Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

Differently from Arabidopsis, ATR activity in maize plays an essential role under control growth conditions, ensuring genome stability during kernel Development.     

Site-directed targeting of plasminogen activator inhibitor-1 as an example for a novel approach in rational drug design

As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition of PAI-1. Since monoclonal antibodies are in most cases not ideal for therapeutic use, the question of whether smaller molecules exert comparable effects is a hot issue. To answer this question, Cys residues were introduced in PAI-1 at positions previously identified as determining the epitope of a PAI-1-inhibiting antibody, MA-8H9D4, resulting in PAI-1-R300C, PAI-1-Q303C, and PAI-1-D305C. Subsequently, low molecular mass sulfhydryl-specific reagents (i.e. BODIPY 530/550 IA (molecular mass 626 Da) and BODIPY FL C(1)-IA (molecular mass 417 Da)) were allowed to react covalently with the cysteine. The functional distribution (inhibitory versus substrate) toward tissue-type plasminogen activator was determined for the labeled and the unlabeled samples. Labeling at position 300 leads to a 1.7- and 2.2-fold increase in SI value for BODIPY 530/550 IA and BODIPY FL C(1)-IA, respectively. Labeling at position 303 results in a 3.3- and 1.9-fold increase of the SI value for the large and the small label, respectively. At position 305, the SI values are 3.1-fold increased for both labels. The effect (on SI and on serpin activity) of the manipulations at these positions is in good agreement with the effect exerted by MA-8H9D4. In conclusion, our study provides proof of concept for the proposed approach in evaluating whether targeting a functional epitope with a small synthetic compound may be a feasible strategy in rational drug design.     

De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination

Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.     

Dolomite limits acidification of a biofilter degrading dimethyl sulphide

The applicability of dolomite particlesto control acidificationin a Hyphomicrobium MS3inoculated biofilter removingdimethyl sulphide (Me₂S) wasstudied. While direct inoculationof the dolomite particles with theliquid microbial culture was notsuccessful, start-up ofMe₂S-degradation in thebiofilter was observed when thedolomite particles were mixed with33% (wt/wt) of Hyphomicrobium MS3-inoculatedcompost or wood bark material.Under optimal conditions, anelimination capacity (EC) of 1680~g Me₂S m^-3 d^-1 wasobtained for the compost/dolomitebiofilter. Contrary to a wood barkor compost biofilter, no reductionin activity due to acidificationwas observed in these biofiltersover a 235 day period because ofthe micro environmentneutralisation of the microbialmetabolite H₂SO₄ with thecarbonate in the dolomite material.However, performance of thebiofilter decreased when themoisture content of the mixedcompost/dolomite material droppedbelow 15%. Next to this, nutrientlimitation resulted in a gradualdecrease of the EC andsupplementation of a nitrogensource was a prerequisite to obtaina long-term high EC (> 250 gMe₂S m^-3 d^-1) forMe₂S. In relation to thisnitrogen supplementation, it wasobserved that stable ECs forMe₂S were obtained when thisnutrient was dosed to the biofilterat a Me₂S-C/NH₄Cl-Nratio of about 10.Abbreviations:DW – dry weight,EC – elimination capacity,Me₂S – dimethyl sulphide,OL – organic loading rate,VS - volatile solids     

A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2

Several nutrient permeases have been identified in yeast, which combine a transport and receptor function, and are called transceptors. The Gap1 general amino acid permease and the Mep2 ammonium permease mediate rapid activation by amino acids and by ammonium, respectively, of the protein kinase A (PKA) pathway in nitrogen-starved cells. Their mode of action is not well understood. Both proteins are subject to complex controls governing their intracellular trafficking. Using a split-ubiquitin yeast two-hybrid screen with Gap1 or Mep2 as bait, we identified proteins putatively interacting with Gap1 and/or Mep2. They are involved in glycosylation, the secretory pathway, sphingolipid biosynthesis, cell wall biosynthesis and other processes. For several candidate interactors, determination of transport and signaling capacity, as well as localization of Gap1 or Mep2 in the corresponding deletion strains, confirmed a functional interaction with Gap1 and/or Mep2. Also common interacting proteins were identified. Transport and signaling were differentially affected in specific deletion strains, clearly separating the two functions of the transceptors and confirming that signaling does not require transport. We identified two new proteins, Bsc6 and Yir014w, that affect trafficking or downregulation of Gap1. Deletion of EGD2, YNL024c or SPC2 inactivates Gap1 transport and signaling, while its plasma membrane level appears normal.. Vma4 is required for Mep2 expression, while Gup1 appears to be required for proper distribution of Mep2 over the plasma membrane. Some of the interactions were confirmed by GST pull-down assay, using the C-terminal tail of Gap1 or Mep2 expressed in E.coli. Our results reveal the effectiveness of split-ubiquitin two-hybrid screening for identification of proteins functionally interacting with membrane proteins. They provide several candidate proteins involved in the transport and signaling function or in the complex trafficking control of the Gap1 and Mep2 transceptors.     

The Effect of Cigarette Smoke Exposure on the Development of Inflammation in Lungs,Gut and Joints of TNFΔARE Mice

The inflammatory cytokine TNF-α is a central mediator in many immune-mediated diseases, such as Crohn’s disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNF^ΔARE mice; in which a systemic TNF-α overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNF^ΔARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNF^ΔARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNF^ΔARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNF^ΔARE mice. The lung responses towards CS in TNF^ΔARE mice however depend on the duration of CS exposure.     

Soluble Tumor Necrosis Factor Receptor 1 and 2 Predict Outcomes in Advanced Chronic Kidney Disease: A Prospective Cohort Study

Background

Soluble tumor necrosis factor receptors 1 (sTNFR1) and 2 (sTNFR2) have been associated to progression of renal failure, end stage renal disease and mortality in early stages of chronic kidney disease (CKD), mostly in the context of diabetic nephropathy. The predictive value of these markers in advanced stages of CKD irrespective of the specific causes of kidney disease has not yet been defined. In this study, the relationship between sTNFR1 and sTNFR2 and the risk for adverse cardiovascular events (CVE) and all-cause mortality was investigated in a population with CKD stage 4-5, not yet on dialysis, to minimize the confounding by renal function.

Patients and methods

In 131 patients, CKD stage 4-5, sTNFR1, sTNFR2 were analysed for their association to a composite endpoint of all-cause mortality or first non-fatal CVE by univariate and multivariate Cox proportional hazards models. In the multivariate models, age, gender, CRP, eGFR and significant comorbidities were included as covariates.

Results

During a median follow-up of 33 months, 40 events (30.5%) occurred of which 29 deaths (22.1%) and 11 (8.4%) first non-fatal CVE. In univariate analysis, the hazard ratios (HR) of sTNFR1 and sTNFR2 for negative outcome were 1.49 (95% confidence interval (CI): 1.28-1.75) and 1.13 (95% CI: 1.06-1.20) respectively. After adjustment for clinical covariables (age, CRP, diabetes and a history of cardiovascular disease) both sTNFRs remained independently associated to outcomes (HR: sTNFR1: 1.51, 95% CI: 1.30-1.77; sTNFR2: 1.13, 95% CI: 1.06-1.20). A subanalysis of the non-diabetic patients in the study population confirmed these findings, especially for sTNFR1.

Conclusion

sTNFR1 and sTNFR2 are independently associated to all-cause mortality or an increased risk for cardiovascular events in advanced CKD irrespective of the cause of kidney disease.     

Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles

Background

Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7^th day and identification of the bacterial isolates was carried out with a molecular technique.

Methods

Self-collected vaginal swabs were obtained daily from 17 non pregnant, menarchal volunteers, and used for daily Gram staining and weekly culture. Bacterial isolates were identified with tDNA-PCR and 16 S rRNA gene sequencing.

Results

Nine women presented with predominantly normal VMF and the 8 others had predominantly disturbed VMF. The overall VMF of each volunteer was characteristic and rather stable. Menses and antimicrobials were the major disturbing factors of the VMF. Disturbances were always accompanied by a rise in Gram positive cocci, which also appeared to be a significant group within the VMF in general.

Conclusions

We observed a huge interindividual variability of predominantly stable VMF types. The importance of Gram positive cocci in VMF is underestimated. L. crispatus was the species that was most negatively affected by the menses, whereas the presence of the other lactobacilli was less variable.