Polyphenols and Phenolic Glucosides in Antibacterial Twig Extracts of Naturally Occurring Salix myrsinifolia (Salisb.), S. phylicifolia (L.) and S. starkeana (Willd.) and the Cultivated Hybrid S. x pendulina (Wender.).

(1) Background: Salix species occurring in Finland have not been well studied for their antimicrobial potential, despite their frequent use for lung and stomach problems in traditional medicine. Thus, twig extracts of three species of Salix that are found naturally in Finland and one cultivated species were screened for their antimicrobial properties against human pathogenic bacteria. S. starkeana and S. x pendulina were screened for antibacterial effects for the first time. (2) Methods: An agar diffusion and a microplate method were used for the screenings. Time-kill effects were measured using a plate-count and a microplate method. A DPPH-method using a qualitative TLC-analysis was used to detect antioxidant compounds in antimicrobial extracts. Metabolites from a S. myrsinifolia extract showing good antibacterial effects were identified using UPLC/QTOF-MS. (3) Results: A methanol extract of S. starkeana was particularly active against B. cereus (MIC 625 µg/mL), and a methanol extract of S. myrsinifolia showed good activity against S. aureus and B. cereus (MIC 1250 µg/mL) and showed bactericidal effects during a 24 h incubation of B. cereus. Moreover, a decoction of S. myrsinifolia resulted in good growth inhibition against P. aeruginosa. Our UPLC/QTOF-MS results indicated that proanthocyanidins (PAs), and especially the dimer procyanidin B1 (m/z 577) and other procyanidin derivatives, including highly polymerized proanthocyanidins, were abundant in S. myrsinifolia methanol extracts. Procyanidin B1 and its monomer catechin, as well as taxifolin and p-hydroxycinnamic acid, all present in S. myrsinifolia twigs, effectively inhibited B. cereus (MIC 250 µg/mL). (4) Conclusions: This study indicates that Finnish Salix species contain an abundance of antibacterial condensed tannins, phenolic acids and other polyphenols that deserve further research for the antibacterial mechanisms of action.

Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria.

Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary oxidative phosphorylation (OXPHOS) deficiency can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, and cellular senescence in the affected organs such as liver and kidney, and a systemic phenotype resembling juvenile-onset progeroid syndromes. Mechanistically, CIII deficiency triggers presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. Transgenic alternative oxidase dampens mitochondrial integrated stress response and the c-MYC induction, suppresses the illicit proliferation, and prevents juvenile lethality despite that canonical OXPHOS-linked functions remain uncorrected. Inhibition of c-MYC with the dominant-negative Omomyc protein relieves the DNA damage in CIII-deficient hepatocytes in vivo. Our results connect primary OXPHOS deficiency to genomic instability and progeroid pathogenesis and suggest that targeting c-MYC and aberrant cell proliferation may be therapeutic in mitochondrial diseases.

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana.

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.

Multi-modal meta-analysis of cancer cell line omics profiles identifies ECHDC1 as a novel breast tumor suppressor.

Molecular and functional profiling of cancer cell lines is subject to laboratory-specific experimental practices and data analysis protocols. The current challenge therefore is how to make an integrated use of the omics profiles of cancer cell lines for reliable biological discoveries. Here, we carried out a systematic analysis of nine types of data modalities using meta-analysis of 53 omics studies across 12 research laboratories for 2,018 cell lines. To account for a relatively low consistency observed for certain data modalities, we developed a robust data integration approach that identifies reproducible signals shared among multiple data modalities and studies. We demonstrated the power of the integrative analyses by identifying a novel driver gene, ECHDC1, with tumor suppressive role validated both in breast cancer cells and patient tumors. The multi-modal meta-analysis approach also identified synthetic lethal partners of cancer drivers, including a co-dependency of PTEN deficient endometrial cancer cells on RNA helicases.

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

Enzymatically synthesized 2'-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity.

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2'-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2'-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2'-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2'-F-cytidine and 2'-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2'-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2'-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2' position, for further antiviral studies in vitro and in vivo.

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTß, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.

Epidermal UV-A absorbance and whole-leaf flavonoid composition in pea respond more to solar blue light than to solar UV radiation.

Plants synthesize phenolic compounds in response to certain environmental signals or stresses. One large group of phenolics, flavonoids, is considered particularly responsive to ultraviolet (UV) radiation. However, here we demonstrate that solar blue light stimulates flavonoid biosynthesis in the absence of UV-A and UV-B radiation. We grew pea plants (Pisum sativum cv. Meteor) outdoors, in Finland during the summer, under five types of filters differing in their spectral transmittance. These filters were used to (1) attenuate UV-B; (2) attenuate UV-B and UV-A < 370 nm; (3) attenuate UV-B and UV-A; (4) attenuate UV-B, UV-A and blue light; and (5) as a control not attenuating these wavebands. Attenuation of blue light significantly reduced the flavonoid content in leaf adaxial epidermis and reduced the whole-leaf concentrations of quercetin derivatives relative to kaempferol derivatives. In contrast, UV-B responses were not significant. These results show that pea plants regulate epidermal UV-A absorbance and accumulation of individual flavonoids by perceiving complex radiation signals that extend into the visible region of the solar spectrum. Furthermore, solar blue light instead of solar UV-B radiation can be the main regulator of phenolic compound accumulation in plants that germinate and develop outdoors.

Multiple roles for UV RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation.

Photomorphogenic responses triggered by low fluence rates of ultraviolet B radiation (UV-B; 280-315 nm) are mediated by the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8). Beyond our understanding of the molecular mechanisms of UV-B perception by UVR8, there is still limited information on how the UVR8 pathway functions under natural sunlight. Here, wild-type Arabidopsis (Arabidopsis thaliana) and the uvr8-2 mutant were used in an experiment outdoors where UV-A (315-400 nm) and UV-B irradiances were attenuated using plastic films. Gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation, and leaf metabolite signatures were analyzed. The results show that UVR8 is required for transcript accumulation of genes involved in UV protection, oxidative stress, hormone signal transduction, and defense against herbivores under solar UV. Under natural UV-A irradiance, UVR8 is likely to interact with UV-A/blue light signaling pathways to moderate UV-B-driven transcript and PDX1 accumulation. UVR8 both positively and negatively affects UV-A-regulated gene expression and metabolite accumulation but is required for the UV-B induction of phenolics. Moreover, UVR8-dependent UV-B acclimation during the early stages of plant development may enhance normal growth under long-term exposure to solar UV.