Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3'-terminal positions of synthesized tails.

Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3'-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3'-terminal extensions in vivo. Here, using high-throughput sequencing of the 3'-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases' substrate preference, which may allow us to predict their specificity.

FAM46 proteins are novel eukaryotic non-canonical poly(A) polymerases.

FAM46 proteins, encoded in all known animal genomes, belong to the nucleotidyltransferase (NTase) fold superfamily. All four human FAM46 paralogs (FAM46A, FAM46B, FAM46C, FAM46D) are thought to be involved in several diseases, with FAM46C reported as a causal driver of multiple myeloma; however, their exact functions remain unknown. By using a combination of various bioinformatics analyses (e.g. domain architecture, cellular localization) and exhaustive literature and database searches (e.g. expression profiles, protein interactors), we classified FAM46 proteins as active non-canonical poly(A) polymerases, which modify cytosolic and/or nuclear RNA 3' ends. These proteins may thus regulate gene expression and probably play a critical role during cell differentiation. A detailed analysis of sequence and structure diversity of known NTases possessing PAP/OAS1 SBD domain, combined with state-of-the-art comparative modelling, allowed us to identify potential active site residues responsible for catalysis and substrate binding. We also explored the role of single point mutations found in human cancers and propose that FAM46 genes may be involved in the development of other major malignancies including lung, colorectal, hepatocellular, head and neck, urothelial, endometrial and renal papillary carcinomas and melanoma. Identification of these novel enzymes taking part in RNA metabolism in eukaryotes may guide their further functional studies.

Diverse cap-binding properties of Drosophila eIF4E isoforms.

The majority of eukaryotic mRNAs are translated in a cap-dependent manner, which requires recognition of the mRNA 5' cap by eIF4E protein. Multiple eIF4E family members have been identified in most eukaryotic organisms. Drosophila melanogaster (Dm) has eight eIF4E related proteins; seven of them belong to Class I and one to Class II. Their biological roles with the exception of Dm eIF4E-1, Dm eIF4E-3 and Dm 4EHP, remain unknown. Here, we compare the molecular basis of Dm eIF4E's interactions with cap and eIF4G peptide by using homology modelling and fluorescence binding assays with various cap analogues. We found that despite the presence of conserved key residues responsible for cap recognition, the differences in binding different cap analogues among Class I Dm eIF4E isoforms are up to 14-fold. The highest affinity for cap analogues was observed for Dm eIF4E-3. We suggest that Dm eIF4E-3 and Dm eIF4E-5 bind the second nucleoside of the cap in an unusual manner via stacking interactions with a histidine or a phenylalanine residue, respectively. Moreover, the analysis of ternary complexes of eIF4G peptide-eIF4E-cap analogue showed cooperativity between eIF4G and cap binding only for Dm eIF4E-4, which exhibits the lowest affinity for cap analogues among all Dm eIF4Es.

Five eIF4E isoforms from Arabidopsis thaliana are characterized by distinct features of cap analogs binding.

The assembly of the ribosome on majority of eukaryotic mRNAs is initiated by the recruitment of eIF4E protein to the mRNA 5' end cap structure. Flowering plants use two eIF4E isoforms, named eIF4E and eIF(iso)4E, as canonical translation initiation factors and possess a homolog of mammalian 4EHP (or eIF4E-2) termed nCBP. Plants from Brassicaceae family additionally conserve a close paralog of eIF4E which in Arabidopsis thaliana has two copies named eIF4E1b and eIF4E1c. In order to assess the efficiency of plant non-canonical (eIF4E1b/1c and nCBP) and canonical (eIF4E and eIF(iso)4E) eIF4E proteins to bind mRNAs we utilized fluorescence titrations to determine accurate binding affinities of five A.thaliana eIF4E isoforms for a series of cap analogs. We found that eIF4E binds cap analogs from 4-fold to 10-fold stronger than eIF(iso)4E, while binding affinities of nCBP and eIF(iso)4E are comparable. Furthermore, eIF4E1c interacts similarly strongly with the cap as eIF4E, but eIF4E1b binds cap analogs ca. 2-fold weaker than eIF4E1c, regardless of the 95% sequence identity between these two proteins. The use of differentially chemically modified cap analogs in binding studies and a detailed analysis of the obtained homology models gave us insight into the molecular characteristic of varying cap-binding abilities of Arabidopsis eIF4E isoforms.

DNAtraffic--a new database for systems biology of DNA dynamics during the cell life.

DNAtraffic (http://dnatraffic.ibb.waw.pl/) is dedicated to be a unique comprehensive and richly annotated database of genome dynamics during the cell life. It contains extensive data on the nomenclature, ontology, structure and function of proteins related to the DNA integrity mechanisms such as chromatin remodeling, histone modifications, DNA repair and damage response from eight organisms: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli and Arabidopsis thaliana. DNAtraffic contains comprehensive information on the diseases related to the assembled human proteins. DNAtraffic is richly annotated in the systemic information on the nomenclature, chemistry and structure of DNA damage and their sources, including environmental agents or commonly used drugs targeting nucleic acids and/or proteins involved in the maintenance of genome stability. One of the DNAtraffic database aim is to create the first platform of the combinatorial complexity of DNA network analysis. Database includes illustrations of pathways, damage, proteins and drugs. Since DNAtraffic is designed to cover a broad spectrum of scientific disciplines, it has to be extensively linked to numerous external data sources. Our database represents the result of the manual annotation work aimed at making the DNAtraffic much more useful for a wide range of systems biology applications.

[Unsuccessful suicidal attempt with use of self-prepared bullet -- case report]. / Nieudana próba samobójcza z zastosowaniem naboju samodzialowego -- opis przypadku.

Gunshot injuries of the viscerocranium are rarely reported during times of peace in Europe. Penetrating wounds to the maxillofacial region pose a significant challenge for surgeons as they often comprise serious soft and bone tissue defects.We present a case report of 38-year-old male with gunshot wound to the viscerocranium after suicidal attempt. The patient's general condition was stable. The inlet wound was found in the submental region in the central line penetrating deep into the floor of the mouth, to the left, avoiding large vessels and hypoglossal nerve. No exit wound was identified. The ophthalmic examination revealed the limitation of motion in the left eyeball and diplopia in the whole field of vision. The revision was performed under general anesthesia. Control CT scan revealed the presence of one metallic fragment wedged in the hard palate. Second look of oral cavity with particular emphasis on the hard palate was performed. Shrapnel proved to be wedged in the bone of the hard palate very firmly and complete removal without damaging the function of the palate was impossible. The decision was made to withdraw from surgical removal of the remaining piece of the projectile. In most cases, it is recommended to remove all foreign material from human body. However, in the illustrated case we decided to leave small debris in the craniofacial skeleton. In our opinion, further surgical revision would result in greater tissue damage, disproportionate to the benefits of the removal of all fragments of the projectile.

Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human.

This article presents a comprehensive review of large and highly diverse superfamily of nucleotidyltransferase fold proteins by providing a global picture about their evolutionary history, sequence-structure diversity and fulfilled functional roles. Using top-of-the-line homology detection method combined with transitive searches and fold recognition, we revised the realm of these superfamily in numerous databases of catalogued protein families and structures, and identified 10 new families of nucleotidyltransferase fold. These families include hundreds of previously uncharacterized and various poorly annotated proteins such as Fukutin/LICD, NFAT, FAM46, Mab-21 and NRAP. Some of these proteins seem to play novel important roles, not observed before for this superfamily, such as regulation of gene expression or choline incorporation into cell membrane. Importantly, within newly detected families we identified 25 novel superfamily members in human genome. Among these newly assigned members are proteins known to be involved in congenital muscular dystrophy, neurological diseases and retinal pigmentosa what sheds some new light on the molecular background of these genetic disorders. Twelve of new human nucleotidyltransferase fold proteins belong to Mab-21 family known to be involved in organogenesis and development. The determination of specific biological functions of these newly detected proteins remains a challenging task.

Predicting proteome dynamics using gene expression data.

While protein concentrations are physiologically most relevant, measuring them globally is challenging. mRNA levels are easier to measure genome-wide and hence are typically used to infer the corresponding protein abundances. The steady-state condition (assumption that protein levels remain constant) has typically been used to calculate protein concentrations, as it is mathematically convenient, even though it is often not satisfied. Here, we propose a method to estimate genome-wide protein abundances without this assumption. Instead, we assume that the system returns to its baseline at the end of the experiment, which is true for cyclic phenomena (e.g. cell cycle) and many time-course experiments. Our approach only requires availability of gene expression and protein half-life data. As proof-of-concept, we predicted proteome dynamics associated with the budding yeast cell cycle, the results are available for browsing online at http://dynprot.cent.uw.edu.pl/ . The approach was validated experimentally by verifying that the predicted protein concentration changes were consistent with measurements for all proteins tested. Additionally, if proteomic data are available as well, we can also infer changes in protein half-lives in response to posttranslational regulation, as we did for Clb2, a post-translationally regulated protein. The predicted changes in Clb2 abundance are consistent with earlier observations.

Paragangliomas of head and neck - A surgical challenge.

Paragangliomas of the head and neck region are rare and predominantly asymptomatic tumors. These neoplasms arise from widely distributed paraganglionic cells, which originate from both mesodermal elements of third branchial arch and the neural crest residues. Despite the rare occurrence of paragangliomas, problems encountered in their diagnosis, unclear malignancy and treatment ensure that they still remain in the focus of head and neck surgeons. This is a retrospective study of the medical records of patients treated in the ENT Department of the 5th Military Hospital in Krakow during the period 2010-2014. All the preoperative, intraoperative and postoperative data were carefully analyzed for each patient. Thirteen patients (16 tumors) were treated during the study period. All the patients with a pre-operative suspicion of paraganglioma underwent computed tomography angiography. The whole cohort of patients was treated surgically. Paraganglioma should be always considered in the differential diagnosis for painless neck masses. These tumors require thorough radiological pre-operative evaluation and skilled operative technique. Surgical treatment occurs to provide good cure rates with minimal recurrence and morbidity rates.