Subdivision of the bacterioferritin comigratory protein family of bacterial peroxiredoxins based on catalytic activity.

© American Chemical Society,2010. Post-print version of article deposited in accordance with SHERPA RoMEO guidelinesPeroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity

Determination of protein thiol reduction potential by isotope labeling and intact mass measurement

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, E⊕, for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists

Tissue specific promoters from rice and wheat for modifying grain characteristics

Trabalho final do 6º Ano Médico com vista à atribuição do grau de mestre no âmbito do ciclo de estudos de Mestrado Integrado em Medicina, apresentada à Faculdade de Medicina da Universidade de CoimbraA tiroidite, inflamação da glândula tiroideia, é, juntamente com o bócio, a afecção endócrina mais frequente, sendo comummente encontrada em medicina ambulatória. Associada a uma função tiroideia normal, aumentada ou diminuída (frequentemente com evolução de uma condição para outra), as circunstâncias da sua descoberta podem ser variadas e a distinção dos seus vários tipos baseia-se, essencialmente, no panorama clínico, rapidez de instalação sintomática, história familiar e presença ou ausência de sintomas prodrómicos e dor no pescoço. Vários critérios podem ser utilizados para a sua classificação, nomeadamente histológicos e clínicos (acompanhada ou não de dor tiroideia), sendo, contudo, a evolução da doença o critério classicamente utilizado. Assim, de acordo com este último, os diferentes subtipos de tiroidite podem ser agrupados em: tiroidite aguda, tiroidite subaguda ou tiroidite crónica. A primeira é uma forma dolorosa de tiroidite extremamente rara causada por uma infecção bacteriana, fúngica ou parasitária da tiróide e surgindo, sobretudo, na criança/adolescente e adulto jovem. Nas tiroidites subagudas, podemos encontrar a tiroidite subaguda granulomatosa ou de De Quervain – causa mais frequente de dor tiroideia, eventualmente, de origem viral – e as tiroidites subagudas linfocíticas – tiroidites esporádica indolor, do pós-parto, iatrogénica (interferão, interleucina-2, lítio), tóxica (amiodarona), por irradiação (iodo 131, irradiação externa) ou traumática (cirurgia, punção, traumatismo externo). Finalmente, nas tiroidites crónicas, caracterizadas, portanto, por um maior tempo de evolução, podemos destacar a tiroidite de Riedel, de natureza fibrótica e fisiopatologia desconhecida, e a tiroidite de Hashimoto, a tiroidite mais frequente e causa mais comum de hipotiroidismo nas regiões com aporte suficiente de iodo e da qual as tiroidites esporádica indolor e do pós-parto se aproximam pelo seu carácter autoimune. O diagnóstico destas afecções é feito pelo contexto e achados clínicos, incluindo a presença ou ausência de dor, textura e autoanticorpos. Adicionalmente, o grau de absorção de iodo radioactivo pela iii glândula é reduzido na maioria dos pacientes com inflamação viral, induzida por radiação, traumática, autoimune ou induzida por drogas. O tratamento é, primeiramente, dirigido ao alívio sintomático da dor tiroideia e restabelecimento do eutiroidismo.Thyroiditis, inflammation of the thyroid gland, is, along with goiter, the most frequent endocrine pathology, being commonly found in ambulatory medicine. Associated with normal, elevated or depressed thyroid function (often with evolution from one condition to another), the circumstances of its discovery are varied and the distinction of its several types is based primarily on the clinical setting, rapidity of symptom onset, family history and presence or absence of prodromal symptoms and neck pain. Different criteria can be used to its classification, namely histological and clinical (with thyroid pain or not). Nevertheless, the evolution of the disease is the classically used criterion. Thus, accordingly with this one, the several subtypes of thyroiditis can be divided into: acute thyroiditis, subacute thyroiditis and chronic thyroiditis. The first one is an extremely rare painful form of thyroiditis cause by a bacterial, fungic or parasitic infection of the thyroid and it in children/adolescents and young adults. In subacute thyroiditis, we can have the subacute granulomatous or de De Quervain thyroiditis – most common cause of thyroid pain, eventually of viral original – and the subacute lymphocytic thyroiditis – sporadic painless, postpartum, iatrogenic (interferon, interleukin-2, lithium), toxic (amiodarone), by irradiation (iodine 131 and external irradiation) or traumatic (surgery, puncture or external traumatism) thyroiditis. Finally, in chronic thyroiditis, characterized, therefore, by a longer time of evolution, we can mention Riedel’s thyroiditis, of fibrotic nature and unknown physiopathology, and Hashimoto’s thyroiditis, the most frequent thyroiditis and the most common cause of hypothyroidism in the regions with an adequate iodine supply, from which the sporadic painful and postpartum thyroiditis are close by their autoimune character. Diagnosis is by clinical context and findings, including the presence or absence of pain, tenderness and autoantibodies. In addition, the degree of radioactive iodine uptake by the gland is reduced in most patients with viral, radiation-induced, traumatic, autoimmune, or drug-induced inflammation of the thyroid. Treatment primarily is directed at symptomatic relief of the thyroid pain and restoration of euthyroidism

The chemical basis of serine palmitoyltransferase inhibition by myriocin

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent, enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular-mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.</p

Interaction of convective organisation with monsoon precipitation, atmosphere, surface and sea: the 2016 INCOMPASS field campaign in India

The INCOMPASS field campaign combines airborne and ground measurements of the 2016 Indian monsoon, towards the ultimate goal of better predicting monsoon rainfall. The monsoon supplies the majority of water in South Asia, but forecasting from days to the season ahead is limited by large, rapidly developing errors in model parametrizations. The lack of detailed observations prevents thorough understanding of the monsoon circulation and its interaction with the land surface: a process governed by boundary‐layer and convective‐cloud dynamics. INCOMPASS used the UK Facility for Airborne Atmospheric Measurements (FAAM) BAe‐146 aircraft for the first project of this scale in India, to accrue almost 100 h of observations in June and July 2016. Flights from Lucknow in the northern plains sampled the dramatic contrast in surface and boundary‐layer structures between dry desert air in the west and the humid environment over the northern Bay of Bengal. These flights were repeated in pre‐monsoon and monsoon conditions. Flights from a second base at Bengaluru in southern India measured atmospheric contrasts from the Arabian Sea, over the Western Ghats mountains, to the rain shadow of southeast India and the south Bay of Bengal. Flight planning was aided by forecasts from bespoke 4 km convection‐permitting limited‐area models at the Met Office and India's NCMRWF. On the ground, INCOMPASS installed eddy‐covariance flux towers on a range of surface types, to provide detailed measurements of surface fluxes and their modulation by diurnal and seasonal cycles. These data will be used to better quantify the impacts of the atmosphere on the land surface, and vice versa. INCOMPASS also installed ground instrumentation supersites at Kanpur and Bhubaneswar. Here we motivate and describe the INCOMPASS field campaign. We use examples from two flights to illustrate contrasts in atmospheric structure, in particular the retreating mid‐level dry intrusion during the monsoon onset

Interaction of convective organisation with monsoon precipitation, atmosphere, surface and sea: the 2016 INCOMPASS field campaign in India

The INCOMPASS field campaign combines airborne and ground measurements of the 2016 Indian monsoon, towards the ultimate goal of better predicting monsoon rainfall. The monsoon supplies the majority of water in South Asia, but forecasting from days to the season ahead is limited by large, rapidly developing errors in model parametrizations. The lack of detailed observations prevents thorough understanding of the monsoon circulation and its interaction with the land surface: a process governed by boundary-layer and convective-cloud dynamics. INCOMPASS used the UK Facility for Airborne Atmospheric Measurements (FAAM) BAe-146 aircraft for the first project of this scale in India, to accrue almost 100 hours of observations in June and July 2016. Flights from Lucknow in the northern plains sampled the dramatic contrast in surface and boundary layer structures between dry desert air in the west and the humid environment over the northern Bay of Bengal. These flights were repeated in pre-monsoon and monsoon conditions. Flights from a second base at Bengaluru in southern India measured atmospheric contrasts from the Arabian Sea, over the Western Ghats mountains, to the rain shadow of southeast India and the south Bay of Bengal. Flight planning was aided by forecasts from bespoke 4km convection-permitting limited-area models at the Met Office and India's NCMRWF. On the ground, INCOMPASS installed eddy-covariance flux towers on a range of surface types, to provide detailed measurements of surface fluxes and their modulation by diurnal and seasonal cycles. These data will be used to better quantify the impacts of the atmosphere on the land surface, and vice versa. INCOMPASS also installed ground instrumentation supersites at Kanpur and Bhubaneswar. Here we motivate and describe the INCOMPASS field campaign. We use examples from two flights to illustrate contrasts in atmospheric structure, in particular the retreating mid-level dry intrusion during the monsoon onset

Nitrate regulates floral induction in Arabidopsis, acting independently of light, gibberellin and autonomous pathways

The transition from vegetative growth to reproduction is a major developmental event in plants. To maximise reproductive success, its timing is determined by complex interactions between environmental cues like the photoperiod, temperature and nutrient availability and internal genetic programs. While the photoperiod- and temperature- and gibberellic acid-signalling pathways have been subjected to extensive analysis, little is known about how nutrients regulate floral induction. This is partly because nutrient supply also has large effects on vegetative growth, making it difficult to distinguish primary and secondary influences on flowering. A growth system using glutamine supplementation was established to allow nitrate to be varied without a large effect on amino acid and protein levels, or the rate of growth. Under nitrate-limiting conditions, flowering was more rapid in neutral (12/12) or short (8/16) day conditions in C24, Col-0 and Laer. Low nitrate still accelerated flowering in late-flowering mutants impaired in the photoperiod, temperature, gibberellic acid and autonomous flowering pathways, in the fca co-2 ga1-3 triple mutant and in the ft-7 soc1-1 double mutant, showing that nitrate acts downstream of other known floral induction pathways. Several other abiotic stresses did not trigger flowering in fca co-2 ga1-3, suggesting that nitrate is not acting via general stress pathways. Low nitrate did not further accelerate flowering in long days (16/8) or in 35S::CO lines, and did override the late-flowering phenotype of 35S::FLC lines. We conclude that low nitrate induces flowering via a novel signalling pathway that acts downstream of, but interacts with, the known floral induction pathways

Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.This work was supported by the Wellcome Trust. We would like to thank the members of the Pathogen Informatics Team and the core sequencing teams at the Wellcome Trust Sanger Institute (Cambridge, UK). We are grateful to D. Harris for work in managing the sequence data