Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features

A Novel Mechanism of Programmed Cell Death in Bacteria by Toxin–Antitoxin Systems Corrupts Peptidoglycan Synthesis

Most genomes of bacteria contain toxin–antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics

Real-world study of children and young adults with myeloproliferative neoplasms: identifying risks and unmet needs

Myeloproliferative neoplasms (MPNs) are uncommon in children/young adults. Here, we present data on unselected patients diagnosed before 25 years of age included from 38 centers in 15 countries. Sequential patients were included. We identified 444 patients, with median follow-up 9.7 years (0-47.8). Forty-nine (11.1%) had a history of thrombosis at diagnosis, 49 new thrombotic events were recorded (1.16% patient per year [pt/y]), perihepatic vein thromboses were most frequent (47.6% venous events), and logistic regression identified JAK2V617F mutation (P = .016) and hyperviscosity symptoms (visual disturbances, dizziness, vertigo, headache) as risk factors (P = .040). New hemorrhagic events occurred in 44 patients (9.9%, 1.04% pt/y). Disease transformation occurred in 48 patients (10.9%, 1.13% pt/y), usually to myelofibrosis (7.5%) with splenomegaly as a novel risk factor for transformation in essential thrombocythemia (ET) (P= .000) in logistical regression. Eight deaths (1.8%) were recorded, 3 after allogeneic stem cell transplantation. Concerning conventional risk scores: International Prognostic Score for Essential Thrombocythemia-Thrombosis and new International Prognostic Score for Essential Thrombocythemia-Thrombosis differentiated ET patients in terms of thrombotic risk. Both scores identified high-risk patients with the same median thrombosis-free survival of 28.5 years. No contemporary scores were able to predict survival for young ET or polycythemia vera patients. Our data represents the largest real-world study of MPN patients age < 25 years at diagnosis. Rates of thrombotic events and transformation were higher than expected compared with the previous literature. Our study provides new and reliable information as a basis for prospective studies, trials, and development of harmonized international guidelines for the specific management of young patients with MPN

Transformations of humic substances, nitrogen and heavy metal forms in sewage sludge composted in a mixture with lignocellulosic waste

Celem pracy była ocena wpływu kompostowania osadów ściekowych z odpadami lignocelulozowymi na przemiany materii organicznej, w tym substancji humusowych, związków azotu oraz zawartość metali ciężkich i ich form chemicznych. Proces prowadzono w układzie dwustopniowym (1o napowietrzany bioreaktor, 2o okresowo przerzucana pryzma). Wykazano, że w wyniku kompostowania nastąpił ubytek materii organicznej z 615,9 g/kg s.m. do 486,6 g/kg s.m., ale wzrosła zawartość substancji humusowych, w tym głównie kwasów huminowych (z 93,3 mg C/g mat. org. do 182,3 mg C/g mat. org). W dojrzałym kompoście azot mineralny występował głównie w postaci azotu azotanowego, a stosunek NNH4/NNO3 kształtował się na poziomie 0,07. Całkowita zawartość miedzi, cynku, niklu, ołowiu i kadmu nie przekraczała wartości normatywnych. W wyniku kompostowania nastąpiła redystrybucja form metali ciężkich, a w dojrzałym kompoście, w porównaniu ze wsadem, największe zmniejszenie mobilności metali (jako Mf) odnotowano dla cynku (z 48,3% do 41,6%) oraz miedzi (z 7,2% do 1,6). Dodatkowo, dla wszystkich metali nastąpiło zwiększenie siły ich wiązania (jako IR) z 0,48 do 0,59 dla Cu, z 0,19 do 0,21 dla Zn, z 0,79 do 0,87 dla Ni oraz z 0,47 do 0,56 dla Pb.The aim of this study was to assess the impact of sewage sludge composting in mixture with lignocellulosic waste on the transformations of organic matter, therein humic substances, nitrogen compounds, heavy metals and their chemical forms. Composting in two-stage system (1o aerated bioreactor, 2o periodically aerated windrow) led to loss of organic matter from 615.9 g/kg d.w. to 486.6 g/kg d.w. and increase of humic substances content, mainly humic acids from 93.3 mg C/g OM to 182.3 mg C/g OM. In mature compost, mineral nitrogen occurred mainly as nitrate and the NNH4/NNO3 ratio was 0.07. Total Cu, Zn, Ni, Pb and Cd concentration was below permissible values. Composting resulted in redistribution of metal forms and in final product the highest lowering of metal mobility (as Mf), compared to the feedstock, was achieved for Zn and Cu. For all metals the increase of metal binding intensity (IR) was observed: 0.48 to 0.59 (Cu), 0.19 to 0.21 (Zn), 0.79 to 0.87 (Ni), 0.47 to 0.56 (Pb)