Infrared mass spectrometric imaging below the diffraction limit

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)1 is an established technique for the analysis of biological macromolecules. Its relative insensitivity to pollutants makes MALDI-MS very suitable for the direct analysis of biological samples. As such, it has facilitated great advances in the field of biomolecular imaging mass spectrometry. Traditionally, MALDI-MS imaging is performed in a scanning microprobe methodology.(2-4) However, in a recent study we have demonstrated an alternative methodology; the so-called microscope mode,(5) where the requirement for a highly focused ionization beam is removed. Spatial details from within the desorption area are conserved during the flight of the ions through the mass analyzer, and a magnified ion image is projected onto a 2D-detector. In this paper, we demonstrate how imaging mass spectrometry benefits from the microscope mode approach. For the first time, high-lateral resolution ion images were recorded using infrared MALDI at 2.94 microm wavelength. The ion optical resolution achieved was well below the theoretical limit of (light-) diffraction for the setup used, which is impossible to achieve in the conventional scanning microprobe approach.     

Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells

Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.     

A Novel HIV Vaccine Adjuvanted by IC31 Induces Robust and Persistent Humoral and Cellular Immunity

The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31®, and the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31® facilitated immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime with IC31® and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune responses.     

Oral health promotion among older persons and their care providers in a nursing home facility

Objectives: To assess oral health status and oral health‐related quality of life (OHRQoL) of residents in an extended care facility and to assess the care providers’ oral health attitudes and knowledge. Methods: Participants included 137 residents (58.1% female, age range 32–94 years, 91% African–American) and 22 care providers. Residents received an oral examination and completed the Oral Health Impact Profile (OHIP‐14), an OHRQoL questionnaire. Care providers completed an oral health knowledge (OHK) questionnaire before and after the on‐site geriatric oral health education and training programme. Results: Oral examinations showed that 58% of the residents had extensive oral health needs. On the OHIP‐14, the mean severity was 9.2 (SD = 12.0), extent (number of items rated as ‘fairly often’ or ‘often’) was 1.2 (SD = 2.6) and prevalence (participants rating at least one item at least ‘fairly often’) was 37.8%. Most prevalent negative impact items were about ‘oral pain’, ‘appearance’ and ‘self‐consciousness’. Regarding OHK, caregivers’ knowledge improved following instruction from 65% correct on the pre‐test to 90% correct on the post‐test (p < 0.05). Subsequent to the eight in‐service workshops, providers reported that physical limitations, fear of getting bitten and time constraints were barriers to providing oral hygiene to their residents. Conclusion: Examination data showed a high level of dental needs among the majority of residents, accompanied by significantly reduced OHRQoL. Although care providers’ OHK improved following the geriatric service programme, they reported specific barriers regarding their provision of oral hygiene care to the residents.     

Adrenergic innervation of the gills, pulmonary arterial plexus, and dorsal aorta in the neotenic salamander, Ambystoma tigrinum

The presence of adrenergic innervation was investigated in four different vascular segments of the neotenic tiger salamander, Ambystoma tigrinum, by histofluorescent staining for catecholamines. The segments were the respiratory section of the gill, the branchial shunt vessels, a vascular plexus in the pulmonary artery, and the dorsal aorta. No adrenergic fibers were detected in the respiratory section of the gill or the pulmonary arterial plexus. In contrast, the branchial shunt vessels contained both adrenergic varicosities and catecholamine-containing cell bodies. These cells resemble Type I cells of the mammalian carotid body and amphibian carotid labyrinth. Adrenergic innervation of the dorsal aorta was sparse and restricted to the adventitia. The results suggest that adrenergic nerves may directly regulate blood flow in the gill, and thus gas exchange, by controlling vascular resistance of the branchial shunts. The contractile state of the dorsal aorta may also be under adrenergic control. In addition, it is suggested that the adrenergic cells of the branchial shunts may serve a receptor function in being sensitive to arterial blood gases.     

An intracerebral, physiological role for angiotensin: effects of central blockade.

Although exogenous angiotensin II (AII) exerts a multitude of effects on the central nervous system, there is little evidence supporting a physiological role for the endogenously produced peptide. Some investigators have tested the hypothesis that AII is physiologically active in the brain with intracerebral infusions of blockers of the renin-angiotensin system. If blocker infusions produce effects that are opposite to exogenous AII infusions, it is evidence supporting a physiological role for endogenously generated angiotensin. Previous work has demonstrated that intraventricular infusion of AII elicits thirst and stimulates antidiuretic hormone and ACTH release. Intracerebral administration of AII also suppresses aldosterone secretion. Experiments that employed the blockers saralasin, a competitive inhibitor of AII, and SQ 20881, a converting enzyme blocker, are presented; results suggest that endogenous AII is involved in the control of thirst and peripheral hormone levels. Infusion of the blockers in the ventricular system led to changes in peripheral hormone concentrations opposite to that observed following infusions of AII.     

Responses of trout gill ion transport systems to acute acidosis.

The response of rainbow trout Na+ and Cl- uptake systems to acute acidosis was tested by slow infusion of lactic acid into anaesthetized animals. Depression of blood pH by 0-4 pH unit had no effect on influx rates for either ion, and we conclude that gill ion uptake systems do not respond rapidly to blood pH changes.