Mass spectrometry-based neurochemical analysis: perspectives for primate research

The analysis of neurochemicals from the brain represents a challenge for current analytical techniques due to a variety of factors, such as compositional complexity, limited amounts of sample and endogenous inferences. Advances in mass spectrometry (MS) provide great opportunities for the sensitive measurement of neurochemicals, offering benefits including simple sample preparation, broad capability for analysis of diverse compounds and rich structural information of analytes. Until recently, however, limited numbers of studies have reported on the analysis of small molecular neurochemicals, such as classical neurotransmitters, in part due to the difficulties in separation of polar molecules by using current chromatography techniques with MS-compatible conditions. By contrast, MS has become an indispensable tool for neuropeptide analysis , offering tremendous potential in the discovery of novel signaling peptides and biomarkers. This review covers recent advances in MS-based neurochemical analysis , including a comparison with related detection techniques, chromatographic separation and neuropeptide discovery. Issues relating to in vivo sample collection and sample preparation are discussed. To provide a wider view of the capability of MS in basic neuroscience and clinical research, we discuss MS-based neurochemical analysis conducted in different animal models and humans. We specifically highlight perspectives for the use of MS for brain functional studies and drug discovery in nonhuman primates.     

Bestrophin-1 enables Ca2+-activated Cl- conductance in epithelia

Epithelial cells express calcium-activated Cl(-) channels of unknown molecular identity. These Cl(-) channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. The family of bestrophins has been suggested to form calcium-activated Cl(-) channels. Here, we demonstrate molecular and functional expression of bestrophin-1 (BEST1) in mouse and human airways, colon, and kidney. Endogenous calcium-activated whole cell Cl(-) currents coincide with endogenous expression of the Vmd2 gene product BEST1 in murine and human epithelial cells, whereas calcium-activated Cl(-) currents are absent in epithelial tissues lacking BEST1 expression. Blocking expression of BEST1 with short interfering RNA or applying an anti-BEST1 antibody to a patch pipette suppressed ATP-induced whole cell Cl(-) currents. Calcium-dependent Cl(-) currents were activated by ATP in HEK293 cells expressing BEST1. Thus, BEST1 may form the Ca2+-activated Cl(-) current, or it may be a component of a Cl(-) channel complex in epithelial tissues.     

Metal complexes of natural melophlins and their cytotoxic and antibiotic activities

Complexes of the natural melophlins A and C with Mg, Zn, Ga, La and Ru were prepared, characterized and tested for antimicrobial and cytotoxic effects. The lanthanum complex La(melophlinato C)₃ and the ruthenium complex chlorido(η⁶-p-cymene)(melophlinato C)ruthenium(II) inhibited cells of human A-498 kidney cancer at IC₅₀ = 0.54 μM and 1.0 μM, respectively, and so distinctly better than free melophlin C. Another synergistic effect of coordinating melophlins to bioactive metals was found in the growth inhibition of the melophlin C-resistant bacterium Micrococcus luteus by Ga, La and Ru complexes of melophlin C.     

Impact of Previous Virological Treatment Failures and Adherence on the Outcome of Antiretroviral Therapy in 2007

Background

Combination antiretroviral treatment (cART) has been very successful, especially among selected patients in clinical trials. The aim of this study was to describe outcomes of cART on the population level in a large national cohort.

Methods

Characteristics of participants of the Swiss HIV Cohort Study on stable cART at two semiannual visits in 2007 were analyzed with respect to era of treatment initiation, number of previous virologically failed regimens and self reported adherence. Starting ART in the mono/dual era before HIV-1 RNA assays became available was counted as one failed regimen. Logistic regression was used to identify risk factors for virological failure between the two consecutive visits.

Results

Of 4541 patients 31.2% and 68.8% had initiated therapy in the mono/dual and cART era, respectively, and been on treatment for a median of 11.7 vs. 5.7 years. At visit 1 in 2007, the mean number of previous failed regimens was 3.2 vs. 0.5 and the viral load was undetectable (<50 copies/ml) in 84.6% vs. 89.1% of the participants, respectively. Adjusted odds ratios of a detectable viral load at visit 2 for participants from the mono/dual era with a history of 2 and 3, 4, >4 previous failures compared to 1 were 0.9 (95% CI 0.4–1.7), 0.8 (0.4–1.6), 1.6 (0.8–3.2), 3.3 (1.7–6.6) respectively, and 2.3 (1.1–4.8) for >2 missed cART doses during the last month, compared to perfect adherence. From the cART era, odds ratios with a history of 1, 2 and >2 previous failures compared to none were 1.8 (95% CI 1.3–2.5), 2.8 (1.7–4.5) and 7.8 (4.5–13.5), respectively, and 2.8 (1.6–4.8) for >2 missed cART doses during the last month, compared to perfect adherence.

Conclusions

A higher number of previous virologically failed regimens, and imperfect adherence to therapy were independent predictors of imminent virological failure.     

RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background

Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/Principal Findings

Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/Significance

Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.     

The Combined Dexamethasone/CRH Test (DEX/CRH Test) and Prediction of Acute Treatment Response in Major Depression

Background

In this study the predictive value of the combined dexamethasone/CRH test (DEX/CRH test) for acute antidepressant response was investigated.

Methodology/Principal Findings

In 114 depressed inpatients suffering from unipolar or bipolar depression (sample 1) the DEX/CRH test was performed at admission and shortly before discharge. During their stay in the hospital patients received different antidepressant treatment regimens. At admission, the rate of nonsuppression (basal cortisol levels >75.3 nmol/l) was 24.6% and was not related to the later therapeutic response. Moreover, 45 out of 114 (39.5%) patients showed an enhancement of HPA axis function at discharge in spite of clinical improvement. In a second sample, 40 depressed patients were treated either with reboxetine or mirtazapine for 5 weeks. The DEX/CRH test was performed before, after 1 week, and after 5 weeks of pharmacotherapy. Attenuation of HPA axis activity after 1 week was associated with a more pronounced alleviation of depressive symptoms after 5-week mirtazapine treatment, whereas downregulation of HPA system activity after 5 weeks was related to clinical response to reboxetine. However, early improvement of HPA axis dysregulation was not necessarily followed by a beneficial treatment outcome.

Conclusions/Significance

Taken together, performance of a single DEX/CRH test does not predict the therapeutic response. The best predictor for response seems to be an early attenuation of HPA axis activity within 1 or 2 weeks. However, early improvement of HPA system dysfunction is not a sufficient condition for a favourable response. Since a substantial part of depressive patients display a persistence of HPA axis hyperactivity at discharge, downregulation of HPA system function is not a necessary condition for acute clinical improvement either. Our data underline the importance of HPA axis dysregulation for treatment outcome in major depression, although restoration of HPA system dysfunction seems to be neither a necessary nor a sufficient determinant for acute treatment response.     

A connection between the mitochondrial permeability transition pore, autophagy, and cerebral amyloidogenesis

In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the beta-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy.     

Conjugates of the fungal cytotoxin illudin M with improved tumour specificity

A simplified procedure for the isolation of gram quantities of illudin M from culture broths of basidiomycete Omphalotus olearius is described. Esters of illudin M with docosahexaenoic acid, chlorambucil, demethylcantharidinic acid (endothall) and 2,2'-bipyridyl-5,5'-dicarboxylic acid were synthesised and tested for cytotoxicity and induction of apoptosis in two clinically relevant tumour cell lines (Panc-1 pancreas carcinoma and HT-29 colon carcinoma) and in non-malignant human foreskin fibroblasts. The demethylcantharidin and the bipyridine conjugates retained the cytotoxicity of the parent illudin M while displaying an improved specificity for the tumour cells over the fibroblasts.