Cryogenic fluorescence spectroscopy of oxazine ions isolated in vacuo.

Recent developments in fluorescence spectroscopy have made it possible to measure both absorption and dispersed fluorescence spectra of isolated molecular ions at liquid-nitrogen temperatures. Absorption is here obtained from fluorescence-excitation experiments and does not rely on ion dissociation. One large advantage of reduced temperature compared to room-temperature spectroscopy is that spectra are narrow, and they provide information on vibronic features that can better be assigned from theoretical simulations. We report on the intrinsic spectroscopic properties of oxazine dyes cooled to about 100 K. They include six cations (crystal violet, darrow red, oxazine-1, oxazine-4, oxazine-170 and nile blue) and one anion (resorufin). Experiments were done with a home-built setup (LUNA2) where ions are stored, mass-selected, cooled, and photoexcited in a cylindrical ion trap. We find that the Stokes shifts are small (14-50 cm-1), which is ascribed to rigid geometries, that is, there are only small geometrical changes between the electronic ground and excited states. However, both the absorption and the emission spectra of darrow-red cations are broader than those of the other ionic dyes, which is likely associated with a less symmetric electronic structure and more non-zero Franck-Condon factors for the vibrational progressions. In the case of resorufin, the smallest ion under study, vibrational features are assigned based on calculated spectra.

Cryogenic Absorption and Emission Spectroscopy of the Oxyluciferin Anion In Vacuo.

Bioluminescence from fireflies, click beetles, and railroad worms ranges in color from green-yellow to orange to red. The keto form of oxyluciferin is considered a key emitter species in the proposed mechanisms to account for color variation. To establish the intrinsic photophysics in the absence of a microenvironment, we present experimental and theoretical gas-phase absorption and emission spectra of the 5,5-dimethyloxyluciferin anion (keto form) at room and cryogenic temperatures as well as lifetime measurements based on fluorescence. The theoretical model includes all 75 vibrational modes. The spectral impact of the large number of excited states at elevated temperatures is captured by an effective state distribution. At low temperature, spectral congestion is greatly reduced, and the observed well-resolved vibrational features are assigned to multiple Franck-Condon progressions involving different vibrational modes. An in-plane ∼60 cm-1 scissoring mode is found to be involved in the dominant progressions.

Association Between Number of Volunteer Responders and Interventions Before Ambulance Arrival for Cardiac Arrest.

BACKGROUND: Volunteer responder (VR) programs for activation of laypersons in out-of-hospital cardiac arrest (OHCA) have been deployed worldwide, but the optimal number of VRs to dispatch is unknown. OBJECTIVES: The purpose of this study was to investigate the association between the number of VRs arriving before Emergency Medical Services (EMS) and the proportion of bystander cardiopulmonary resuscitation (CPR) and defibrillation. METHODS: We included OHCAs not witnessed by EMS with VR activation from the Capital Region (September 2, 2017, to May 14, 2019) and the Central Region of Denmark (November 5, 2018, to December 31, 2019). We created 4 groups according to the number of VRs arriving before EMS: 0, 1, 2, and 3 or more. Using a logistic regression model adjusted for EMS response time, we examined associations between the number of VRs arriving before EMS and bystander CPR and defibrillation. RESULTS: We included 906 OHCAs. The adjusted ORs for bystander CPR were 2.40 (95% CI: 1.42-4.05), 3.18 (95% CI: 1.39-7.26), and 2.70 (95% CI: 1.32-5.52) when 1, 2, or 3 or more VRs arrived before EMS (reference), respectively. The adjusted OR for bystander defibrillation increased when 1 (1.97 [95% CI: 1.12-3.52]), 2 (2.88 [95% CI: 1.48-5.58]), or 3 or more (3.85 [95% CI: 2.11-7.01]) VRs arrived before EMS (reference). The adjusted OR of bystander defibrillation increased to 1.95 (95% CI: 1.18-3.22) when ≥3 VRs arrived first compared with 1 VR arriving first (reference). CONCLUSIONS: We found an association of increased bystander CPR and defibrillation when 1 or more VRs arrived before the EMS with a trend toward increased bystander defibrillation with increasing number of VRs arriving first.

Contemporary levels of cardiopulmonary resuscitation training in Denmark.

Aim: Many efforts have been made to train the Danish population in cardiopulmonary resuscitation (CPR) and automated external defibrillator (AED) use. We assessed CPR and AED training levels among the broad Danish population and volunteer responders. Methods: In November 2018, an electronic cross-sectional survey was sent to (1) a representative sample of the general Danish population (by YouGov) and (2) all volunteer responders in the Capital Region of Denmark. Results: A total of 2,085 people from the general population and 7,768 volunteer responders (response rate 36%) completed the survey. Comparing the general Danish population with volunteer responders, 81.0% (95% CI 79.2-82.7%) vs. 99.2% (95% CI 99.0-99.4%) p < 0.001 reported CPR training, and 54.0% (95% CI 51.8; 56.2) vs. 89.5% (95% CI 88.9-90.2) p < 0.001 reported AED training, at some point in life.In the general population, the unemployed and the self-employed had the lowest proportion of training with CPR training at 71.9% (95% CI 68.3-75.4%) and 65.4% (95% CI 53.8-75.8%) and AED training at 39.0% (95% CI 35.2-42.9%) and 34.6% (95% CI 24.2-46.2%), respectively.Applicable to both populations, the workplace was the most frequent training provider. Among 18-29-year-olds in the general population, most reported training when acquiring a driver's license. Conclusions: A large majority of the Danish population and volunteer responders reported previous CPR/AED training. Mandatory training when acquiring a driver's license and training through the workplace seems to disseminate CPR/AED training effectively. However, new strategies reaching the unemployed and self-employed are warranted to ensure equal access.

Ocular abnormalities in the Icelandic horse with a focus on equine recurrent uveitis: 112 Icelandic horses living in Denmark and 26 Icelandic horses living in the United States.

PURPOSE: To describe the most common ocular abnormalities in the Icelandic horse with focus on equine recurrent uveitis (ERU) and association between ocular abnormalities and summer eczema and coat colors. METHODS: A descriptive cross-sectional study. A complete physical and ophthalmic examination as well as measurement of serum titers for Leptospira serovariants was performed on Icelandic horses from Denmark (DK) and the United States (USA). RESULTS: One hundred and twelve Icelandic horses living in DK and 26 Icelandic horses living in the United States were included in this study (total of 138 horses, 274 eyes). The three most common ocular abnormalities were follicular conjunctivitis (55.8%, 153/274 eyes), cataracts (27.4%, 75/274 eyes), and multifocal chorioretinopathy ("bullet holes"; 19%, 52/274 eyes). The prevalence for ERU among Icelandic horses aged "eight years and older" was 8% (6/75 horses). The "insidious ERU" type was found in 83% of the ERU cohort (5/6 horses), while "classic ERU" was found in 17% (1/6 horses). A correlation between ERU and cataracts was found in the population (p < .01). Leptospiral serology results were not associated with ERU in either DK or US-Icelandic horses (p = 1.00, p = 1.00, respectively). ERU was not associated with summer eczema (p = .49), and no coat colors were associated with the ocular abnormalities found in the Icelandic horse (all p-values > .05). CONCLUSION: Follicular conjunctivitis, cataracts, and multifocal chorioretinopathy were the three most common ocular abnormalities findings. Icelandic horses who were 8 years or older had an 8% prevalence for ERU. Summer eczema and coat color were not associated with evidence of ERU or other ocular abnormalities.

Attenuated Deuterium Stabilization of Hydrogen-Bound Complexes at Room Temperature.

We have observed nine bimolecular hydrogen- or deuterium-bound complexes at room temperature using Fourier transform infrared (FTIR) spectroscopy. The complexes were formed using methanol or ethanol as hydrogen bond donors, as well as deuterated isotopologues of these, in order to study isotopic effects on hydrogen bonds. The complexes were formed using either a dimethylether- (O) or trimethylamine (N) acceptor, to facilitate comparison of two different types of hydrogen or deuterium bonds, OH(D)·O and OH(D)·N. For each complex, the characteristic OH- or OD-stretching fundamental band in the bimolecular complex was observed. The Gibbs energy of complex formation was determined at room temperature for each complex to compare the relative stability of hydrogen- and deuterium-bound bimolecular complexes. It is well known that deuterium-bound complexes are more stable at low temperatures because of the lower frequency of its intermolecular modes and thus a lower zero point vibrational energy. However, at room temperature, entropic contributions to the stability should also be considered. At room temperature, we find the Gibbs energy of complex formation for each pair of corresponding hydrogen- and deuterium-bound complex to be similar. The similar values of the Gibbs energies at room temperature is explained from a difference in the entropy, upon complexation, which favors the formation of the hydrogen-bound complex more than the deuterium-bound complex at higher temperatures.

The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp.

In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration. We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1

Host size-dependent anisakid infection in Baltic cod Gadus morhua associated with differential food preferences.

A significant increase in the infection level of Baltic cod Gadus morhua with the anisakid nematode larvae Contracaecum osculatum and Pseudoterranova decipiens has been recorded during recent years due to the expanding local population of grey seals Halichoerus grypus, which act as final hosts for these parasites. Here, we report from an investigation of 368 cod (total length [TL] 6-49 cm; caught in ICES Subdivision 25) that the infection level of juvenile cod (TL 6-30 cm) with larvae of C. osculatum and P. decipiens is absent or very low, whereas it increases drastically in larger cod (TL 31-48 cm). A third nematode Hysterothylacium aduncum was rarely found. The study indicates that the prey animals for large cod act as transport hosts for the parasite larvae. Analyses of stomach contents of cod caught in the same area (2007-2014) showed that small benthic organisms (including polychaetes Harmothoë sarsi) are preferred food items by small cod, the isopod Saduria entomon is taken by all size classes, and sprat Sprattus sprattus are common prey items for cod larger than 30 cm. Parasitological investigations (microscopic and molecular analyses) of H. sarsi (100 specimens) and S. entomon (40 specimens) did not reveal infection in these invertebrates, but 11.6% of sprat (265 specimens examined) was shown to be infected with 1-8 C. osculatum third stage larvae per fish. Analyses of sprat stomach contents confirmed that copepods and cladocerans are the main food items of sprat. These observations suggest that the C. osculatum life cycle in the Baltic Sea includes grey seals as final hosts, sprat as the first transport host and cod as second transport host. It may be speculated that sprat obtain infection by feeding on copepods and/or cladocerans, which could serve as the first intermediate hosts. One cannot exclude the possibility that the size-dependent C. osculatum infection of cod may contribute (indirectly or directly) to the differential mortality of larger cod (>38 cm) compared to smaller cod (<30 cm) recently recorded in the Baltic cod population.

The Parasite Fauna of the Gyrfalcon (Falco rusticolus) in Iceland.

We examined 46 Gyrfalcon (Falco rusticolus) carcasses from Iceland for parasites, including 29 first-year birds and 17 second-year birds and older. Endoparasites observed were the trematodes Cryptocotyle lingua (prevalence 8%), Cryptocotyle concavum (4%), and Strigea sp. (8%); the cestode Mesocestoides sp. (27%); and the nematodes Eucoleus contortus (76%) and Serratospiculum guttatum (7%). Ectoparasites included the astigmatan mite Dubininia accipitrina (47%), a mesostigmatan rhynonyssid mite (4%), the tick Ixodes caledonicus (20%), the mallophagans Degeeriella rufa (90%) and Nosopon lucidum (7%), the flea Ceratophyllus vagabundus (7%), and the louse fly Ornithomya chloropus (7%). Cryptocotyle lingua, C. concavum, S. guttatum, D. accipitrina, I. caledonicus, and N. lucidum are new host records. Of the five most common parasites (prevalence ≥ 20%) only Mesocestoides sp. showed a significant age relationship, being more prevalent in adult falcons (P = 0.021). Eucoleus contortus was also more prevalent in adults with marginal statistical significance (P = 0.058). Frounce, caused by E. contortus (possibly also by Trichomonas gallinae, which was not searched for in the survey) was highly prevalent (43%), but did not show a relationship with host age (P = 0.210). Birds with frounce were in poorer body condition than healthy birds (P = 0.015).

Histological assessment of PAXgene tissue fixation and stabilization reagents.

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world.

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.

Synthesis of hydroxymethyl branched [3.2.0]bicyclic nucleosides using a regioselective oxetane ring-formation.

Two [3.2.0]bicyclic nucleosides, 35 and 34, with one and two hydroxymethyl substituents, respectively, have been efficiently synthesized. A protected (3'-C-vinyl-beta-D-allofuranosyl)thymine derivative 28 was easily prepared from diacetone-D-glucose and the thymine moiety was protected with a BOM-group. After the introduction of a leaving group in the 2'-position, the subsequent nucleoside 31 was used as the substrate for a stereoselective dihydroxylation and a regioselective oxetane ring-formation to give after deprotection the bicyclic nucleoside 34. The surprisingly efficient formation of an oxetane was first discovered by serendipity on a corresponding methylfuranoside derivative. The allo-configured bicyclic nucleoside 34 was easily shortened to a ribo-configured analogue 35 by a diol-cleaving reaction and subsequent reduction. Both 34 and 35 are conformationally restricted in the important intermediate 04'-endo conformation.

Bi- and tricyclic nucleoside derivatives restricted in S-type conformations and obtained by RCM-reactions.

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.

Alpha-LNA (alpha-D-configured locked nucleic acid).

Two pyrimidine alpha-LNA nucleoside monomers have been synthesised and incorporated into alpha-configured oligonucleotides. A fully modified mixed alpha-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows alpha-LNA:RNA to form an extended duplex with a very broad major groove.

NMR solution structure of dsDNA containing a bicyclic D-arabino-configured nucleotide fixed in an O4'-endo sugar conformation.

[3.2.0]bcANA is a D-arabino-configured bicyclic nucleotide with a 2'-O,3'-C-methylene bridge. We here present the high-resolution NMR structure of a [3.2.0]bcANA modified dsDNA nonamer with one modified nucleotide incorporated. NOE restraints were obtained by analysis of NOESY cross peak intensities using a full relaxation matrix approach, and subsequently these restraints were incorporated into a simulated annealing scheme for the structure determination. In addition, the furanose ring puckers of the deoxyribose moieties were determined by analysis of COSY cross peaks. The modified duplex adopts a B-like geometry with Watson-Crick base pairing in all base pairs and all glycosidic angles in the anti range. The stacking arrangement of the nucleobases appears to be unperturbed relative to the normal B-like arrangement. The 2'-O,3'-C-methylene bridge of the modified nucleotide is located at the brim of the major groove where it fits well into the B-type duplex framework. The sugar pucker of the [3.2.0]bcANA nucleotide is O4'-endo and this sugar conformation causes a change in the delta backbone angle relative to the C2'-endo deoxyribose sugar pucker. This change is absorbed locally by slight changes in the epsilon and zeta angles of the modified nucleotide. Overall, the [3.2.0]bcANA modifications fits very well into a B-like duplex framework and only small and local perturbations are observed relative to the unmodified dsDNA of identical base sequence.

Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides.

Phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides were prepared via a solid-phase synthesis strategy. Under Reformatsky reaction conditions, novel esterified acetic acid phosphinodiamidites were synthesized and condensed with appropriately protected 5'-O-(4, 4'-dimethoxytrityl)-2'-deoxynucleosides to yield 3'-O-phosphinoamidite reactive monomers. These synthons when activated with tetrazole were used with an automated DNA synthesizer to prepare phosphonoacetic acid modified internucleotide linkages on controlled pore glass. The phosphinoacetate coupling products were quantitatively oxidized at each step with (1S)-(+)-(10-camphorsulfonyl)oxaziridine or 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce mixed sequence phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides with an average per cycle coupling efficiency of greater than 97%. Completely deprotected, modified oligodeoxynucleotides were purified by reverse-phase HPLC and characterized by ion exchange HPLC, (31)P NMR, and MALDI/TOF mass spectroscopy. Both analogues were stable toward hydrolysis with snake venom phosphodiesterase and stimulated RNase H1 activity.

Alpha-LNA (locked nucleic acid with alpha-D-configuration): synthesis and selective parallel recognition of RNA.

Alpha-LNA is presented as a stereoisomer of LNA (locked nucleic acid) with alpha-D-configuration. Three different approaches towards the thymine alpha-LNA monomer as well as the 5-methylcytosine alpha-LNA monomer are presented. Different alpha-LNA sequences have been synthesised and their hybridisation with complementary DNA and RNA has been evaluated by means of thermal stability experiments and circular dichroism spectroscopy. In a mixed pyrimidine sequence, alpha-LNA displays unprecedented parallel-stranded and selective RNA binding. Furthermore, a remarkable selectivity for hybridisation with RNA over DNA is indicated.