Electronic Coherence Dephasing in Excitonic Molecular Complexes: Role of Markov and Secular Approximations

We compare four different types of equations of motion for reduced density matrix of a system of molecular excitons interacting with thermodynamic bath. All four equations are of second order in the linear system-bath interaction Hamiltonian, with different approximations applied in their derivation. In particular we compare time-nonlocal equations obtained from so-called Nakajima-Zwanzig identity and the time-local equations resulting from the partial ordering prescription of the cummulant expansion. In each of these equations we alternatively apply secular approximation to decouple population and coherence dynamics from each other. We focus on the dynamics of intraband electronic coherences of the excitonic system which can be traced by coherent two-dimensional spectroscopy. We discuss the applicability of the four relaxation theories to simulations of population and coherence dynamics, and identify features of the two-dimensional coherent spectrum that allow us to distinguish time-nonlocal effects.Comment: 14 pages, 8 figure

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F0- and the Fm-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 μm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed

Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model

MUC5B levels in submandibular gland saliva of patients treated with radiotherapy for head-and-neck cancer: A pilot study

<p>Abstract</p> <p>Background</p> <p>The salivary mucin MUC5B, present in (sero)mucous secretions including submandibular gland (SMG) saliva, plays an important role in the lubrication of the oral mucosa and is thought to be related to the feeling of dry mouth. We investigated if MUC5B levels in SMG saliva could distinguish between the presence or absence of severe dry mouth complaints 12 months after radiotherapy (RT) for head-and-neck cancer (HNC).</p> <p>Findings</p> <p>Twenty-nine HNC patients with a residual stimulated SMG secretion rate of ≥0.2 ml/10 min at 12 months after RT were analyzed. MUC5B (in U; normalized to 1) and total protein levels (mg/ml) were measured in SMG saliva at baseline and 12 months after RT using ELISA and BCA protein assay, respectively. Overall, median MUC5B levels decreased after RT from 0.12 to 0.03 U (<it>p</it> = 0.47). Patients were dichotomized into none/mild xerostomia (n = 12) and severe xerostomia (n = 17) based on a questionnaire completed at 12 months. SMG and whole saliva flow rates decreased after RT but were comparable in both groups. The median MUC5B level was higher in patients with no or mild xerostomia compared to patients with severe xerostomia (0.14 vs 0.01 U, <it>p</it> = 0.22). Half of the patients with severe xerostomia had no detectable MUC5B at 12 months after RT. No differences in total protein levels were observed.</p> <p>Conclusions</p> <p>Qualitative saliva parameters like MUC5B need further investigation in RT-induced xerostomia. This pilot study showed a trend towards lower MUC5B levels in the SMG saliva of patients with severe xerostomia 12 months after RT for HNC.</p

On the Relationship between Sialomucin and Sulfomucin Expression and Hydrogenotrophic Microbes in the Human Colonic Mucosa

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases

Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples

<p>Abstract</p> <p>Background</p> <p>Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva.</p> <p>Methods</p> <p>Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-<it>0</it>-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses.</p> <p>Results</p> <p>The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible.</p> <p>Conclusions</p> <p>Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.</p

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X7 knockout mice or in cells pretreated with a P2X7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro

Quantifying the Evolution of Vascular Barrier Disruption in Advanced Atherosclerosis with Semipermeant Nanoparticle Contrast Agents

Acute atherothrombotic occlusion in heart attack and stroke implies disruption of the vascular endothelial barrier that exposes a highly procoagulant intimal milieu. However, the evolution, severity, and pathophysiological consequences of vascular barrier damage in atherosclerotic plaque remain unknown, in part because quantifiable methods and experimental models are lacking for its in vivo assessment.To develop quantitative nondestructive methodologies and models for detecting vascular barrier disruption in advanced plaques.Sustained hypercholesterolemia in New Zealand White (NZW) rabbits for >7-14 months engendered endothelial barrier disruption that was evident from massive and rapid passive penetration and intimal trapping of perfluorocarbon-core nanoparticles (PFC-NP: ∼250 nm diameter) after in vivo circulation for as little as 1 hour. Only older plaques (>7 mo), but not younger plaques (<3 mo) demonstrated the marked enhancement of endothelial permeability to these particles. Electron microscopy revealed a complex of subintimal spongiform channels associated with endothelial apoptosis, superficial erosions, and surface-penetrating cholesterol crystals. Fluorine ((19)F) magnetic resonance imaging and spectroscopy (MRI/MRS) enabled absolute quantification (in nanoMolar) of the passive permeation of PFC-NP into the disrupted vascular lesions by sensing the unique spectral signatures from the fluorine core of plaque-bound PFC-NP.The application of semipermeant nanoparticles reveals the presence of profound barrier disruption in later stage plaques and focuses attention on the disrupted endothelium as a potential contributor to plaque vulnerability. The response to sustained high cholesterol levels yields a progressive deterioration of the vascular barrier that can be quantified with fluorine MRI/MRS of passively permeable nanostructures. The possibility of plaque classification based on the metric of endothelial permeability to nanoparticles is suggested

A Recombinant Influenza A Virus Expressing Domain III of West Nile Virus Induces Protective Immune Responses against Influenza and West Nile Virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 105 TCID50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines