pmoA Primers for detection of anaerobic methanotrophs

Published pmoA primers do not match the pmoA sequence of "Candidatus Methylomirabilis oxyfera," a bacterium that performs nitrite-dependent anaerobic methane oxidation. Therefore, new pmoA primers for the detection of "Ca. Methylomirabilis oxyfera"-like methanotrophs were developed and successfully tested on freshwater samples from different habitats. These primers expand existing molecular tools for the study of methanotrophs in the environment.     

p24 Proteins from the same subfamily are functionally nonredundant

The p24 proteins function in early secretory pathway transport processes, but their exact role is unclear. In physiologically activated Xenopus melanotrope cells, a representative of each p24 subfamily (p24α₃, -β₁, -γ₃, -δ₂) is upregulated coordinately with the major melanotrope cargo, proopiomelanocortin (POMC), whereas two other p24s (p24γ₂ and -δ₁) are also expressed, but not coordinately with POMC. Using melanotrope-specific transgene expression, we here find that the roles of both p24γ₂ and p24δ₁ in the transport, glycosylation, sulphation and cleavage of POMC are different from those of their upregulated subfamily relatives (p24γ₃ and p24δ₂, respectively). Thus, even p24 proteins from the same subfamily have distinct functions in secretory cargo biosynthesis.     

Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice

Scavenger receptor class B type I (SR-BI) has been identified as a functional HDL binding protein that can mediate the selective uptake of cholesteryl ester (CE) from HDL. To quantify the in vivo role of SR-BI in the process of selective uptake, HDL was labeled with cholesteryl ether ([(3)H] CEt-HDL) and (125)I-tyramine cellobiose ([(125)I]TC-HDL) and injected into SR-BI knockout (KO) and wild-type (WT) mice. In SR-BI KO mice, the clearance of HDL-CE from the blood circulation was greatly diminished (0.043 +/- 0.004 pools/h for SR-BI KO mice vs. 0.106 +/- 0.004 pools/h for WT mice), while liver and adrenal uptake were greatly reduced. Utilization of double-labeled HDL ([(3)H]CEt and [(125)I]TC) indicated the total absence in vivo of the selective decay and liver uptake of CE from HDL in SR-BI KO mice. Parenchymal cells isolated from SR-BI KO mice showed similar association values for [(3)H]CEt and [(125)I]TC in contrast to WT cells, indicating that in parenchymal liver cells SR-BI is the only molecule exerting selective CE uptake from HDL. Thus, in vivo and in vitro, SR-BI is the sole molecule mediating the selective uptake of CE from HDL by the liver and the adrenals, making it the unique target to modulate reverse cholesterol transport.     

The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development

Ent‐kaurenoic acid oxidase (KAO), a class of cytochrome P450 monooxygenases of the subfamily CYP88A, catalyzes the conversion of ent‐kaurenoic acid (KA) to gibberellin (GA) GA₁₂, the precursor of all GAs, thereby playing an important role in determining GA concentration in plants. Past work has demonstrated the importance of KAO activity for growth in various plant species. In Arabidopsis, this enzyme is encoded by two genes designated KAO1 and KAO2. In this study, we used various approaches to determine the physiological roles of KAO1 and KAO2 throughout plant development. Analysis of gene expression pattern reveals that both genes are mainly expressed in germinating seeds and young developing organs, thus suggesting functional redundancy. Consistent with this, kao1 and kao2 single mutants are indistinguishable from wild‐type plants. By contrast, the kao1 kao2 double mutant exhibits typical non‐germinating GA‐dwarf phenotypes, similar to those observed in the severely GA‐deficient ga1‐3 mutant. Phenotypic characterization and quantitative analysis of endogenous GA contents of single and double kao mutants further confirm an overlapping role of KAO1 and KAO2 throughout Arabidopsis development.     

Identifying Ca2+-Binding Sites in Proteins by Liquid Chromatography-Mass Spectrometry Using Ca2+-Directed Dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.Calcium-dependent protein interactions mostly organized in protein networks are responsible for the regulation of cell cycle progression, cell growth, differentiation, secretion, and cytoskeletal organization (1–3). As many of these proteins are linked to various pathological conditions, they are clinically important. The speed at which calcium can have an interplay between various cellular components is impressive and comes notably detectable in neurological processes and in muscle contraction. Calcium binding sites in proteins can be determined by NMR spectroscopy (4, 5). For example, by such NMR measurements, the Ca²⁺-binding sites of the tellurite-resistance protein TerD from Klebsiella pneumoniae were found to be formed in part by a highly conserved motif of 13 residues specified by the sequence GDN(R/L)TG(E/A)GDGDDE (4).Although NMR is the gold standard to study calcium binding in proteins, this approach has several drawbacks. For instance, protein size is limited (< 30 kDa) and proteins should be pure and isotopically labeled. In addition, although the information content is high, NMR is relatively insensitive compared with other techniques such as MS and fluorescence spectroscopy, and relatively large quantities of material (typically 0.5 ml at 0.5–1.0 mm in biological samples) are needed, although efforts are devoted to improve sensitivity in NMR, such as stripline NMR (6).In bottom-up proteomics, proteolytic peptides, generated by enzymatic digestion of complex protein mixtures, are sequenced by MS-based methods (MS/MS (7, 8)) using collision-induced dissociations. Because of the even higher complexity of these peptide mixtures, liquid chromatography (LC)¹ is used to separate the peptides prior to sequencing. In such an LC-MS/MS procedure, many peptides can be identified belonging to the same protein. It has been stated (9) that by this procedure more peptides are analyzed than strictly necessary for identification purposes, but it can equally well be argued that such large coverages enable more reliable protein identifications; moreover, these larger coverages allow the detection of post-translational modifications, including specific calcium complexation as described here.Considering the need of identifying calcium-bound proteins in complex biological samples at low concentrations, we set out to develop a novel method for detecting Ca²⁺-binding sites in proteins based on LC-MS.     

Transcriptional profile of Salmonella enterica subsp. enterica serovar Weltevreden during alfalfa sprout colonization

Sprouted seeds represent a great risk for infection by human enteric pathogens because of favourable growth conditions for pathogens during their germination. The aim of this study was to identify mechanisms of interactions of Salmonella enterica subsp. enterica Weltevreden with alfalfa sprouts. RNA-seq analysis of S. Weltevreden grown with sprouts in comparison with M9-glucose medium showed that among a total of 4158 annotated coding sequences, 177 genes (4.3%) and 345 genes (8.3%) were transcribed at higher levels with sprouts and in minimal medium respectively. Genes that were higher transcribed with sprouts are coding for proteins involved in mechanisms known to be important for attachment, motility and biofilm formation. Besides gene expression required for phenotypic adaption, genes involved in sulphate acquisition were higher transcribed, suggesting that the surface on alfalfa sprouts may be poor in sulphate. Genes encoding structural and effector proteins of Salmonella pathogenicity island 2, involved in survival within macrophages during infection of animal tissue, were higher transcribed with sprouts possibly as a response to environmental conditions. This study provides insight on additional mechanisms that may be important for pathogen interactions with sprouts.     

Age Related Differences in Dynamics of Specific Memory B Cell Populations after Clinical Pertussis Infection

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (B_mem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and B_mem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of B_mem cell populations; higher levels of B_mem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects'' levels of specific B_mem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific B_mem cell and plasma IgG levels.     

Glucose Kinetics in the Collagen-Induced Arthritis Model: An All-in-One Model to Assess Both Efficacy and Metabolic Side Effects of Glucocorticoids

Prednisolone and other glucocorticoids (GCs) are potent anti-inflammatory drugs, but chronic use is hampered by metabolic side effects. Therefore, there is an urgent medical need for improved GCs that are as effective as classical GCs but have a better safety profile. A well-established model to assess anti-inflammatory efficacy is the chronic collagen-induced arthritis (CIA) model in mice, a model with features resembling rheumatoid arthritis. Models to quantify undesired effects of glucocorticoids on glucose kinetics are less well-established. Recently, we have described a model to quantify basal blood glucose kinetics using stably-labeled glucose. In the present study, we have integrated this blood glucose kinetic model in the CIA model to enable quantification of both efficacy and adverse effects in one animal model. Arthritis scores were decreased after treatment with prednisolone, confirming the anti-inflammatory properties of GCs. Both inflammation and prednisolone induced insulin resistance as insulin secretion was strongly increased whereas blood glucose concentrations and hepatic glucose production were only slightly decreased. This insulin resistance did not directly resulted in hyperglycemia, indicating a highly adaptive compensatory mechanism in these mice. In conclusion, this ‘all-in-one’ model allows for studying effects of (novel) GC compounds on the development of arthritis and glucose kinetics in a single animal. This integrative model provides a valuable tool for investigating (drug-induced) metabolic dysregulation in an inflammatory setting.     

The Burden Attributable to Mental and Substance Use Disorders as Risk Factors for Suicide: Findings from the Global Burden of Disease Study 2010

Background

The Global Burden of Disease Study 2010 (GBD 2010) identified mental and substance use disorders as the 5^th leading contributor of burden in 2010, measured by disability adjusted life years (DALYs). This estimate was incomplete as it excluded burden resulting from the increased risk of suicide captured elsewhere in GBD 2010''s mutually exclusive list of diseases and injuries. Here, we estimate suicide DALYs attributable to mental and substance use disorders.

Methods

Relative-risk estimates of suicide due to mental and substance use disorders and the global prevalence of each disorder were used to estimate population attributable fractions. These were adjusted for global differences in the proportion of suicide due to mental and substance use disorders compared to other causes then multiplied by suicide DALYs reported in GBD 2010 to estimate attributable DALYs (with 95% uncertainty).

Results

Mental and substance use disorders were responsible for 22.5 million (14.8–29.8 million) of the 36.2 million (26.5–44.3 million) DALYs allocated to suicide in 2010. Depression was responsible for the largest proportion of suicide DALYs (46.1% (28.0%–60.8%)) and anorexia nervosa the lowest (0.2% (0.02%–0.5%)). DALYs occurred throughout the lifespan, with the largest proportion found in Eastern Europe and Asia, and males aged 20–30 years. The inclusion of attributable suicide DALYs would have increased the overall burden of mental and substance use disorders (assigned to them in GBD 2010 as a direct cause) from 7.4% (6.2%–8.6%) to 8.3% (7.1%–9.6%) of global DALYs, and would have changed the global ranking from 5^th to 3^rd leading cause of burden.

Conclusions

Capturing the suicide burden attributable to mental and substance use disorders allows for more accurate estimates of burden. More consideration needs to be given to interventions targeted to populations with, or at risk for, mental and substance use disorders as an effective strategy for suicide prevention.