1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish, the common carp (Cyprinus carpio)

All 20.000 different fish species vary greatly in their ability to tolerate and survive fluctuating oxygen concentrations in the water. Especially fish of the genus Carassius, e.g. the crucian carp and the goldfish, exhibit a remarkable tolerance to limited/absent oxygen concentrations. The metabolic changes of anoxia-tolerant crucian carp were recently studied and published. Contrary to crucian carp, the hypoxia-tolerant common carp cannot survive a complete lack of oxygen (anoxia). Therefore, we studied the ¹H-NMR-based metabolomics of brain, heart, liver and white muscle extracts of common carp, subjected to anoxia (0 mg O₂ l^?1) and hypoxia (0.9 mg O₂ l^?1) at 5 °C. Specifically, fish were exposed to normoxia (i.e. 9 mg O₂ l^?1; controls 24 h, 1 week and 2 weeks), acute hypoxia (24 h), chronic hypoxia (1 week) and chronic hypoxia (1 week) with normoxic reoxygenation (1 week). Additionally, we also investigated the metabolic responses of fish to anoxia for 2 h. Both anoxia and hypoxia significantly changed the tissue levels of standard energy metabolites as lactate, glycogen, ATP/ADP and phosphocreatine. Remarkably, anoxia induced increased lactate levels in all tissues except for the heart whereas hypoxia resulted in decreased lactate concentrations in all tissues except for brains. Furthermore, hypoxia and anoxia influenced amino acids (alanine, valine/(iso)leucine) and neurotransmitters levels (GABA, glutamate). Lastly, we also detected ‘other’ i.e. previously not reported compounds to play a role in the present context. Scyllo-inositol levels changed significantly in heart, liver and muscle, providing novel insights into the anoxia/hypoxic responses of the common carp.     

Multiple Inflammatory Biomarker Detection in a Prospective Cohort Study: A Cross-Validation between Well-Established Single-Biomarker Techniques and an Electrochemiluminescense-Based Multi-Array Platform

Background

In terms of time, effort and quality, multiplex technology is an attractive alternative for well-established single-biomarker measurements in clinical studies. However, limited data comparing these methods are available.

Methods

We measured, in a large ongoing cohort study (n = 574), by means of both a 4-plex multi-array biomarker assay developed by MesoScaleDiscovery (MSD) and single-biomarker techniques (ELISA or immunoturbidimetric assay), the following biomarkers of low-grade inflammation: C-reactive protein (CRP), serum amyloid A (SAA), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1). These measures were realigned by weighted Deming regression and compared across a wide spectrum of subjects’ cardiovascular risk factors by ANOVA.

Results

Despite that both methods ranked individuals’ levels of biomarkers very similarly (Pearson’s r all≥0.755) absolute concentrations of all biomarkers differed significantly between methods. Equations retrieved by the Deming regression enabled proper realignment of the data to overcome these differences, such that intra-class correlation coefficients were then 0.996 (CRP), 0.711 (SAA), 0.895 (sICAM-1) and 0.858 (sVCAM-1). Additionally, individual biomarkers differed across categories of glucose metabolism, weight, metabolic syndrome and smoking status to a similar extent by either method.

Conclusions

Multiple low-grade inflammatory biomarker data obtained by the 4-plex multi-array platform of MSD or by well-established single-biomarker methods are comparable after proper realignment of differences in absolute concentrations, and are equally associated with cardiovascular risk factors, regardless of such differences. Given its greater efficiency, the MSD platform is a potential tool for the quantification of multiple biomarkers of low-grade inflammation in large ongoing and future clinical studies.     

Is the Improvement of CF Patients,Hospitalized for Pulmonary Exacerbation,Correlated to a Decrease in Bacterial Load?

Background

Cystic Fibrosis (CF) patients are vulnerable to airway colonization with Pseudomonas aeruginosa. In case eradication fails after antibiotic treatment, patients become chronically colonized with P. aeruginosa, with recurrent pulmonary exacerbation, for which patients typically are hospitalized for 2 weeks and receive intravenous antibiotic treatment. Normally, improvement of the patients'' health is established.

Aim

Determination of the correspondence between patient improvement and changes of the P. aeruginosa and total bacterial load in the sputum.

Methods

Eighteen CF patients with exacerbation were included for a total of 27 hospitalization episodes. At day 1, 8 and 15, inflammation and lung function parameters were determined, together with the P. aeruginosa load in the sputum using culture, quantitative PCR (qPCR) and propidium monoazide qPCR.

Results

Patients improved during hospitalization (decrease in levels of C-reactive protein, white blood cell counts and erythrocyte sedimentation rate, increase of FEV₁), reaching normal values already after one week. Also the P. aeruginosa load and the total bacterial load decreased during the first week of antibiotic treatment (p<0.05), except for patients with a low lung function (FEV₁≤39.4%), for whom no significant decrease of P. aeruginosa was established. Comparison of culture-based and propidium monoazide qPCR-based quantification of P. aeruginosa showed that at the end of the treatment on average 62% of the P. aeruginosa cells are not cultivable, indicating that many cells are alive but dormant, or dead but still structurally intact.

Conclusion

Improvement of the clinical status is accompanied with a decrease of the P. aeruginosa load, whereby both occur mainly during the first week of antibiotic treatment. However, for patients with a low lung function, no decrease of the P. aeruginosa load is observed. Comparison of detection techniques shows that a large amount of noncultivable or dead bacteria are present in the samples.     

Resting State fMRI Reveals Diminished Functional Connectivity in a Mouse Model of Amyloidosis

Introduction

Functional connectivity (FC) studies have gained immense popularity in the evaluation of several neurological disorders, such as Alzheimer’s disease (AD). AD is a complex disorder, characterised by several pathological features. The problem with FC studies in patients is that it is not straightforward to focus on a specific aspect of pathology. In the current study, resting state functional magnetic resonance imaging (rsfMRI) is applied in a mouse model of amyloidosis to assess the effects of amyloid pathology on FC in the mouse brain.

Methods

Nine APP/PS1 transgenic and nine wild-type mice (average age 18.9 months) were imaged on a 7T MRI system. The mice were anesthetized with medetomidine and rsfMRI data were acquired using a gradient echo EPI sequence. The data were analysed using a whole brain seed correlation analysis and interhemispheric FC was evaluated using a pairwise seed analysis. Qualitative histological analyses were performed to assess amyloid pathology, inflammation and synaptic deficits.

Results

The whole brain seed analysis revealed an overall decrease in FC in the brains of transgenic mice compared to wild-type mice. The results showed that interhemispheric FC was relatively preserved in the motor cortex of the transgenic mice, but decreased in the somatosensory cortex and the hippocampus when compared to the wild-type mice. The pairwise seed analysis confirmed these results. Histological analyses confirmed the presence of amyloid pathology, inflammation and synaptic deficits in the transgenic mice.

Conclusions

In the current study, rsfMRI demonstrated decreased FC in APP/PS1 transgenic mice compared to wild-type mice in several brain regions. The APP/PS1 transgenic mice had advanced amyloid pathology across the brain, as well as inflammation and synaptic deficits surrounding the amyloid plaques. Future studies should longitudinally evaluate APP/PS1 transgenic mice and correlate the rsfMRI findings to specific stages of amyloid pathology.     

Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides

Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.     

The Household Costs of Visceral Leishmaniasis Care in South-eastern Nepal

Background and objectives

Visceral leishmaniasis (VL) is an important public health problem in south-eastern Nepal affecting very poor rural communities. Since 2005, Nepal is involved in a regional initiative to eliminate VL. This study assessed the economic impact of VL on households and examined whether the intensified VL control efforts induced by the government resulted in a decrease in household costs.

Methods

Between August and September 2010, a household survey was conducted among 168 patients that had been treated for VL within 12 months prior to the survey in five districts in south-eastern Nepal. We collected data on health-seeking behaviour, direct and indirect costs and coping strategies.

Results

The median total cost of one episode of VL was US$ 165 or 11% of annual household income. The median delay between the onset of symptoms and presentation to a qualified provider was 25 days. Once the patient presented to a qualified provider, the delay to correct diagnosis was minimal (median 3 days). Direct and indirect costs (income losses) represented 47% and 53% of total costs respectively. Households used multiple strategies to cope with the cost of illness, mainly mobilizing cash/savings (71%) or taking a loan (56%).

Conclusions

The provision of free VL diagnosis and drugs by the Nepalese control programme has been an important policy measure to reduce the cost of VL to households. But despite the free VL drugs, the economic burden is still important for households. More effort should be put into reducing indirect costs, in particular the length of treatment, and preventing the transmission of VL through vector control.     

Burden of Mycobacterium ulcerans Disease (Buruli Ulcer) and the Underreporting Ratio in the Territory of Songololo,Democratic Republic of Congo

Background

Cutaneous infection by Mycobacterium ulcerans, also known as Buruli ulcer (BU), represents the third most common mycobacterial disease in the world after tuberculosis and leprosy. Data on the burden of BU disease in the Democratic Republic of Congo are scanty. This study aimed to estimate the prevalence rate and the distribution of BU in the Songololo Territory, and to assess the coverage of the existing hospital-based reporting system.

Methods

We conducted a cross-sectional survey (July–August 2008) using the door-to-door method simultaneously in the two rural health zones (RHZ) of the Songololo Territory (RHZ of Kimpese and Nsona-Mpangu), each containing twenty health areas. Cases were defined clinically as active BU and inactive BU in accordance with WHO-case definitions.

Results

We detected 775 BU patients (259 active and 516 inactive) in a total population of 237,418 inhabitants. The overall prevalence of BU in Songololo Territory was 3.3/1000 inhabitants, varying from 0 to 27.5/1000 between health areas. Of the 259 patients with active BU, 18 (7%) had been reported in the hospital-based reporting system at Kimpese in the 6–8 months prior to the survey.

Conclusion

The survey demonstrated a huge variation of prevalence between health areas in Songololo Territory and gross underreporting of BU cases in the hospital-based reporting system. Data obtained may contribute to better targeted and improved BU control interventions, and serve as a baseline for future assessments of the control program.