Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.     

A Systematic Review and Meta-Analysis on the Safety of Vascular Endothelial Growth Factor (VEGF) Inhibitors for the Treatment of Retinopathy of Prematurity

Introduction

Laser photocoagulation is the current gold standard treatment for proliferative retinopathy of prematurity (ROP). However, it permanently reduces the visual field and might induce myopia. Vascular endothelial growth factor (VEGF) inhibitors for the treatment of ROP may enable continuing vascularization of the retina, potentially allowing the preservation of the visual field. However, for their use in infants concern remains. This meta-analysis explores the safety of VEGF inhibitors.

Methods

The Ovid Interface was used to perform a systematic review of the literature in the databases PubMed, EMBASE and the Cochrane Library.

Results

This meta-analysis included 24 original reports (including 1.457 eyes) on VEGF inhibitor treatment for ROP. The trials were solely observational except for one randomized and two case-control studies. We estimated a 6-month risk of retreatment per eye of 2.8%, and a 6-month risk of ocular complication without the need of retreatment of 1.6% per eye. Systemic complications were only reported as isolated incidents.

Discussion

VEGF inhibitors seem to be associated with low recurrence rates and ocular complication rates. They may have the benefit of potentially allowing the preservation of visual field and lower rates of myopia. Due to the lack of data, the risk of systemic side effects cannot be assessed.     

DNA Methylation of the Serotonin Transporter Gene in Peripheral Cells and Stress-Related Changes in Hippocampal Volume: A Study in Depressed Patients and Healthy Controls

Serotonin plays an important role in the etiology of depression. Serotonin is also crucial for brain development. For instance, animal studies have demonstrated that early disruptions in the serotonin system affect brain development and emotion regulation in later life. A plausible explanation is that environmental stressors reprogram the serotonin system through epigenetic processes by altering serotonin system gene expression. This in turn may affect brain development, including the hippocampus, a region with dense serotonergic innervations and important in stress-regulation. The aim of this study was to test whether greater DNA methylation in specific CpG sites at the serotonin transporter promoter in peripheral cells is associated with childhood trauma, depression, and smaller hippocampal volume. We were particularly interested in those CpG sites whose state of methylation in peripheral cells had previously been associated with in vivo measures of brain serotonin synthesis. Thirty-three adults with Major Depressive Disorder (MDD) (23 females) and 36 matched healthy controls (21 females) were included in the study. Depressive symptoms, childhood trauma, and high-resolution structural MRI for hippocampal volume were assessed. Site-specific serotonin transporter methylation was assessed using pyrosequencing. Childhood trauma, being male, and smaller hippocampal volume were independently associated with greater peripheral serotonin transporter methylation. Greater serotonin transporter methylation in the depressed group was observed only in SSRI-treated patients. These results suggest that serotonin transporter methylation may be involved in physiological gene-environment interaction in the development of stress-related brain alterations. The results provide some indications that site-specific serotonin transporter methylation may be a biomarker for serotonin-associated stress-related psychopathology.     

Sequential expression of maternally inherited phosphoglycerate kinase-1 in the early mouse embryo

Enzyme activities of X-linked phosphoglycerate kinase (PGK-1) and autosomal glucose phosphate isomerase (GPI-1) were determined in intact mouse blastocysts and isolated inner cell masses (ICMs). Blastocysts were recovered from the uterus on day 4 of gestation and cultured overnight in vitro. ICMs were isolated by treatment with calcium ionophore A23187. On day 4, approximately 35% of the total activity of both PGK-1 and GPI-1 was located in the ICM. After overnight culture, the PGK-1 activity of the whole blastocyst nearly doubled, due to the activation of only the maternally derived gene coding for PGK-1. In the ICM, however, a pronounced decrease of PGK-1 activity was measured: only 10% of the total PGK-1 activity was measured in the ICM on day 5. In contrast to PGK-1, GPI-1 activity of the intact blastocyst remained stable from day 4 to day 5. In the ICM, the GPI-1 activity did decline, but to a lesser extent than PGK-1 activity: 20% of total GPI-1 activity was found in the ICM on day 5. These results, when compared with the data of Handyside and Hunter, suggest that the decline in GPI-1 activity in the ICM is due to a change in the ratio of trophectoderm (TE) to ICM cells. The greater reduction of PGK-1 activity in the ICM cannot, however, be explained solely by this mechanism. To explain the observed additional decrease, we postulate that Pgk-1 is not activated in the ICM prior to day 6. This implies that on day 4 maternal Pgk-1 is activated in the TE exclusively.     

Viscosity dynamics and the production of extracellular polymeric substances and soluble microbial products during anaerobic digestion of pulp and paper mill wastewater sludges

Bioprocess and Biosystems Engineering - The production processes of the pulp and paper industry often run in campaigns, leading to large variations in the composition of wastewaters and waste...     

Development of a Transgenic Plasmodium berghei Line (Pbpfpkg) Expressing the P. falciparum cGMP-Dependent Protein Kinase,a Novel Antimalarial Drug Target

With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known.     

Tau as a marker for Alzheimer's disease

Alzheimer's Disease (AD) is an age-related dementia that has received increasing attention in recent years. The disease appears to be specific for humans and, moreover, its causes are unknown. Studies of the disease are complicated by the fact that a reliable diagnosis depends on post-mortem analysis of brain tissue, and useful animal or cell models are not yet available. This article discusses the possibility of using tau (a protein associated with the neurofibrillary tangles that are an indicator of AD) as a tool for further pursuing the diagnosis and treatment of the disease.     

Mutational Analysis of Sclerostin Shows Importance of the Flexible Loop and the Cystine-Knot for Wnt-Signaling Inhibition

The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured β-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.     

Relationships between primary production and vertical particle export at the Atlantic-Arctic boundary (Fram Strait, HAUSGARTEN)

The lack of extended dataset has so far prevented an inclusive understanding of the long-term relationships between primary production (PP) and vertical export in the Arctic Ocean. It is urgent to investigate these connections as Arctic ecosystems are on the verge of climate-related shifts, which could be caused by the combined effects of increase in Pacific and Atlantic inflow, climate warming, and sea ice decline. For a period of 6 years we investigated the degree of coupling between PP and export by making use of modelled PP rates and vertical particle fluxes collected with sediment traps moored at ~300 m depth in the eastern Fram Strait. Our analyses indicate that total and new simulated PP averaged for different areas centered on the mooring location (5–200 km radius) explain at best 20–44% of the observed biogenic particle fluxes at 300 m, when applying extended time-lags (55–90 days) between PP and vertical fluxes. Based on this phasing, we define a conceptual framework that presents the temporal dimension as a prime determinant of the maximum strength of the PP-export coupling at a given depth. Our results support that planktonic food webs in the Fram Strait process heavily biogenic material in the epipelagic zone, but we further suggest that Atlantic-Arctic water interactions induce a particular ecological setting responsible for the extended turn-over. In conclusion, we hypothesize that global warming could promote a transition toward a more retentive ecosystem in the Fram Strait region despite the likely increase of pelagic PP in the Arctic Ocean.