Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane‐aerated biofilm reactor (MABR) and the composition of the initial ammonia‐ and nitrite‐oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat‐sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO₂^‐‐N/NH₄⁺‐N) were obtained in the Inoculum B‐ (55.2–56.4%) versus the Inoculum A‐ (20.2–22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100–150 µm), but the AOB profiles [based on 16S rRNA gene targeted real‐time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B‐ versus A‐initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A‐ and B‐initiated reactors were Nitrosospira spp. (48.9–61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8–63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell^?1 h^?1 versus 0.79 fmol cell^?1 h^?1). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.     

Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the bchI, -D, and -H Gene Products of the Green Sulfur Bacterium Chlorobium vibrioforme Expressed in Escherichia coli

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg²⁺ into protoporphyrin IX. Three genes, designated bchI, -D, and -H, from the strictly anaerobic and obligately phototrophic green sulfur bacterium Chlorobium vibrioforme show a significant level of homology to the magnesium chelatase-encoding genes bchI, -D, and -H and chlI, -D, and -H of Rhodobacter sphaeroides and Synechocystis strain PCC6803, respectively. These three genes were expressed in Escherichia coli; the subsequent purification of overproduced BchI and -H proteins on an Ni²⁺-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase of C. vibrioforme is similar to the magnesium chelatases of the distantly related bacteria R. sphaeroides and Synechocystis strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the C. vibrioforme BchI and -D proteins and the Synechocystis strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the bchI gene product, were expressed in E. coli, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in C. vibrioforme.     

Fibrin Clot Structure and Platelet Aggregation in Patients with Aspirin Treatment Failure

Background

Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF.

Methods

We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics.

Results

Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31–0.35, p-values<0.001) and CRP levels (r = 0.60, p<0.001).

Conclusions

Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.     

NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase

The chloroplast-localized NADPH-dependent thioredoxin reductase (NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding protochlorophyllide formation. A specific involvement of NTRC during biosynthesis of protochlorophyllide is indicated from in vitro aerobic cyclase assays in which the conversion of Mg-protoporhyrin monomethyl ester into protochlorophyllide is stimulated by addition of the NTRC/2-Cys peroxiredoxin system. These findings support the hypothesis that this NADPH-dependent hydrogen peroxide scavenging system is particularly important during periods with limited reducing power from photosynthesis, e.g. under chloroplast biogenesis.     

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.     

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

Background

The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.

Results

We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.

Conclusions

Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.     

No evidence of inhibition of horizontal gene transfer by CRISPR–Cas on evolutionary timescales

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)–Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR–Cas maintenance and one of the causes of the patchy distribution of CRISPR–Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR–Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR–Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.