Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

We applied a ¹⁵N dilution technique called “Integrated Total Nitrogen Input” (ITNI) to quantify annual atmospheric N input into a peatland surrounded by intensive agricultural practices over a 2-year period. Grass species and grass growth effects on atmospheric N deposition were investigated using Lolium multiflorum and Eriophorum vaginatum and different levels of added N resulting in increased biomass production. Plant biomass production was positively correlated with atmospheric N uptake (up to 102.7 mg N pot⁻¹) when using Lolium multiflorum. In contrast, atmospheric N deposition to Eriophorum vaginatum did not show a clear dependency to produced biomass and ranged from 81.9 to 138.2 mg N pot⁻¹. Both species revealed a relationship between atmospheric N input and total biomass N contents. Airborne N deposition varied from about 24 to 55 kg N ha⁻¹ yr⁻¹. Partitioning of airborne N within the monitor system differed such that most of the deposited N was found in roots of Eriophorum vaginatum while the highest share was allocated in aboveground biomass of Lolium multiflorum. Compared to other approaches determining atmospheric N deposition, ITNI showed highest airborne N input and an up to fivefold exceedance of the ecosystem-specific critical load of 5–10 kg N ha⁻¹ yr⁻¹.     

A COLQ Missense Mutation in Sphynx and Devon Rex Cats with Congenital Myasthenic Syndrome

An autosomal recessive neuromuscular disorder characterized by skeletal muscle weakness, fatigability and variable electromyographic or muscular histopathological features has been described in the two related Sphynx and Devon Rex cat breeds (Felis catus). Collection of data from two affected Sphynx cats and their relatives pointed out a single disease candidate region on feline chromosome C2, identified following a genome-wide SNP-based homozygosity mapping strategy. In that region, we further identified COLQ (collagen-like tail subunit of asymmetric acetylcholinesterase) as a good candidate gene, since COLQ mutations were identified in affected humans and dogs with endplate acetylcholinesterase deficiency leading to a synaptic form of congenital myasthenic syndrome (CMS). A homozygous c.1190G>A missense variant located in exon 15 of COLQ, leading to a C397Y substitution, was identified in the two affected cats. C397 is a highly-conserved residue from the C-terminal domain of the protein; its mutation was previously shown to produce CMS in humans, and here we confirmed in an affected Sphynx cat that it induces a loss of acetylcholinesterase clustering at the neuromuscular junction. Segregation of the c.1190G>A variant was 100% consistent with the autosomal recessive mode of inheritance of the disorder in our cat pedigree; in addition, an affected, unrelated Devon Rex cat recruited thereafter was also homozygous for the variant. Genotyping of a panel of 333 cats from 14 breeds failed to identify a single carrier in non-Sphynx and non-Devon Rex cats. Finally, the percentage of healthy carriers in a European subpanel of 81 genotyped Sphynx cats was estimated to be low (3.7%) and 14 control Devon Rex cats were genotyped as wild-type individuals. Altogether, these results strongly support that the neuromuscular disorder reported in Sphynx and Devon Rex breeds is a CMS caused by a unique c.1190G>A missense mutation, presumably transmitted through a founder effect, which strictly and slightly disseminated in these two breeds. The presently available DNA test will help owners avoid matings at risk.     

Is There a Link between Exertional Heat Stroke and Susceptibility to Malignant Hyperthermia?

Objective

The identification of a predisposition toward malignant hyperthermia (MH) as a risk factor for exertional heat stroke (EHS) remains a matter of debate. Such a predisposition indicates a causal role for MH susceptibility (MHS) after EHS in certain national recommendations and has led to the use of an in vitro contracture test (IVCT) to identify the MHS trait in selected or unselected EHS patients. The aim of this study was to determine whether the MHS trait is associated with EHS.

Methods

EHS subjects in the French Armed Forces were routinely examined for MHS after experiencing an EHS episode. This retrospective study compared the features of IVCT-diagnosed MHS (iMHS) EHS subjects with those of MH-normal EHS patients and MH patients during the 2004–2010 period. MHS status was assessed using the European protocol.

Results

During the study period, 466 subjects (median age 25 years; 31 women) underwent MHS status investigation following an EHS episode. None of the subjects reported previous MH events. An IVCT was performed in 454 cases and was diagnostic of MHS in 45.6% of the study population, of MH susceptibility to halothane in 18.5%, of MH susceptibility to caffeine in 9.9%, and of MH susceptibility to halothane and caffeine in 17.2%. There were no differences in the clinical features, biological features or outcomes of iMHS EHS subjects compared with those of MH-normal or caffeine or halothane MHS subjects without known prior EHS episode. The recurrence rate was 12.7% and was not associated with MH status or any clinical or biological features. iMHS EHS patients exhibited a significantly less informative IVCT response than MH patients.

Conclusions

The unexpected high prevalence of the MHS trait after EHS suggested a latent disturbance of calcium homeostasis that accounted for the positive IVCT results. This study did not determine whether EHS patients have an increased risk of MH, and it could not determine whether MH susceptibility is a risk factor for EHS.     

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3’ end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.     

Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.     

Experience Sampling-Based Personalized Feedback and Positive Affect: A Randomized Controlled Trial in Depressed Patients

Objectives

Positive affect (PA) plays a crucial role in the development, course, and recovery of depression. Recently, we showed that a therapeutic application of the experience sampling method (ESM), consisting of feedback focusing on PA in daily life, was associated with a decrease in depressive symptoms. The present study investigated whether the experience of PA increased during the course of this intervention.

Design

Multicentre parallel randomized controlled trial. An electronic random sequence generator was used to allocate treatments.

Settings

University, two local mental health care institutions, one local hospital.

Participants

102 pharmacologically treated outpatients with a DSM-IV diagnosis of major depressive disorder, randomized over three treatment arms.

Intervention

Six weeks of ESM self-monitoring combined with weekly PA-focused feedback sessions (experimental group); six weeks of ESM self-monitoring combined with six weekly sessions without feedback (pseudo-experimental group); or treatment as usual (control group).

Main outcome

The interaction between treatment allocation and time in predicting positive and negative affect (NA) was investigated in multilevel regression models.

Results

102 patients were randomized (mean age 48.0, SD 10.2) of which 81 finished the entire study protocol. All 102 patients were included in the analyses. The experimental group did not show a significant larger increase in momentary PA during or shortly after the intervention compared to the pseudo-experimental or control groups (χ² (2) =0.33, p=.846). The pseudo-experimental group showed a larger decrease in NA compared to the control group (χ² (1) =6.29, p=.012).

Conclusion

PA-focused feedback did not significantly impact daily life PA during or shortly after the intervention. As the previously reported reduction in depressive symptoms associated with the feedback unveiled itself only after weeks, it is conceivable that the effects on daily life PA also evolve slowly and therefore were not captured by the experience sampling procedure immediately after treatment.

Trial Registration

Trialregister.nl/trialreg/index.asp. NTR1974     

Transcriptome Analysis of Poplar during Leaf Spot Infection with Sphaerulina spp.

Diseases of poplar caused by the native fungal pathogen Sphaerulina musiva and related species are of growing concern, particularly with the increasing interest in intensive poplar plantations to meet growing energy demands. Sphaerulina musiva is able to cause infection on leaves, resulting in defoliation and canker formation on stems. To gain a greater understanding of the different responses of poplar species to infection caused by the naturally co-evolved Sphaerulina species, RNA-seq was conducted on leaves of Populus deltoides, P. balsamifera and P. tremuloides infected with S. musiva, S. populicola and a new undescribed species, Ston1, respectively. The experiment was designed to contain the pathogen in a laboratory environment, while replicating disease development in commercial plantations. Following inoculation, trees were monitored for disease symptoms, pathogen growth and host responses. Genes involved in phenylpropanoid, terpenoid and flavonoid biosynthesis were generally upregulated in P. balsamifera and P. tremuloides, while cell wall modification appears to play an important role in the defense of P. deltoides. Poplar defensive genes were expressed early in P. balsamifera and P. tremuloides, but their expression was delayed in P. deltoides, which correlated with the rate of disease symptoms development. Also, severe infection in P. balsamifera led to leaf abscission. This data gives an insight into the large differences in timing and expression of genes between poplar species being attacked by their associated Sphaerulina pathogen.