DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least–squares to latent structures–discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.The study was financially supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement No. 606895 (http://cordis.europa.eu/project/rcn/109122_en.html) as well as a minor contribution from Bergthora and Thorsteinn Scheving Thorsteinsson Fund. The funders provided support in the form of salaries for author [MX], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author is articulated in the ‘author contributions’ section. The commercial affiliation Arctic Mass has no financial or competing interests in this study but two of the authors MT and FFE are partially affiliated there. Arctic Mass had the role of providing access to instruments (mass spectrometers) used in the study.Peer Reviewe

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials

Maternal milk feedings and cytomegalovirus infection in preterm infants in sweden

In Sweden, preterm infants are preferably fed human milk. Very preterm infants (< 32 weeks), who are unable to breastfeed, are fed with expressed maternal milk via a nasogastric tube. Mothers of these infants often experience difficulties in establishing and maintaining lactation. The majority of women excrete cytomegalovirus (CMV) in their breast milk. CMV transmitted through maternal milk can cause symptomatic infection in preterm infants presenting as a sepsis like syndrome, pneumonitis, hepatopathy or enterocolitis. Routine freezing of maternal milk decreases the CMV load in breast milk and is used in some neonatal centers to reduce CMV transmission to preterm infants. The aims of the studies in this thesis were to document existing routines pertaining to breast milk use for preterm infants in Sweden, to investigate predictors of maternal milk feedings in extremely preterm infants (EPIs, < 28 weeks), to evaluate the rate and clinical expression of postnatal CMV infection in EPIs, to evaluate the effect of routine freezing of maternal milk on CMV transmission rate, CMV associated disease and neonatal morbidity and mortality in EPIs and to evaluate the prevalence of CMV infection in intestinal specimens from infants with necrotizing enterocolitis (NEC), spontaneous intestinal perforation (SIP) and related surgical conditions. In a national cross sectional study in 2006 in Sweden, we found that 27 of 36 (75%) neonatal units had their own milk bank. Milk donors were screened for human immunodeficiency virus, human T-lymphotropic virus, and hepatitis B and C viruses by 27 (100%), 14 (52%), and 22 (81%) of the milk banks, respectively. Bacterial culture was performed on donor milk in 24 (89%) milk banks. Donor milk was pasteurized in 22 (81%) milk banks. In 11 of the 36 (31%) neonatal units maternal milk was frozen to reduce the risk ofCMV transmission. Nutritional analysis of donor and/or maternal milk was performed in 25 (69%) units. In a prospective cohort study at the neonatal units in Stockholm, including 97 mothers and their singleton EPIs, predictors of maternal milk feedings in EPIs during the first 6 weeks of life and at discharge were evaluated. Favorable predictors of maternal milk feedings the first 6 weeks of life were high maternal milk feedings (>90%) at second week of life, maternal university education and Nordic origin of the mother. The proportion of maternal milk feedings the first 6 weeks of life and maternal age were positively associated to the provision of maternal milk feedings at discharge while maternal overweight was an unfavorable predictor. High maternal milk feedings (>90%) at second week of life, assisted reproduction technology and maternal employment were predictive factors for exclusive maternal milk feedings at discharge. Ten EPIs and their 6 mothers were included in a pilot study at the neonatal unit, Astrid Lindgrens Children´s hospital to evaluate the rate and clinical expression of breast milk induced CMV infection. Five (83%) mothers were CMV-seropositive; of these, 4 (80%) excreted CMV-DNA in breast milk and 2 (40%) had a positive CMV culture. CMV was detected in the urine of 2/7 (29%) EPIs fed with CMV-positive milk; both were fed with breast milk positive for CMV culture. One EPI, later diagnosed with cystic fibrosis, developed hepatic affection concurrent with CMV urine excretion. In a randomized study at the neonatal units in Stockholm, evaluating the effect of routine freezing of maternal milk on postnatal CMV infection and neonatal outcome, 140 EPIs were randomized to be fed only freeze-thawed maternal milk (intervention group, IG) or both fresh maternal milk and freeze-thawed maternal milk (control group, CG). Outcome measures were CMV transmission rate and symptomatic infection in EPIs, neonatal mortality and morbidity during hospital stay. Fifty-six EPIs in the IG and 65 EPIs in the CG were included in the final per protocol analysis. We observed an overall low CMV transmission rate (8%) to EPIs from mothers with detectable CMV in breast milk. Routine freezing of maternal milk did not reduce the rate of CMV transmission (9% in IG vs 6% in CG). Congenital CMV infection was detected in 2% of screened infants. No infected EPI presented with clinical symptoms of CMV infection. Mortality rates were similar; 7% in the IG and 6% in the CG. Neonatal morbidity did not differ except for late onset Candida sepsis; the incidence was 12% in the CG while no case was observed in the IG. In a retrospective observational study, we investigated the occurrence of the CMV in 70 intestinal specimens from 61 infants with NEC, SIP and related surgical conditions at the Karolinska University Hospital Solna and Uppsala University Hospital. Ten intestinal specimens from autopsied infants without bowel disease were controls. By using immunohistochemistry (IHC), we detected the CMV specific proteins CMV-immediate early antigen (CMV-IEA) in 81% (57/70) and CMV-late antigen (CMV-LA) in 64% (45/70) of the intestinal specimens; 2/10 (20%) of the control specimens were positive for both antigens. Although CMV antigens were prevalent irrespective of pathologic diagnosis, they were most frequent in specimens with the pathologic diagnosis NEC and intestinal perforation; 95% and 89% ofthese tissue specimens were positive for CMV-IEA and CMV-LA, respectively. CMV infection was confirmed by CMV-DNA analysis in 4/10 (40%) CMV-IHC-positive intestinal samples using Taqman PCR after laser capture microdissection and in 13/13 (100%) CMV-IHC-positive intestinal samples by in situ hybridization. To conclude, human milk handling routines vary between neonatal units in Sweden and need to be standardized. Mothers of EPIs should aim for a high breast milk production immediately after delivery to optimize lactation success. Mothers who are young, overweight, ofnon-Nordic origin or without university education may need special lactation support. Postnatal CMV transmission from mothers excreting CMV in breast milk to EPIs was low (8%) and was not reduced by routine freezing of maternal milk. However, congenital CMV infection in EPIs was unexpectedly high (2%). No EPI infected by CMV presented with clinical symptoms. Routine freezing of maternal milk did not affect neonatal death in EPIs although it may have protected against fungal late onset sepsis. CMV infection was prevalent in intestinal specimens from infants with NEC, SIP and related surgical condition implicating a possible role of the virus in disease pathogenesis. More studies are needed to further evaluate the risk/benefit ratio ofmaternal milk feedings in EPIs with regard to the short-term and long-term effects of postnatal CMV infection

A report on operating a nationwide human milk bank in Korea

Purpose : A human milk bank collects, processes, eliminates, and stores breast milk from donors and provides breast milk to those in need. The authors hereby present the experiences and the objective lessons obtained through operating a nationwide human milk bank over a period of 2 years. Methods : The characteristics of the donors and the recipients and the amounts of breast milk donated, processed, and received at the East-West Neo Medical Center Human Milk Bank were investigated from August 2007 to August 2009. Results : The donor pool consisted of 131 first-time donors and 39 repeat donors who made 341 and 127 donations, respectively. Seventy-nine percent of the donors resided in the Seoul-Kyunggi area, and 60&#37; of the donors were in their 30s. Most information and motivation came from the Internet (66&#37;) or television (14&#37;). A total of 2,736 L of breast milk was collected, and 1,979 L were processed. The cumulative number of recipients was 160 preterm or full-term infants and 21 adults, each group receiving the breast milk 337 and 41 times, respectively. In total, infants received 1,663 L and adults received 179 L. Conclusion : Through the present study, the role and importance of a human milk bank in collecting, pasteurizing, and storing surplus breast milk in through sanitary, medically proven methods and providing this breast milk to recipients could be appreciated and reevaluated. The authors believe that a national support system is necessary to expand this practice to a nationwide scale

Icelanders’ perspectives on security and foreign affair

Afstaða Íslendinga til öryggismála hefur lítið verið rannsökuð frá því í lok kalda stríðsins. Í þessari grein eru kynntar niðurstöður könnunar um afstöðu til og hugmyndir um utanríkis- og öryggismál, en Félagsvísindastofnun HÍ vann könnunina í nóvember og desember 2016. Niðurstöður könnunarinnar eru settar í samhengi við þróun í öryggisfræðum, þá sérstaklega öryggisgeira (e. security sectors) verufræðilegt öryggi (e. ontological security) og öryggisvæðingu (e. securitization). Helstu niðurstöður eru að almenningur á Íslandi telur öryggi sínu helst stafa ógn af efnahagslegum og fjárhagslegum óstöðugleika og náttúruhamförum, en telur litlar líkur á því að hernaðarátök eða hryðjuverkaárásir snerti landið beint. Þessar niðurstöður eru í takmörkuðu samræmi við helstu áherslur stjórnvalda í öryggismálum og því mikilvægt að stjórnvöld átti sig á því hvernig hægt er að tryggja það að almenningur sé meðvitaður um þær forsendur sem áhættumat og öryggisstefna grundvallast á.Icelanders’ views on security and foreign affairs since the end of the Cold War are an understudied issue. This article presents the findings of a large scale survey on the position and ideas about foreign affairs and security. The survey was conducted by the Social Science Research Institute of the University of Iceland in November and December 2016. The results of the survey are placed in the context of developments in security studies, with an emphasis on security sectors, ontological security, and securitization. The main findings are that the Icelandic public believes that its security is most threatened by economic and financial instability, as well as natural hazards, but thinks there is a very limited chance of military conflict or terrorist attacks directly affecting the country. These findings are incongruent with the main emphases of Icelandic authorities, as they appear in security policy and political discourse. It is therefore important that the authorities understand how to engage with the public about the criteria upon which risk assessments and security policies are based.Peer Reviewe

Phylogenetic diversity of the lichenized algal genus Trebouxia (Trebouxiophyceae, Chlorophyta): a new lineage and novel insights from fungal-algal association patterns of Icelandic cetrarioid lichens (Parmeliaceae, Ascomycota)

Publisher's version (útgefin grein)Lichens have high tolerance to harsh environmental conditions, where lichen symbiont interactions (e.g. myco- and photobionts) may play a crucial role. The characterization of fungal-algal association patterns is essential to understand their symbiotic interactions. This study investigated fungal-algal association patterns in Icelandic cetrarioid lichens using a multi-locus phylogenetic framework, including fungal nrITS, MCM7, mtSSU, RPB1 and RPB2 and algal nrITS, nrLSU, rbcL and mtCOXII data. Most Icelandic cetrarioid lichenized fungi were found to be specifically associated to the known Trebouxia clade “S” (Trebouxia simplex/suecica group), whereas the lichen-forming fungus Cetrariella delisei forms a symbiosis with a previously unrecognized lineage of Trebouxia, provisionally named as the “D” clade. This new Trebouxia lineage is supported by maximum likelihood and Bayesian phylogenetic analyses using all four included algal loci.This project was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration (grant agreement number 606895) to FP7-MCA-ITN MedPlant, “Phylogenetic Exploration of Medicinal Plant Diversity”. The Icelandic Research Fund (grant number 185442051) and the Bergthora and Thorsteinn Scheving Thorsteinsson Fund are also acknowledged for financial support.Peer Reviewe

Docosahexaenoic Acid Modulates NK Cell Effects on Neutrophils and Their Crosstalk.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadNatural killer (NK) cells and neutrophils engage in crosstalk that is important in inflammation and likely also for resolution of inflammation. NK cells activate neutrophils and induce their infiltration to the inflamed sites but may also influence their apoptosis and their subsequent efferocytosis by macrophages. Several studies indicate that docosahexaenoic acid (DHA) can inhibit NK cell cytotoxicity but the effects of DHA on the ability of NK cells to engage in crosstalk with neutrophils and affect their functions have not been described. This study explored the kinetics of the effects of NK cells and NK cells pre-treated with DHA on neutrophil surface molecule expression and apoptosis, as well as the ability of NK cells to affect other neutrophil functions. In addition, the study explored the effects of neutrophils on NK cell phenotype and function. Primary NK cells were pre-incubated with or without DHA, then stimulated and co-cultured with freshly isolated neutrophils. When co-cultured with NK cells, neutrophils had higher expression levels of CD11b and CD47; secreted more IL-8, IL-1ra, and CXCL10; had increased phagocytic ability; and their apoptosis was increased early after initiation of the co-culture while dampened at a later time-point. Pre-incubation of NK cells with DHA attenuated NK cell-induced upregulation of CD11b and CD47 on neutrophils, had minor effects on NK cell induction of cytokine/chemokine secretion or their phagocytic ability. Neutrophils also affected the function of NK cells, lowering the frequency of NKp46+ and CXCR3+ NK cells and increasing the concentrations of IFN-γ, TNF-α, and GM-CSF in the co-cultures. Pre-incubation of NK cells with DHA further decreased the frequency of NKp46+ NK cells in the co-culture with neutrophils and decreased the concentrations of IFN-γ, CCL3 and GM-CSF. These findings indicate that NK cells have mostly pro-inflammatory effects on neutrophils and that DHA can attenuate some of these pro-inflammatory effects. Neutrophils had both anti- and pro-inflammatory effects on NK cells. When NK cells had been pre-treated with DHA, the anti-inflammatory effects were increased and some of the pro-inflammatory effects attenuated. Overall, the results suggest that DHA may lead to a more anti-inflammatory microenvironment for NK cell and neutrophil crosstalk.Icelandic Research Fund University of Iceland Research Fund Landspitali University Hospital Research Fund Memorial Fund of Helga Jonsdottir and Sigurlidi Kristjansso

Alkaloid fingerprinting resolves Huperzia selago genotypes in Iceland

Pre-print (óritrýnt handrit)The club moss family Lycopodiaceae produces a diverse array of bioactive lycopodium alkaloids (LAs). In particular, the alkaloid huperzine A (hupA) has grasped attention since it is a potent acetylcholinesterase inhibitor of medical interest in Alzheimer's disease. Although the structural diversity and bioactivities of LAs have been studied to some extent, their chemotaxonomic value is mostly unexplored, especially to a lower taxonomic unit (e.g. subspecies or genotypes). This study focused on previously reported Icelandic Huperzia selago genotypes, and aimed to evaluate the chemotaxonomic value of LAs in resolving them. Using liquid chromatography-mass spectrometry (LC-MS), alkaloid fingerprints of H. selago taxa were subjected to principal component analysis (PCA). Our results revealed that each genotype tends to have its own alkaloid profile. Genotype 1 and 3 form distinct groups in a PCA plot, where genotype 2 is an intermediate between the other two genotypes. HupA and its derivative, huperzine B, both contribute to the differentiation of genotype 3 from the others. Therefore, our study demonstrated the potential of alkaloid fingerprints in resolving deep taxonomic groups and selecting plant taxa of medicinal importance.This work was supported by The People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 (grant number 606895); Icelandic Research Fund (grant number 152604051); and the Bergthora and Thorsteinn Scheving Thorsteinsson Fund

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex

Publisher's version (útgefin grein)The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimerʼs disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 – 679 µg/g) than genotype 1 (20 – 180 µg/g), where the former shows a typical green and reflexed “selago” morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 – 599 µg/g). The content of huperzine B in Icelandic taxa (6 – 13 µg/g) is much lower than that in Chinese H. serrata (79 – 207 µg/g).The People Programme (Marie Curie Actions) of the European Unionʼs Seventh Framework Programme FP7/2007–2013 (grant number 606895) and the Icelandic Research Fund (grant number 152604051) are acknowledged for financial support. This study was also funded from the Bergthora and Thorsteinn Scheving Thorsteinsson Fund.Peer Reviewe