Recent insights into noncanonical 5' capping and decapping of RNA.

The 5' N7-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5' capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as "no-cap decay." It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.

Diadenosine tetraphosphate modulated quorum sensing in bacteria treated with kanamycin.

BACKGROUND: The dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress. RESULTS: Here, we labeled Ap4A with biotin and incubated the labeled Ap4A with the total proteins extracted from kanamycin-treated Escherichia coli to identify the Ap4A binding protein in bacteria treated with kanamycin. Liquid chromatography‒mass spectrometry (LCMS) and bioinformatics were used to identify novel proteins that Ap4A interacts with that are involved in biofilm formation, quorum sensing, and lipopolysaccharide biosynthesis pathways. Then, we used the apaH knockout strain of E. coli K12-MG1655, which had increased intracellular Ap4A, to demonstrate that Ap4A affected the expression of genes in these three pathways. We also found that the swarming motility of the apaH mutant strain was reduced compared with that of the wild-type strain, and under kanamycin treatment, the biofilm formation of the mutant strain decreased. CONCLUSIONS: These results showed that Ap4A can reduce the survival rate of bacteria treated with kanamycin by regulating quorum sensing (QS). These effects can expand the application of kanamycin combinations in the treatment of multidrug-resistant bacteria.

Np4A alarmones function in bacteria as precursors to RNA caps.

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5' end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Bronchial artery embolization for the treatment of haemoptysis in pulmonary hypertension.

PURPOSE: To test the efficacy of bronchial artery embolization (BAE) to treat haemoptysis in pulmonary hypertension (PH). METHODS: 33 patients were treated by BAE for haemoptysis associated with PH (PH group = 21) or non-associated with PH (control group = 12). The details of procedure, outcome, and rate of relapse were compared between the two groups. Within the PH group, the comparison was operated between subjects with congenital heart disease-associated pulmonary artery hypertension (CHD-APAH subgroup = 12) and non-CHD (non-CHD-APAH subgroup = 9). RESULTS: The rate of relapse at 30 and 90-days was similar between the PH group and control group. BAE in the PH group was more challenging (median 2 arteries embolized per procedure) compared to the control group (median 1 artery embolized per procedure; p = 0.001). Bleeding arteries were more heterogeneous in the PH group, while a single right bronchial artery was the only clinical finding in 66.7% of controls (p = 0.001). Within the PH group, the CHD subgroup showed higher survival rate compared to the non-CHD-APAH group (p = 0.007). CONCLUSION: BAE is effective and safe for the treatment of haemoptysis in PH, yet more challenging than other conditions. In PH-associated haemoptysis, BAE provides higher survival rate for subjects with PH associated with CHD.

Assessment of bacterial biotransformation of alkylnaphthalene lubricating base oil component 1-butylnaphthalene by LC/ESI-MS(/MS).

Alkylnaphthalene lubricating oils are synthetic Group V base oils that are utilized in wide-ranging industrial applications and which are composed of polyalkyl chain-alkylated naphthalenes. Identification of alkylnaphthalene biotransformation products and determination of their mass spectrometry (MS) fragmentation signatures provides valuable information for predicting their environmental fates and for development of analytical methods to monitor their biodegradation. In this work, laboratory-based environmental petroleomics was applied to investigate the catabolism of the alkylnaphthalene, 1-butylnaphthalene (1-BN), by liquid chromatography electrospray ionization MS data mapping and targeted collision-induced dissociation (CID) analyses. Comparative mapping revealed that numerous catabolites were produced from soil bacterium, Sphingobium barthaii KK22. Targeted CID showed unique patterns of production of even-valued deprotonated fragments that were found to originate from specific classes of bacterial catabolites. Based upon results of CID analyses of catabolites and authentic standards, MS signatures were proposed to occur through formation of distonic radical anions from bacterially-produced alkylphenol biotransformation products. Finally, spectra interpretation was guided by CID results to propose chemical structures for twenty-two 1-BN catabolites resulting in construction of 1-BN biotransformation pathways. Multiple pathways were identified that included aromatic ring-opening, alkyl chain-shortening and production of α,ß-unsaturated aldehydes from alkylated phenols. Until now, α,ß-unsaturated aldehydes have not been a class of compounds much reported from alkylated polycyclic aromatic hydrocarbon (APAH) and PAH biotransformation. This work provides a new understanding of alkylnaphthalene biotransformation and proposes MS markers applicable to monitoring APAH biotransformation in the form of alkylated phenols, and by extension, α,ß-unsaturated aldehydes, and toxic potential during spilled oil biodegradation.

Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia.

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future.

Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?

BACKGROUND: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. RESULTS: We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. CONCLUSIONS: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored.

The complex enzymology of mRNA decapping: Enzymes of four classes cleave pyrophosphate bonds.

The 5' ends of most RNAs are chemically modified to enable protection from nucleases. In bacteria, this is often achieved by keeping the triphosphate terminus originating from transcriptional initiation, while most eukaryotic mRNAs and small nuclear RNAs have a 5'→5' linked N7 -methyl guanosine (m7 G) cap added. Several other chemical modifications have been described at RNA 5' ends. Common to all modifications is the presence of at least one pyrophosphate bond. To enable RNA turnover, these chemical modifications at the RNA 5' end need to be reversible. Dependent on the direction of the RNA decay pathway (5'→3' or 3'→5'), some enzymes cleave the 5'→5' cap linkage of intact RNAs to initiate decay, while others act as scavengers and hydrolyse the cap element of the remnants of the 3'→5' decay pathway. In eukaryotes, there is also a cap quality control pathway. Most enzymes involved in the cleavage of the RNA 5' ends are pyrophosphohydrolases, with only a few having (additional) 5' triphosphonucleotide hydrolase activities. Despite the identity of their enzyme activities, the enzymes belong to four different enzyme classes. Nudix hydrolases decap intact RNAs as part of the 5'→3' decay pathway, DXO family members mainly degrade faulty RNAs, members of the histidine triad (HIT) family are scavenger proteins, while an ApaH-like phosphatase is the major mRNA decay enzyme of trypanosomes, whose RNAs have a unique cap structure. Many novel cap structures and decapping enzymes have only recently been discovered, indicating that we are only beginning to understand the mechanisms of RNA decapping. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA Processing > Capping and 5' End Modifications.

Quality of life among pulmonary hypertension patients in Finland.

BACKGROUND: The purpose of the study was to examine pulmonary hypertension (PH) patients' quality of life (QOL) for the first time in Finland. METHODS: This was a non-interventional, cross-sectional study. The SF-36v2 questionnaire was sent to the PH patients who had been referred to or followed up on at the Helsinki University Central Hospital's pulmonary clinic for idiopathic pulmonary arterial hypertension, associated pulmonary arterial hypertension (APAH), or chronic thromboembolic PH (CTEPH). The patients were on pulmonary arterial hypertension (PAH) - specific drugs, were at least 18 years old, and had signed an informed consent. RESULTS: There were 62 patients who fulfilled the inclusion criteria, and 53% of respondents rated their health as moderate. Similarly, 55% of respondents rated their health status approximately the same compared to their situation 1 year ago. QOL was impaired in all other subscales, except for the mental health and mental component score. A majority of patients suffered from PH symptoms, which worsened their QOL. The greatest impact on their QOL was associated with a high World Health Organization (WHO) functional class (FC), poor performance in a 6-min walking test (6MWT), symptoms, oxygen therapy, elevated pro-brain natriuretic peptide, pericardial effusion, APAH etiology, and being retired from work. CONCLUSIONS: The respondents had a reduced QOL, compared to the general population, in all other subscales, except for mental health. APAH patients had the worst QOL. Good results in functional capacity measures (WHO FC, 6MWT) were associated with a better QOL. Patients' QOL can be improved by reducing the symptoms of PAH.

Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus.

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn(2+), PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co(2+) PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn(2+) was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co(2+) was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively.

Sequence-based prediction of linear autoepitopes involved in pathogenesis of IPAH and the corresponding organism sources of molecular mimicry.

We proposed here a sequence-based approach predicting some microorganisms as possible sources of autoantigen-related molecular mimicry concerning Idiopathic Pulmonary Arterial Hypertension (IPAH) and related hypertension mostly accompanying autoimmune diseases and AIDS (APAH). This approach (SPECIES_VALENCE) processes the database occurrences of linear autoepitope-related short Dense Quasi-Pattern Sequences (DQPA) generated based on identities of important autoantigenic sequences. The corresponding enumeration comprises two types of statistical evaluations performed in each of eight proposed models. Based on this enumeration, we selected nine microorganisms, whereas revaluation of the obtained scoring values restricted Pseudomonas aeruginosa, Aspergillus fumigatus and the two co-infecting herpes viruses (Epstein Barr virus and cytomegalovirus) as most favourable. The results are discussed in terms of (a) the validity of increased DQPA occurrence in functionally correlated sequences, (b) the possible mechanisms leading to autoantibody response, (c) selected additional pathogenic effects of predicted microorganisms and (d) possible effects of cross-reactivities and immune tolerance.

Pediatric pulmonary hypertension.

Pulmonary hypertension (PH) is a rare disease in newborns, infants, and children that is associated with significant morbidity and mortality. In the majority of pediatric patients, PH is idiopathic or associated with congenital heart disease and rarely is associated with other conditions such as connective tissue or thromboembolic disease. Incidence data from the Netherlands has revealed an annual incidence and point prevalence of 0.7 and 4.4 for idiopathic pulmonary arterial hypertension and 2.2 and 15.6 for pulmonary arterial hypertension, respectively, associated with congenital heart disease (CHD) cases per million children. The updated Nice classification for PH has been enhanced to include a greater depth of CHD and emphasizes persistent PH of the newborn and developmental lung diseases, such as bronchopulmonary dysplasia and congenital diaphragmatic hernia. The management of pediatric PH remains challenging because treatment decisions continue to depend largely on results from evidence-based adult studies and the clinical experience of pediatric experts.

Updated treatment algorithm of pulmonary arterial hypertension.

The demands on a pulmonary arterial hypertension (PAH) treatment algorithm are multiple and in some ways conflicting. The treatment algorithm usually includes different types of recommendations with varying degrees of scientific evidence. In addition, the algorithm is required to be comprehensive but not too complex, informative yet simple and straightforward. The type of information in the treatment algorithm are heterogeneous including clinical, hemodynamic, medical, interventional, pharmacological and regulatory recommendations. Stakeholders (or users) including physicians from various specialties and with variable expertise in PAH, nurses, patients and patients' associations, healthcare providers, regulatory agencies and industry are often interested in the PAH treatment algorithm for different reasons. These are the considerable challenges faced when proposing appropriate updates to the current evidence-based treatment algorithm.The current treatment algorithm may be divided into 3 main areas: 1) general measures, supportive therapy, referral strategy, acute vasoreactivity testing and chronic treatment with calcium channel blockers; 2) initial therapy with approved PAH drugs; and 3) clinical response to the initial therapy, combination therapy, balloon atrial septostomy, and lung transplantation. All three sections will be revisited highlighting information newly available in the past 5 years and proposing updates where appropriate. The European Society of Cardiology grades of recommendation and levels of evidence will be adopted to rank the proposed treatments.