Absorption and fluorescence spectroscopy of cold proflavine ions isolated in the gas phase.

Proflavine, a fluorescent cationic dye with strong absorption in the visible, has been proposed as a potential contributor to diffuse interstellar bands (DIBs). To investigate this hypothesis, it is essential to examine the spectra of cold and isolated ions for comparison. Here, we report absorption spectra of proflavine ions, trapped in a liquid-nitrogen-cooled ion trap filled with helium-buffer gas, as well as fluorescence spectra to provide further information on the intrinsic photophysics. We find absorption- and fluorescence-band maxima at 434.2 ± 0.1 and 434.7 ± 0.3 nm, corresponding to a Stokes shift of maximum 48 cm-1, which indicates minor differences between ground-state and excited-state geometries. Based on time-dependent density functional theory, we assign the emitting state to S2 as its geometry closely resembles that of S0, whereas the S1 geometry differs from that of S0. As a result, simulated spectra involving S1 exhibit long Franck-Condon progressions, absent in the experimental spectra. The latter displays well-resolved vibrational features, assigned to transitions involving in-plane vibrational modes where the vibrational quantum number changes by one. Dominant transitions are associated with vibrations localized on the NH2 moieties. Experiments repeated at room temperature yield broader spectra with maxima at 435.5 ± 1 nm (absorption) and 438.0 ± 1 nm (fluorescence). We again conclude that prevalent fluorescence arises from S2, i.e., anti-Kasha behavior, in agreement with previous work. Excited-state lifetimes are 5 ± 1 ns, independent of temperature. Importantly, we exclude the possibility that a narrow DIB at 436.4 nm originates from cold proflavine cations as the band is redshifted compared to our absorption spectra.

Empirical Calibration of a Cylindrical Ion Trap for Mass-Selected Gas-Phase Fluorescence Spectroscopy.

The ion motion in a quadrupole ion trap of hyperbolic geometry is well described by the Mathieu equations. A simpler cylindrical ion trap has also gained significance and has been used by us for fluorescence-spectroscopy experiments. This design allows for the easy replacement of the end-cap with a mesh, enhancing the photon collection. It is crucial to obtain a firm understanding of the ion motion in cylindrical ion traps and their capability as mass spectrometers. We present here an empirical method of calibrating a cylindrical ion trap based on fluorescence detection. This can be done nearly background-free in a pulsed experiment. The ions are located at the center of the trap, where the field is primarily quadrupolar, and here an effective Mathieu description is found through an effective geometry parameter. In spectroscopy experiments, high buffer-gas pressures are needed to efficiently cool the ions, which complicates the ions' motion and hence their stability. Still, simulations show that the stability diagram closely aligns with the Mathieu diagram, albeit shifted due to collisions. We map the stability diagram for six molecular ions by fluorescence collection from four cations and two anions spanning m/z from 212 to 647. The stability diagram is parametrized through the Mathieu functions with an m/z-dependent effective geometry parameter and a q-dependent shrinkage of the diagram. Based on the calibration, we estimate the mass resolution to be +7/-3 Da for ions with masses in the hundreds of Da.

Gas-phase Förster resonance energy transfer in mass-selected and trapped ions.

Förster Resonance Energy transfer (FRET) is a nonradiative process that may occur from an electronically excited donor to an acceptor when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. FRET experiments have been done in the gas phase based on specially designed mass-spectroscopy setups with the goal to obtain structural information on biomolecular ions labeled with a FRET pair (i.e., donor and acceptor dyes) and to shed light on the energy-transfer process itself. Ions are accumulated in a radio-frequency ion trap or a Penning trap where mass selection of those of interest takes place, followed by photoexcitation. Gas-phase FRET is identified from detection of emitted light either from the donor, the acceptor, or both, or from a fragmentation channel that is specific to the acceptor when electronically excited. The challenge associated with the first approach is the collection and detection of photons emitted from a thin ion cloud that is not easily accessible while the second approach relies both on the photophysical and chemical behavior of the acceptor. In this review, we present the different instrumentation used for gas-phase FRET, including a discussion of advantages and disadvantages, and examples on how the technique has provided important structural information that is not easily obtainable otherwise. Furthermore, we describe how the spectroscopic properties of the dyes are affected by nearby electric fields, which is readily discernable from experiments on simple model systems with alkyl or π-conjugated bridges. Such spectral changes can have a significant effect on the FRET efficiency. Ideas for new directions are presented at the end with special focus on cold-ion spectroscopy.

Effect of Freezing out Vibrational Modes on Gas-Phase Fluorescence Spectra of Small Ionic Dyes.

While action spectroscopy of cold molecular ions is a well-established technique to provide vibrationally resolved absorption features, fluorescence experiments are still challenging. Here we report the fluorescence spectra of pyronin-Y and resorufin ions at 100 K using a newly constructed setup. Spectra narrow upon cooling, and the emission maxima blueshift. Temperature effects are attributed to the population of vibrational excited levels in S1, and that frequencies are lower in S1 than in S0. This picture is supported by calculated spectra based on a Franck-Condon model that not only predicts the observed change in maximum, but also assigns Franck-Condon active vibrations. In-plane vibrational modes that preserve the mirror plane present in both S0 and S1 of resorufin and pyronin Y account for most of the observed vibrational bands. Finally, at low temperatures, it is important to pick an excitation wavelength as far to the red as possible to not reheat the ions.

Triangular Rhodamine Triads and Their Intrinsic Photophysics Revealed from Gas-Phase Ion Fluorescence Experiments.

When ionic dyes are close together, the internal Coulomb interaction may affect their photophysics and the energy-transfer efficiency. To explore this, we have prepared triangular architectures of three rhodamines connected to a central triethynylbenzene unit (1,3,5-tris(buta-1,3-diyn-1-yl)benzene) based on acetylenic coupling reactions and measured fluorescence spectra of the isolated, triply charged ions in vacuo. We find from comparisons with previously reported monomer and dimer spectra that while polarization of the π-system causes redshifted emission, the separation between the rhodamines is too large for a Stark shift. This picture is supported by electrostatic calculations on model systems composed of three linear and polarizable ionic dyes in D3h configuration: The electric field that each dye experiences from the other two is too small to induce a dipole moment, both in the ground and the excited state. In the case of heterotrimers that contain either two rhodamine 575 (R575) and one R640 or one R575 and two R640, emission is almost purely from R640 although the polarization of the π-system expectedly diminishes the dipole-dipole interaction.

A new setup for low-temperature gas-phase ion fluorescence spectroscopy.

Here, we present a new instrument named LUNA2 (LUminescence iNstrument in Aarhus 2), which is purpose-built to measure dispersed fluorescence spectra of gaseous ions produced by electrospray ionization and cooled to low temperatures (<100 K). LUNA2 is, as an earlier room-temperature setup (LUNA), optimized for a high collection efficiency of photons and includes improvements based on our operational experience with LUNA. The fluorescence cell is a cylindrical Paul trap made of copper with a hole in the ring electrode to permit laser light to interact with the trapped ions, and one end-cap electrode is a mesh grid combined with an aspheric condenser lens. The entrance and exit electrodes are both in physical contact with the liquid-nitrogen cooling unit to reduce cooling times. Mass selection is done in a two-step scheme where, first, high-mass ions are ejected followed by low-mass ions according to the Mathieu stability region. This scheme may provide a higher mass resolution than when only one DC voltage is used. Ions are irradiated by visible light delivered from a nanosecond 20-Hz pulsed laser, and dispersed fluorescence is measured with a spectrometer combined with an iCCD camera that allows intensification of the signal for a short time interval. LUNA2 contains an additional Paul trap that can be used for mass selection before ions enter the fluorescence cell, which potentially is relevant to diminishing RF heating in the cold trap. Successful operation of the setup is demonstrated from experiments with rhodamine dyes and oxazine-4, and spectral changes with temperature are identified.

Tumescent technique versus electrocautery mastectomy: A randomized controlled trial.

BACKGROUND: Breast cancer is the most common cancer in Danish women. In 2016 about 1450 (31%) Danish breast cancer patients had a mastectomy. The aim was to compare the frequency of postoperative complications in two methods of surgery, electrocautery dissection and tumescent technique, when performing a mastectomy. METHODS: Open randomized controlled trial of all consecutive primary breast cancer or DCIS female patients who underwent either a simple mastectomy or a modified radical mastectomy in Vejle Hospital, from January 2012 to October 2017. Primary outcomes were seroma production, bleeding, infection and necrosis. Secondary outcomes were duration of surgery and delay of adjuvant treatment caused by complications. Categorical outcome variables were compared between randomization groups using chi-square of Fisher exact test and continuous outcome variables by using Wilcoxon rank test. All analyses were performed at a 5% two-sided significance level. RESULTS: 357 patients met the inclusion criteria. 14 had bilateral mastectomy, i.e. 371 breasts. The two randomization groups consisted of 105 patients/107 breasts operated by tumescent technique and 98 patients/102 breasts operated by electrocautery technique. Tumescent technique produced more seroma though not significant (p = 0.631) (mean 605 vs. 630 ml). Bleeding in the tumescent group was 10.3% vs. 5.9% in the electrocautery group (p = 0.245). Infection (5.9% vs. 7.5% p = 0.645) and necrosis (4.9% vs. 4.7% p = 0.938) was uncommon with no difference between the intervention groups. Infection was most common cause of delay of adjuvant treatment; 3.9% in the electrocautery technique group. No significant difference in duration of surgery (p = 0.392). CONCLUSION: Both techniques are equally safe for simple and modified radical mastectomy.

Color tuning of chlorophyll a and b pigments revealed from gas-phase spectroscopy.

Chlorophyll (Chl) pigments are responsible for vital mechanisms in photosynthetic proteins: light harvesting, energy transfer and charge separation. A complex interplay between the Chl molecule and its microenvironment determines its transition energy. Interactions such as excitonic coupling with one or more pigments (Chls or carotenoids), axial ligation to the magnesium center, or electrostatic interactions between Chl and nearby amino-acid residues all influence the photophysical properties. Here we use time-resolved photodissociation action spectroscopy to determine transition energies of Chla/b complexes in vacuo to directly compare the impact of a negatively charged axial ligand (formate) to that of exciton coupling between two Chls. Experiments carried out at the electrostatic ion storage ring ELISA allow dissociation to be sampled on hundreds of milliseconds time scale. Absorption-band maxima of Chla-formate complexes are found at 433 ± 4 nm/2.86 ± 0.03 eV (Soret band) and in the region 654-675 nm/1.84-1.90 eV (Q band) and those of Chla dimers tagged by a quaternary ammonium ion at 419 ± 5 nm/2.96 ± 0.04 eV (Soret band) and 647 nm/1.92 eV (Q band). The axial ligand strongly affects the Chla transition energies causing redshifts of 0.21 eV of the Soret band and 0.04-0.1 eV of the Q band compared to Chla tagged by a quaternary ammonium. Slightly smaller shifts were found in case of Chlb. The redshifts are approximately twice that induced by excitonic coupling between two Chlas, also tagged by a quaternary ammonium ion. Axial ligation brings the absorption by isolated Chls very close to that of photosynthetic proteins.

Gas-Phase Ion Fluorescence Spectroscopy of Tailor-made Rhodamine Homo- and Heterodyads: Quenching of Electronic Communication by π-Conjugated Linkers.

While many key photophysical features are understood for electronic communication between chromophores in neutral compounds, there is limited information on the effect of charges in practically relevant ionic chromo/fluorophores. Here we have chosen positively charged rhodamines and prepared a selection of homo- and heterodimers with alkyl or π-conjugated, acetylenic bridges. Protonated molecules were transferred as isolated ions to gas phase where there is no solvent screening of charges, and fluorescence spectra were measured with a custom-made ion-trap setup. Our work reveals strong polarization of the π-spacer (induced dipole/quadrupole) when it experiences the electric field from one/ two dyes. Hence, π-spacers provide efficient shielding of charges by reducing the Coulomb interaction, whereas two dye cations polarize each other when connected by an alkyl. The screening influences the Förster resonance energy transfer efficiency that relies on the dipole-dipole interaction.

Gas-phase Förster resonance energy transfer in mass-selected ions with methylene or peptide linkers between two dyes: a concerted dance of charges.

Förster Resonance Energy Transfer (FRET) between a photoexcited and a ground-state dye is dictated by how far apart the two dyes are compared to the Förster distance. While there is a significant number of studies on the process for biomacromolecules in solution, there are only a few reports on gas-phase FRET. Here we report on a simple gas-phase model system, synthesized with the rhodamine 575 (R575+) and rhodamine 640 (R640+) FRET pair and a covalent linker with four methylenes, R575+-(CH2)4-R640+. Each dye carries a positive charge which allows for mass-spectroscopy experiments. We have recorded gas-phase dispersed fluorescence spectra of the mass-selected dications excited at different wavelengths using the homebuilt LUNA (LUminescence iNstrument in Aarhus) setup and find in all cases that emission is exclusively from the R640+ acceptor dye. The linker does not interfere electronically with the dyes and simply acts as a spacer. We can therefore establish the direct effect of the interaction between the two dyes when it comes to emission band maximum. Indeed, we find that R640+ experiences a significant shift in its maximum from 560 ± 1 nm for the monomer cation to 577 ± 2 nm in the presence of R575+, independent of initial excitation of R575+ or R640+. This redshift is ascribed to the large polarizability along the long axis of the xanthene core structure, and that this polarizability is larger in the excited state than in the ground state. Experiments were also done on a triply charged 11-mer peptide labelled with the same two dyes, R575+-(Gly-Gln)5-Lys-R640+ + H+ (Gly = glycine, Gln = glutamine, and Lys = lysine) where the extra positive charge is located on the peptide. Again a redshifted emission spectrum of the donor is observed with maximum at 582 ± 2 nm. Our work clearly demonstrates strong sensitivity of the photophysics of one dye to the nearby environment, and that caution is needed when using the energy transfer efficiency to infer dye-dye separations in gas-phase experiments.

Gas-phase action and fluorescence spectroscopy of mass-selected fluorescein monoanions and two derivatives.

Gaseous fluorescein monoanions are weakly fluorescent; they display a broad fluorescence spectrum and a large Stokes shift. This contrasts with the situation in aqueous solution. One explanation of the intriguing behavior in vacuo is based on internal proton transfer from the pendant carboxyphenyl group to one of the xanthene oxygens in the excited state; another that rotation of the carboxyphenyl group relative to the xanthene leads to a partial charge transfer from one chromophore (xanthene) to the other (carboxyphenyl) when the π orbitals start to overlap. To shed light on the mechanism at play, we synthesized two fluorescein derivatives where the carboxylic acid group is replaced with either an ester or a tertiary amide functionality and explored their gas-phase ion fluorescence using the home-built LUminescence iNstrument in Aarhus (LUNA) setup. Results on the fluorescein methyl ester that has no acidic proton clearly disprove the former explanation: The spectrum remains broad, and the band center (at 605 nm) is shifted even more to the red than that of fluorescein (590 nm). Experiments on the other variant that contains a piperidino amide are also in favor of the second explanation as here the piperidino already causes the dihedral angle between the planes defining the xanthene and the benzene ring to be less than 90° in the ground state (i.e., 63°), according to density functional theory calculations. As a result of the closer similarity between the ground-state and excited-state structures, the fluorescence spectrum is narrower than those of the other two ions, and the band maximum is further to the blue (575 nm). In accordance with a more delocalized ground state of the amide derivative, action spectra associated with photoinduced dissociation recorded at another setup show that the absorption-band maximum for the amide derivative is redshifted compared to that of fluorescein (538 nm vs. 525 nm).

Aspects of cAMP Signaling in Epileptogenesis and Seizures and Its Potential as Drug Target.

Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all patients become seizure free upon medical treatment. Thus, there is a need for new pharmacological approaches including novel drug targets for the management of epilepsy. Despite the fact that a role for cAMP signaling in epileptogenesis and seizures was first suggested some four decades ago, none of the current medications target the cAMP signaling system. The reasons for this are probably many including limited knowledge of the underlying biology and pathology as well as difficulties in designing selective drugs for the different components of the cAMP signaling system. This review explores selected aspects of cAMP signaling in the context of epileptogenesis and seizures including cAMP response element binding (CREB)-mediated transcriptional regulation. We discuss the therapeutic potential of targeting cAMP signaling in epilepsy and point to an increased knowledge of the A-kinase anchoring protein-based signaling hubs as being of seminal importance for future drug discovery within the field. Further, in terms of targeting CREB, we argue that targeting upstream cAMP signals might be more fruitful than targeting CREB itself. Finally, we point to astrocytes as cellular targets in epilepsy since cAMP signals may regulate astrocytic K+ clearance affecting neuronal excitability.

Somatostatin Analogue Treatment Primarily Induce miRNA Expression Changes and Up-Regulates Growth Inhibitory miR-7 and miR-148a in Neuroendocrine Cells.

Somatostatin (SST) analogues are used to control the proliferation and symptoms of neuroendocrine tumors (NETs). MicroRNAs (miRNA) are small non-coding RNAs that modulate posttranscriptional gene expression. We wanted to characterize the miRNAs operating under the control of SST to elucidate to what extent they mediate STT actions. NCI-H727 carcinoid cell line was treated with either a chimeric SST/dopamine analogue; a SST or dopamine analogue for proliferation assays and for identifying differentially expressed miRNAs using miRNA microarray. The miRNAs induced by SST analogue treatment are investigated in carcinoid cell lines NCI-H727 and CNDT2 using in situ hybridization, qPCR and proliferation assays. SST analogues inhibited the growth of carcinoid cells more potently compared to the dopamine analogue. Principal Component Analysis (PCA) of the samples based on miRNA expression clearly separated the samples based on treatment. Two miRNAs which were highly induced by SST analogues, miR-7 and miR-148a, were shown to inhibit the proliferation of NCI-H727 and CNDT2 cells. SST analogues also produced a general up-regulation of the let-7 family members. SST analogues control and induce distinct miRNA expression patterns among which miR-7 and miR-148a both have growth inhibitory properties.

Gas-Phase Ion Spectroscopy of Congo Red Dianions and Their Complexes with Betaine.

Congo Red (CR) is an azo dye that is negatively charged in aqueous solutions. Here we report on the intrinsic electronic properties of CR dianions from mass spectroscopy experiments on bare dianions and their complexes with betaine (B). As betaine is a zwitterion, it possesses a large dipole moment and is a good reporter on the sensitivity of CR to microenvironmental changes. Photoexcitation of CR2- in the visible region resulted in several fragment ions after absorption of at least three photons, with major fragmentation routes due to breakage of one or both C-NN bonds, one azo linkage, and/or the bonds to sulfite. Their yields as a function of excitation wavelength reveal a broad absorption in the visible region with the lowest-energy band located at ∼500 nm. Features are observed with a spacing of ∼1500 cm-1. One photon was sufficient to dissociate CR2-·B, and its action spectrum was almost identical to those of CR2- in accordance with previous findings that a symmetric ion is essentially unaffected by changes in its microenvironment. Electron detachment occurs in the UV with threshold energy of 3.6 ± 0.1 eV for CR2- and 3.81 ± 0.06 eV for CR2-·B. Attempts to measure fluorescence from photoexcited CR2- were unsuccessful.

Strong Impact of an Axial Ligand on the Absorption by Chlorophyll a and b Pigments Determined by Gas-Phase Ion Spectroscopy Experiments.

The microenvironments in photosynthetic proteins affect the absorption by chlorophyll (Chl) pigments. It is, however, a challenge to disentangle the impact on the transition energies of different perturbations, for example, the global electrostatics of the protein (nonbonded environmental effects), exciton coupling between Chl's, conformational variations, and binding of an axial ligand to the magnesium center. This is needed to distinguish between the two most commonly proposed mechanisms for energy transport in photosynthetic proteins, relying on either weakly or strongly coupled pigments. Here, on the basis of photodissociation action spectroscopy, we establish that the redshift of the Soret absorption band due to binding of a negatively charged carboxylate (as present in aspartic acid and glutamic acid residues) is 0.1-0.2 eV for Chl a and b. This effect is almost enough to reproduce the well-known green color of plants and can account for the strong spectral variation between Chl's. The experimental data serve to benchmark future high-level calculations of excited-state energies. Finally, we demonstrate that complexes between Chl a and histidine, tagged by a quaternary ammonium ion, can be made in the gas phase by electrospray ionization, but more work is needed to produce enough ions for gas-phase spectroscopy.

Sentinel Lymph Node Dissection in Locally Recurrent Breast Cancer.

BACKGROUND: The aim of this study was to investigate the use of sentinel lymph node dissection (SLND) in the treatment of patients with locally recurrent breast cancer. METHODS: A total of 147 patients with locally recurrent breast cancer were included from five different breast surgery departments in Denmark. Data on previous breast and axillary surgery, adjuvant treatment, second operation in the breast and axilla, and lymphoscintigraphy were collected retrospectively from the original patient files. RESULTS: SLND after recurrence (SLNDAR) was successful in 72 of 144 patients (50 %). The detection rate was significantly higher after previous SLND (66 %) compared with previous ALND (34 %) [p = 0.0001]. Thirty-seven patients (51 %) who had previous SLND had a negative sentinel node. These patients could be spared an ALND. Six patients (8 %) who had a previous ALND had a metastatic sentinel node at recurrence; 17 % of patients had a sentinel node located outside the ipsilateral axilla; and eight patients with negative sentinel node at SLNDAR underwent completion ALND. None of these patients had metastases at completion ALND, corresponding to a false negative rate of 0 %. CONCLUSIONS: SLNDAR seems to be a feasible procedure in locally recurrent breast cancer and can spare a clinically significant number of patients an unnecessary ALND and the following risk of sequelae. In patients who had previous ALND, SLNDAR identified metastases that would have been overlooked following the current guidelines. A large proportion of patients had aberrant drainage, suggesting a need for lymphoscintigraphy.

Successful breast reconstruction using acellular dermal matrix can be recommended in healthy non-smoking patients.

INTRODUCTION: We present Scandinavia's first series of immediate alloplastic breast reconstructions with an acellular dermal matrix. MATERIAL AND METHODS: Data were collected retrospectively in 76 cases of immediate breast reconstruction using an acellular dermal matrix (ADM) and an implant. RESULTS: A total of 59 women were reconstructed between June 2011 and January 2013. Cases included 42 unilateral and 17 bilateral reconstructions. A large number of patients had adjuvant therapy, hormone therapy (34), radiation therapy (27) or chemotherapy (38). The median age was 51 years (30-70 years) and the median follow-up period was 326 days (68-624 days). The co-morbidity factors included hypertension (n = 11), diabetes (n = 2) and 19 patients were smokers. Unsuccessful reconstructions counted ten cases (13%), eight of these due to necrosis and/or wound dehiscence (10%) and two due to infection (3%). The failure rate in non-smokers was 2/52 (4%) compared with 8/24 (33%) in smokers, p = 0.001. In hypertensive patients, the failure rate was 6/12 (50%) compared with 4/64 (6%)in normotensive patients, p = 0.001. 70% of the failed reconstructions occurred in patients older than 65 years of age. CONCLUSION: Immediate alloplastic breast reconstruction using an ADM can be recommended to healthy non-smoking patients. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

[Mastitis-like changes as debut symptom of Kaposi's sarcoma and AIDS]. / Mastitislignende forandring som debutsymptom for Kaposis sarkom og aids.

Kaposi's sarcoma of the breast is rare but has been reported to imitate breast lumps or gynaecomastia, usually accompanied by Kaposi's sarcoma of the skin. We report a case of a patient presenting with mastitis of the breast as the first symptom of Kaposi's sarcoma and AIDS. Although uncommon, the diagnosis should be considered in patients with mastitis without other obvious causes.