Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods

Total number of bacteria, viable counts of aerobic and anaerobic heterotrophic bacteria and 16S rRNA gene diversity were investigated during drilling of three boreholes in the walls of the Äspö hard rock laboratory tunnel, at depths ranging from 380 to 446 m below sea level. Water samples were taken from the drill water source, the drilling equipment and from the drilled boreholes. The drill water was kept under nitrogen atmosphere and all equipment was steam cleaned before the start of a new drilling. Total and viable counts of bacteria in the drilled boreholes were several orders of magnitude lower than in the samples from the drilling equipment, except for sulphate reducing bacteria. A total of 158 16S rRNA genes that were cloned from the drill water source, the drilling equipment and the drilled boreholes were partially sequenced. The drilled boreholes generally had a 16S rRNA diversity that differed from what was found in samples from the drilling equipment. Several of the sequences obtained could be identified on genus level as one of the genera Acinetobacter, Methylophilus, Pseudomonas and Shewanella. In conclusion, the tubing used for drill water supply constituted a source of bacterial contamination to the rest of the drilling equipment and the boreholes. The results show, using molecular and culturing methods, that although large numbers of contaminating bacteria were introduced to the boreholes during drilling, they did not establish in the borehole groundwater at detectable levels.     

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lotta Koskinen and Jihane Romanos are authors with equal contribution.     

New potent C2-symmetric malaria plasmepsin I and II inhibitors

A series of malaria plasmepsin (Plm) I and II inhibitors containing a C(2)-symmetric core structure have been synthesised and tested for protease inhibition activity. These compounds can be prepared using a straightforward synthesis involving a phenol nucleophilic ring opening of a diepoxide. Exemplar compounds synthesised exhibited remarkable inhibitory activity against both Plm I and II, notably 15c with K(i) values of 2.7nM and 0.25nM respectively, as well as showing >100-fold selectivity against Cathepsin D.     

Neuropilin1 is a direct downstream target of Nurr1 in the developing brain stem

The orphan nuclear receptor Nurr1 is expressed in the developing and adult central nervous system. Previous studies have shown that Nurr1 is essential for the generation of midbrain dopamine neurons. Furthermore, Nurr1 is critical for respiratory functions associated with the brain stem. Very few Nurr1 regulated genes have been identified and it remains unclear how Nurr1 influences the function and development of neurons. To identify novel Nurr1 target genes we have searched for regulated genes in the dopaminergic MN9D cell line. These experiments identified Neuropilin-1 (Nrp1), a receptor protein involved in axon guidance and angiogenesis, as a novel Nurr1 target gene. Nrp1 expression was rapidly up-regulated by Nurr1 in MN9D cells and in situ hybridization analysis showed that Nrp1 was coexpressed with Nurr1 in the brain stem dorsal motor nucleus. Importantly, Nrp1 expression was down-regulated in this area in Nurr1 null mice. Moreover, two functional Nurr1 binding sites were identified in the Nrp1 promoter and Nurr1 was found to be recruited to these sites in MN9D cells, further supporting that Nrp1 is a direct downstream target of Nurr1. Taken together, our findings suggest that Nurr1 might influence the processes of axon guidance and/or angiogenesis via the regulation of Nrp1 expression.     

Structure-activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0^4,6]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K_i = 0.36 nM) and viral replication (replicon EC₅₀ = 7.8 nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

Light-dependent induction of cFos during subjective day and night in PACAP-containing ganglion cells of the retinohypothalamic tract.

Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.     

Long-term warming effects on root morphology, root mass distribution, and microbial activity in two dry tundra plant communities in northern Sweden

Effects of warming on root morphology, root mass distribution and microbial activity were studied in organic and mineral soil layers in two alpine ecosystems over>10 yr, using open-top chambers, in Swedish Lapland. Root mass was estimated using soil cores. Washed roots were scanned and sorted into four diameter classes, for which variables including root mass (g dry matter (g DM) m(-2)), root length density (RLD; cm cm(-3) soil), specific root length (SRL; m g DM(-1)), specific root area (SRA; m2 kg DM(-1)), and number of root tips m(-2) were determined. Nitrification (NEA) and denitrification enzyme activity (DEA) in the top 10 cm of soil were measured. Soil warming shifted the rooting zone towards the upper soil organic layer in both plant communities. In the dry heath, warming increased SRL and SRA of the finest roots in both soil layers, whereas the dry meadow was unaffected. Neither NEA nor DEA exhibited differences attributable to warming. Tundra plants may respond to climate change by altering their root morphology and mass while microbial activity may be unaffected. This suggests that carbon may be incorporated in tundra soils partly as a result of increases in the mass of the finer roots if temperatures rise.     

Identification of a novel idiopathic epilepsy locus in Belgian Shepherd dogs

Epilepsy is the most common neurological disorder in dogs, with an incidence ranging from 0.5% to up to 20% in particular breeds. Canine epilepsy can be etiologically defined as idiopathic or symptomatic. Epileptic seizures may be classified as focal with or without secondary generalization, or as primary generalized. Nine genes have been identified for symptomatic (storage diseases) and one for idiopathic epilepsy in different breeds. However, the genetic background of common canine epilepsies remains unknown. We have studied the clinical and genetic background of epilepsy in Belgian Shepherds. We collected 159 cases and 148 controls and confirmed the presence of epilepsy through epilepsy questionnaires and clinical examinations. The MRI was normal while interictal EEG revealed abnormalities and variable foci in the clinically examined affected dogs. A genome-wide association study using Affymetrix 50K SNP arrays in 40 cases and 44 controls mapped the epilepsy locus on CFA37, which was replicated in an independent cohort (81 cases and 88 controls; combined p = 9.70×10⁻¹⁰, OR = 3.3). Fine mapping study defined a ∼1 Mb region including 12 genes of which none are known epilepsy genes or encode ion channels. Exonic sequencing was performed for two candidate genes, KLF7 and ADAM23. No variation was found in KLF7 but a highly-associated non-synonymous variant, G1203A (R387H) was present in the ADAM23 gene (p = 3.7×10⁻⁸, OR = 3.9 for homozygosity). Homozygosity for a two-SNP haplotype within the ADAM23 gene conferred the highest risk for epilepsy (p = 6.28×10⁻¹¹, OR = 7.4). ADAM23 interacts with known epilepsy proteins LGI1 and LGI2. However, our data suggests that the ADAM23 variant is a polymorphism and we have initiated a targeted re-sequencing study across the locus to identify the causative mutation. It would establish the affected breed as a novel therapeutic model, help to develop a DNA test for breeding purposes and introduce a novel candidate gene for human idiopathic epilepsies.     

Impact of Ten-Valent Pneumococcal Conjugate Vaccination on Invasive Pneumococcal Disease in Finnish Children – A Population-Based Study

Background

The ten-valent pneumococcal conjugate vaccine (PCV10) was introduced into the Finnish National Vaccination Program (NVP) in September 2010 with a 2+1 schedule (3, 5, 12 months) without catch-up vaccinations. We evaluated the direct and indirect effects of PCV10 on invasive pneumococcal disease (IPD) among children ≤5 years of age during the first three years after NVP introduction.

Methods

We conducted a population-based, observational follow-up study. The cohort of vaccine-eligible children (all children born June 1, 2010 or later) was followed from 3 months of age until the end of 2013. For the indirect effect, another cohort of older children ineligible for PCV10 vaccination was followed from 2011 through 2013. Both cohorts were compared with season- and age-matched reference cohorts before NVP introduction. National, population-based laboratory surveillance data were used to compare culture-confirmed serotype-specific IPD rates in the vaccine target and reference cohorts by using Poisson regression models.

Results

The overall IPD rate among vaccine-eligible children was reduced by 80% (95%CI 72 to 85); the reduction in vaccine-type IPD was 92% (95%CI 86 to 95). However, a non-significant increase in non-vaccine type IPD was observed. During 2012–2013, we also observed a 48% (95%CI 18 to 69) reduction in IPD among unvaccinated children 2 to 5 years of age, which was mostly attributable to the ten vaccine serotypes.

Conclusions

This is the first population-based study investigating the impact of PCV10 introduction without prior PCV7 use. A substantial decrease in IPD rates among vaccine-eligible children was observed. A smaller and temporally delayed reduction among older, unvaccinated children suggests that PCV10 also provides indirect protection against vaccine-type IPD. Changes in serotype distribution warrant continuous monitoring of potential increases in non-vaccine serotypes.