The use of a mobile laboratory unit in support of patient management and epidemiological surveillance during the 2005 Marburg Outbreak in Angola

Background

Marburg virus (MARV), a zoonotic pathogen causing severe hemorrhagic fever in man, has emerged in Angola resulting in the largest outbreak of Marburg hemorrhagic fever (MHF) with the highest case fatality rate to date.

Methodology/Principal Findings

A mobile laboratory unit (MLU) was deployed as part of the World Health Organization outbreak response. Utilizing quantitative real-time PCR assays, this laboratory provided specific MARV diagnostics in Uige, the epicentre of the outbreak. The MLU operated over a period of 88 days and tested 620 specimens from 388 individuals. Specimens included mainly oral swabs and EDTA blood. Following establishing on site, the MLU operation allowed a diagnostic response in <4 hours from sample receiving. Most cases were found among females in the child-bearing age and in children less than five years of age. The outbreak had a high number of paediatric cases and breastfeeding may have been a factor in MARV transmission as indicated by the epidemiology and MARV positive breast milk specimens. Oral swabs were a useful alternative specimen source to whole blood/serum allowing testing of patients in circumstances of resistance to invasive procedures but limited diagnostic testing to molecular approaches. There was a high concordance in test results between the MLU and the reference laboratory in Luanda operated by the US Centers for Disease Control and Prevention.

Conclusions/Significance

The MLU was an important outbreak response asset providing support in patient management and epidemiological surveillance. Field laboratory capacity should be expanded and made an essential part of any future outbreak investigation.     

Long-term changes in bryophyte diversity of central European managed forests depending on site environmental features

Cryptogamic diversity is a reliable indicator of the state of forest ecosystems. In this study we analysed the variations in both bryophyte species richness overall and number of hemerophobic bryophyte species in Central European managed forests over a 20-year time span, based on data collected in 132 plots scattered across Poland. We tested differences in species richness among five temporal replicates, as well as among site types grouped based on elevation a.s.l., dominant tree species and stand age. The analyses revealed no significant trend in species richness across years. Meanwhile, species richness significantly increased along with elevation a.s.l., especially in broadleaved forests. No significant difference in species number between spruce and pine dominated stands emerged for mature stands, while there was a strong difference for young stands, with spruce forest hosting a much higher number of species. Species richness exhibited a slight, but not significant, increase over time in broadleaved forests, no significant variations in pine dominated stands and significant fluctuations in spruce dominated stands, yet without a significant trend. Out of the tested drivers, dominant tree species exhibited the strongest impact on species community composition. Number of hemerophobic and strongly hemerophobic species did not undergo significant variations across years either. The lack of bryophyte diversity trends highlighted in this study suggests Central European managed forests are in an equilibrium sate, maintained by the opposing effects of climate changes, on one side and of more sustainable forest management and pollutant deposition decline, from the other.

     

Deregulation of Genes Related to Iron and Mitochondrial Metabolism in Refractory Anemia with Ring Sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.     

The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.     

Arterial Hypertension Is Characterized by Imbalance of Pro-Angiogenic versus Anti-Angiogenic Factors

ObjectiveHypertension is the most common cardiovascular disease and the main risk factor for stroke, peripheral arterial disease, arterial aneurysms and kidney disease. It has been reported recently that hypertensive patients and animals are characterized by decreased density of arterioles and capillaries in the tissues, called rarefaction. Rarefaction significantly increases peripheral resistance which results in elevated blood pressure, leads to vessel damage and induction of inflammation. Therefore, we hypothesized that hypertension is associated with decreased serum concentration of physiological pro-angiogenic factors and concomitant increased production of angiogenesis inhibitors.ResultsHypertensive patients were characterized by increased serum concentration of endostatin which is an anti-angiogenic factor. In addition, hypertension was associated with decreased levels of physiological pro-angiogenic mediators such as: angiogenin and bFGF. The hypertensive group was also characterized by elevated levels of CRP, VEGF and IL-8 that are the hallmarks of inflammation.ConclusionsPresented results show that hypertension is characterized by imbalance of pro-angiogenic and anti-angiogenic factors in the background of inflammation.     

Histamine Recycling Is Mediated by CarT,a Carcinine Transporter in Drosophila Photoreceptors

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.     

Colostral Proline-Rich Polypeptide Complexes. Comparative Study of the Antioxidant Properties,Cytokine-Inducing Activity,and Nitric Oxide Release of Preparations Produced by a Laboratory and a Large-Scale Method

Colostral proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimer’s disease (AD). As the laboratory method of isolating PRP is expensive, laborious, and time consuming, a new large-scale methanol method of PRP isolation was developed. The proline-rich polypeptide complex obtained by this new method (named methanol PRP–MPRP) from both ovine or bovine colostra shows psychotropic activity and inhibitory effect on amyloid β aggregation similar to those produced by the laboratory-scale method. The comparative study of the antioxidant properties, cytokine-inducing activity, and nitric oxide release of PRP and MPRP showed that preparations are biologically active, moreover MPRP should be used at concentration higher than 100 µg/ml to obtain results comparable to PRP.

     

Effects of venlafaxine on the expression level and methylation status of genes involved in oxidative stress in rats exposed to a chronic mild stress

Recent human and animal studies indicate that oxidative and nitrosative stress may play a role in the aetiology and pathogenesis of depression. This study investigates the effect of chronic administration of the serotonin-norepinephrine reuptake inhibitor, venlafaxine, on the expression and methylation status of SOD1, SOD2, GPx1, GPx4, CAT, NOS1 and NOS2 in the brain and blood of rats exposed to a chronic mild stress (CMS) model of depression. Separate groups of animals were exposed to CMS for 2 or 7 weeks; the second group received saline or venlafaxine (10 mg/kg/d, IP) for 5 weeks. After completion of both stress conditions and drug administration, the mRNA and protein expression of selected genes and the methylation status of their promoters were measured in peripheral mononuclear blood cells (PBMCs) and in brain structures (hippocampus, amygdala, hypothalamus, midbrain, cortex, basal ganglia) with the use of TaqMan Gene Expression Assay, Western blot and methylation-sensitive high-resolution melting techniques. CMS caused a decrease in sucrose consumption, and this effect was normalized by fluoxetine. In PBMCs, SOD1, SOD2 and NOS2 mRNA expression changed only after venlafaxine administration. In brain, CAT, Gpx1, Gpx4 and NOS1 gene expression changed following CMS or venlafaxine exposure, most prominently in the hippocampus, midbrain and basal ganglia. CMS increased the methylation of the Gpx1 promoter in PBMCs, the second Gpx4 promoter in midbrain and basal ganglia, and SOD1 and SOD2 in hippocampus. The CMS animals treated with venlafaxine displayed a significantly higher CAT level in midbrain and cerebral cortex. CMS caused an elevation of Gpx4 in the hippocampus, which was lowered in cerebral cortex by venlafaxine. The results indicate that CMS and venlafaxine administration affect the methylation of promoters of genes involved in oxidative and nitrosative stress. They also indicate that peripheral and central tissue differ in their response to stress or antidepressant treatments. It is possible that that apart from DNA methylation, a crucial role of expression level of genes may be played by other forms of epigenetic regulation, such as histone modification or microRNA interference. These findings provide strong evidence for thesis that analysis of the level of mRNA and protein expression as well as the status of promoter methylation can help in understanding the pathomechanisms of mental diseases, including depression, and the mechanisms of action of drugs effective in their therapy.     

Changes in the nitric oxide level in the rat liver as a response to brain injury

Liver disturbances stimulate inflammatory reaction in the brain but little is known if injury to the brain can significantly influence liver metabolism. This problem is crucial in modern transplantology, as the condition of the donor brain seems to strongly affect the quality (viability) of the graft, which is often obtained from brain-dead donors, usually after traumatic brain injury. Because nitric oxide is one of the significant molecules in brain and liver biology, we examined if brain injury can affect NO level in the liver. Liver samples of Wistar rats were collected and studied with EPR NO-metry to detect NO level changes at different time points after brain injury. Shortly after the trauma, NO level in the liver was similar to the control. However, later there was a significant increase in the NO content in the livers starting from the 2nd day after brain injury and lasting up to the 7th day. It seems that the response to a mechanical brain injury is of the systemic, rather than local character. Therefore brain metabolism disturbances can influence liver metabolism at least by stimulating the organ to produce NO.