered multibody interactions. Then, we present an algorithm to extract those interactions from data capturing the system-level dynamics of node states and a measure to characterize the complexity of interaction ensembles. Finally, we experimentally validate the robustness of our algorithm against statistical errors and its efficiency at inferring parsimonious interaction ensembles.

Bayesian inference of transition matrices from incomplete graph data with a topological prior.

Many network analysis and graph learning techniques are based on discrete- or continuous-time models of random walks. To apply these methods, it is necessary to infer transition matrices that formalize the underlying stochastic process in an observed graph. For weighted graphs, where weighted edges capture observations of repeated interactions between nodes, it is common to estimate the entries of such transition matrices based on the (relative) weights of edges. However in real-world settings we are often confronted with incomplete data, which turns the construction of the transition matrix based on a weighted graph into an inference problem. Moreover, we often have access to additional information, which capture topological constraints of the system, i.e. which edges in a weighted graph are (theoretically) possible and which are not. Examples include transportation networks, where we may have access to a small sample of passenger trajectories as well as the physical topology of connections, or a limited set of observed social interactions with additional information on the underlying social structure. Combining these two different sources of information to reliably infer transition matrices from incomplete data on repeated interactions is an important open challenge, with severe implications for the reliability of downstream network analysis tasks. Addressing this issue, we show that including knowledge on such topological constraints can considerably improve the inference of transition matrices, especially in situations where we only have a small number of observed interactions. To this end, we derive an analytically tractable Bayesian method that uses repeated interactions and a topological prior to perform data-efficient inference of transition matrices. We compare our approach against commonly used frequentist and Bayesian approaches both in synthetic data and in five real-world datasets, and we find that our method recovers the transition probabilities with higher accuracy. Furthermore, we demonstrate that the method is robust even in cases when the knowledge of the topological constraint is partial. Lastly, we show that this higher accuracy improves the results for downstream network analysis tasks like cluster detection and node ranking, which highlights the practical relevance of our method for interdisciplinary data-driven analyses of networked systems.

Variation in the chemical profiles of three foxglove species in the central Balkans.

The aim of this study was to determine intra- and interspecies variation in the qualitative and quantitative composition of methanol-soluble metabolites in the leaves of three Digitalis species (D. lanata, D. ferruginea, and D. grandiflora) from the central Balkans. Despite the steady use of foxglove constituents for human health as valuable medicinal products, populations of the genus Digitalis (Plantaginaceae) have been poorly investigated to describe their genetic and phenetic variation. Following untargeted profiling using UHPLC-LTQ Orbitrap MS, by which we identified a total of 115 compounds, 16 compounds were quantified using the UHPLC(-)HESI-QqQ-MS/MS approach. In total, 55 steroid compounds, 15 phenylethanoid glycosides, 27 flavonoids, and 14 phenolic acid derivatives were identified across the samples with D. lanata and D. ferruginea showing a great similarity, while 15 compounds were characteristic only for D. grandiflora. The phytochemical composition of methanol extracts, considered here as complex phenotypes, are further examined along multiple levels of biological organization (intra- and interpopulation) and subsequently subjected to chemometric data analysis. The quantitative composition of the selected set of 16 chemomarkers belonging to the classes of cardenolides (3 compounds) and phenolics (13 compounds) pointed to considerable differences between the taxa studied. D. grandiflora and D. ferruginea were found to be richer in phenolics as compared to cardenolides, which otherwise predominate in D. lanata over other compounds. PCA revealed lanatoside C, deslanoside, hispidulin, and p-coumaric acid to be the main compounds contributing to the differences between D. lanata on one side and D. grandiflora and D. ferruginea on the other, while p-coumaric acid, hispidulin, and digoxin contribute to the diversification between D. grandiflora and D. ferruginea. However, quantitative variation in the metabolite content within species was faint with mild population diversification visible in D. grandiflora and particularly in D. ferruginea. This pointed to the highly conserved content and ratio of targeted compounds within the analyzed species, which was not severely influenced by the geographic origin or environmental conditions. The presented metabolomics approach might have, along with morphometrics and molecular genetics studies, a high information value for further elucidation of the relationships among taxa within the genus Digitalis.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklic & Milojevic and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5ßRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Functional Characterization of Genes Coding for Novel ß-D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Centaurium erythraea Rafn.

Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with ß-D glucose linked at C1 position. Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes ß-glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two ß-D-glucosidases (named CeBGlu1 and CeBGlu2) from centaury (Centaurium erythraea Rafn; fam. Gentianaceae), belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using in vitro assays with recombinant proteins, following their heterologous expression in E. coli and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid C. erythraea plants. Transient overexpression of CeBGlu1 and CeBGlu2 in C. erythraea leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI - MS2 profiling of leaves of diploid and tetraploid C. erythraea genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when CeBGlu1 and CeBGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that in planta metabolic engineering adopting transient overexpression of CeBGlu1 and CeBGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of C. erythraea.

Characteristics of warfarin under-prescription in older adults with atrial fibrillation.

BACKGROUND: The prevalence of atrial fibrillation (AF) and atrial flutter (AFl) increases with age. Under-prescription of anticoagulants in older adults can lead to increased morbidity and mortality. We analyzed warfarin prescription patterns in older adults. METHODS: In this observational single-center study, we analyzed 2179 consecutive patients with admission diagnosis of AF or AFl. Patients were divided into "older" (≥ 75 years old) and "younger" (<75 years old) groups. Prescription patterns of warfarin were analyzed. Patients discharged from the hospital on a non-warfarin anticoagulation were excluded. RESULTS: Of the 1988 patients analyzed, 46.9% were ≥75 years old, of which 50.8% were prescribed warfarin. There was no association between mean CHA2DS2-VASc score and warfarin prescription on discharge (OR = 1.06 (95% CI 0.93-1.21), p = 0.388) in the older group. After adjusting for hypertension, renal function, and Black race, warfarin prescription in older adults was independently associated with lower aspirin prescription rates (OR = 0.57 (95% CI 0.43-0.75), p < 0.001), lower body mass index (OR = 1.03 (95% CI 1.01-1.06), p = 0.018), and lower hemoglobin levels (OR = 1.11 (95% CI 1.04-1.19), p = 0.002). CONCLUSIONS: In our study, older adults (≥75 years old) with AF and AFl tended to have lower rates of warfarin prescription despite higher CHA2DS2-VASc score and higher risk of thromboembolic events. Anemia, lower body weight, and aspirin use were characteristics associated with warfarin under-prescription.

Phantom Shocks Associated With a Wearable Cardioverter Defibrillator.

Wearable cardioverter defibrillators (WCDs) are external devices capable of continuous cardiac rhythm monitoring as well as automatic detection and defibrillation of potentially life-threatening arrhythmias such as ventricular tachycardia (VT) and ventricular fibrillation (VF). They are an alternative approach for patients when an implantable cardioverter defibrillator (ICD) is not appropriate. Although treatment with ICD is considered highly effective for the primary and secondary prevention of sudden cardiac death (SCD) in high-risk patients susceptible to VT and VF, patients may still experience psychological difficulties such as fear of shock, avoidance of normal behaviors and reduced quality of life. One of these phenomena is phantom shock (PS), which is defined as a perception of having received a shock with no evidence of recorded defibrillation upon device interrogation. While PS has been reported in the ICD literature, to the best of our knowledge, we present the first known case of WCD-related PS. We also present a review of the current literature to explore the prevalence of PS, the factors associated with its pathogenesis and interventional studies aimed at reducing its occurrence. We highlight this case because PS is considered a phenomenon that few recognize, which should be discriminated from real device shocks before clinicians initiate treatment, device reprogramming or device discontinuation. We describe the psychosocial factors associated with PS to emphasize the importance of managing any associated psychiatric disorders and psychosocial factors both before and after initiation of device treatment.

Alterations in nepetalactone metabolism during polyethylene glycol (PEG)-induced dehydration stress in two Nepeta species.

A number of Nepeta species (fam. Lamiaceae) are interesting medicinal crops for arid and semi-arid areas, due to their ability to maintain essential developmental and physiological processes and to rationalize their specialized metabolism under water deficit growth conditions. The present research is, to our knowledge, the first attempt to investigate the molecular background of the dehydration-induced changes in specialized metabolism of Nepeta species, which will help to understand relations between dehydration stress on one hand and biomass production and yield of nepetalactone (NL) on the other. During the 6 days exposure of Nepeta rtanjensis Diklic & Milojevic and Nepeta argolica Bory & Chaub. ssp. argolica plants to PEG-induced dehydration stress under experimental in vitro conditions, decrease in transcript levels of the majority of 10 NL biosynthetic genes, and some of the 5 transcription factors (TFs) were recorded, simultaneously with the initial reduction in NL content. The two model species evidently employ similar strategies in response to severe dehydration stress; however N. rtanjensis is highlighted as the species more efficient in maintaining NL amounts in tissues. The results suggest trichome-specific and co-ordinately regulated NL biosynthesis at the level of gene expression, with trichome enriched MYC2 and YABBY5 TFs being the potential positive regulators. Manipulation of such TFs can be effective for engineering the NL biosynthetic pathway, and for the increased production of cis,trans-NL in N. argolica ssp. argolica and trans,cis-NL in N. rtanjensis.