Designing conformational control of human tissue transglutaminase

Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.

Traditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.

One such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.

To better understand the effects of TG2’s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation’s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2’s activities are believed to elicit pathological effects.

SAMUDRA Report No. 57, November 2010

Mackerel mayhem: On the ongoing dispute in northern Europe over the boom in mackerel stocks. Small and mighty: The Banjul civil society declaration on sustainable livelihoods in African fisheries. Building partnerships: The case of Red Sea fisheries management shows how fishers’ rights can be strengthened. The write stuff: The website of Comité Local des Pêches Le Guilvinec celebrates its second anniversary. Frankenfish salmon: The United States is close to approving genetically engineered salmon. Trawl brawl: Indian and Sri Lankan fishermen have worked to co-exist in the Palk Bay. Tsunami recovery: On the traditional tenure system of the fishing community of Juan Fernández. New goals from Nagoya: The Nagoya meeting of the Convention on Biological Diversity made some progress. Beyond Bangkok: The civil society workshop in Costa Rica focused on small-scale fishers in Latin America. Securing small-scale fisheries. Recommendations adopted at the San José FAO workshop dealt with small-scale fisheries. (PDF contains 56 pages)

Exciplexes in fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines

Strong quenching of the fluorescence of aromatic hydrocarbons by tertiary aliphatic amines has been observed in solution at room temperature. Accompanying the fluorescence quenching of aromatic hydrocarbons, an anomalous emission is observed. This new emission is very broad, structureless and red-shifted from the original hydrocarbon fluorescence.

Kinetic studies indicate that this anomalous emission is due to an exciplex formed by an aromatic hydrocarbon molecule in its lowest excited singlet state with an amine molecule. The fluorescence quenching of the aromatic hydrocarbons is due to the depopulation of excited hydrocarbon molecules by the formation of exciplexes, with subsequent de-excitation of exciplexes by either radiative or non-radiative processes.

Analysis of rate constants shows the electron-transfer nature of the exciplex. Through the study of the effects on the frequencies of exciplex emissions of substituents on the hydrocarbons, it is concluded that partial electron transfer from the amine molecule to the aromatic hydrocarbon molecule in its lowest excited singlet state occurs in the formation of exciplex. Solvent effects on the exciplex emission frequencies further demonstrate the polar nature of the exciplex.

A model based on this electron-transfer nature of exciplex is proposed and proves satisfactory in interpreting the exciplex emission phenomenon in the fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines.

Catalisadores para hidroisomerização de n-hexadecano

No processo de hidrocraqueamento para a produção de lubrificantes ocorre a formação de uma corrente rica em compostos parafínicos que possuem alto ponto de fluidez, apesar dos mesmos apresentarem excelentes desempenhos em termos de estabilidade térmica e oxidativa. A transformação das n-parafinas obtidas nestas correntes em isoparafinas e compostos naftênicos, os quais possuem menores pontos de fluidez, se faz necessária a fim de enquadrar esta propriedade. Uma das rotas catalíticas mais importantes neste sentido é a hidroisodesparafinação ou HIDW (hydroisodewaxing) que consiste na conversão de n-parafinas nas respectivas isoparafinas, onde são empregados catalisadores bifuncionais zeolíticos com a ocorrência de seletividade de forma. No caso dos catalisadores industriais, se faz necessária a dispersão da fase metálica e da zeólita em uma matriz amorfa para viabilizar sua conformação e melhorar a resistência mecânica do catalisador final. Neste cenário, o objetivo deste trabalho foi preparar e analisar o desempenho de uma série de catalisadores à base de zeólita beta inseridos numa matriz de alumina, variando-se o teor de zeólita e o tipo de precursor de Pt utilizado. Os catalisadores foram avaliados na reação de hidroisomerização de um composto modelo, no caso, n-hexadecano. Os testes realizados para avaliação da atividade e seletividade foram conduzidos em um reator de fluxo contínuo em alta pressão e fase líquida em unidade de laboratório. Os catalisadores foram testados em condições operacionais que proporcionassem uma ampla faixa de conversões do n-C16. Verificou-se que as atividades dos catalisadores foram proporcionais ao teor de zeólita no catalisador, indicando que a função ácida, neste catalisador bifuncional, é a etapa limitante do processo. Quanto à natureza do precursor de Pt, o catalisador preparado com ácido cloroplatínico foi sensivelmente mais ativo que os preparados com o complexo aminplatina. No entanto, para todos os catalisadores, a distribuição de produtos em função da conversão foi similar, independente do teor de zeólita e da natureza do precursor de platina. Foi também determinado o ponto de fluidez de uma série de produtos de reação, obtendo-se valores entre 17,5 C (n-hexadecano) e - 41 C (produto com 98% de conversão). Obteve-se uma boa correlação entre o ponto de fluidez e a composição dos produtos, considerando-se a presença de isômeros mono, di e tri-substituídos e compostos de menor peso molecular que C16

Effect of environmental conditions on the distribution of Pacific mackerel (Scomber japonicus) larvae in the California Current system

We modeled the probability of capturing Pacif ic mackerel (Scomber japonicus) larvae as a function of environmental variables for the Southern California Bight (SCB) most years from 1951 through 2008 and Mexican waters offshore of Baja California from 1951 through 1984. The model exhibited acceptable fit, as indicated by the area under a receiver-operating-characteristic curve of 0.80 but was inconsistent with the zero catches that occurred frequently in the 2000s. Two types of spawners overlapped spatially within the survey area: those that exhibited peak spawning during April in the SCB at about 15.5°C and a smaller group that exhibited peak spawning in August near Punta Eugenia, Mexico, at 20°C or greater. The SCB generally had greater zooplankton than Mexican waters but less appropriate (lower) geostrophic f lows. Mexican waters generally exhibited greater predicted habitat quality than the SCB in cold years. Predicted quality of the habitat in the SCB was greater from the 1980s to 2008 than in the earlier years of the survey primarily because temperatures and geostrophic flows were more appropriate for larvae. However, stock size the previous year had a larger effect on predictions than any environmental variable, indicating that larval Pacific mackerel did not fully occupy the suitable habitat during most years.

Uma leitura dos textos epistolares da Nancy Huston

Este trabalho tem como objetivo discutir e analisar os dois textos epistolares de Nancy Huston: À lAmour comme à la guerre (1984) e Lettres parisiennes (1986). Os dois livros são escritos em momentos diferentes da vida da autora e possuem temáticas diferenciadas. Os dois volumes se constituem como troca de cartas com dois correspondentes: Sam Kinser e Leïla Sebbar, respectivamente. A análise presente nesta dissertação se divide em três momentos. No primeiro, examinamos a evolução da carta ao longo do tempo. No segundo, destacamos alguns temas tratados no corpus, buscando estabelecer relações com outros textos da autora e evidenciar o espaço que ocupam na obra de Nancy Huston. No terceiro momento, estudamos as (in)definições de gênero literário e as características da autobiografia e do ensaio, cotejando-as com os textos epistolares do corpus a fim de analisarmos em que medida essas cartas se configuram como autobiografia e ensaio. O percurso de pesquisa busca, pois, localizar os textos epistolares na produção crítica e literária de Nancy Huston

Effect of towing speed on retention of zooplankton in bongo nets

Long-term time series of zooplankton data provide invaluable information about the fluctuations of species abundance and the stability of marine community structure. These data have demonstrated that environmental variability have a profound effect on zooplankton communities across the Atlantic basin (Beaugrand et al., 2002; Frank et al., 2005; Pershing et al., 2005). The value of these time series increases as they lengthen, but so does the likelihood of changes in sampling or processing methods. Sam-pling zooplankton with nylon nets is highly selective and biased because of mesh selectivity, net avoidance, and damage to fragile organisms. One sampling parameter that must be standardized and closely monitored is the speed of the net through the water column. Tow speed should be as fast as possible to minimize net avoid-ance by the organisms, but not so fast as to damage soft bodied zooplankters or extrude them through the mesh (Tranter et al., 1968; Anderson and Warren, 1991).

A Single-armed Manta-board as a New Diver-controlled Planing Board and Its Use for Underwater Surveys

Due to inadequacies of previous underwater towing techniques and the special needs of a recent underwater survey, a modified mania-board technique was developed. With this new technique, the diver holds on to the manta-board with one arm; consequently, the board is referred to as a single-armed manta-board (sam-board). The sam-board proved inexpensive and highly maneuverable, allowing the divers to freely collect samples or record information. Through some experimenting with the board and changing some of the variables, such as rope lengths, towing speeds, etc., a highly efficient towing method can be achieved. Preplanning and strict diving safety procedures must, however, be implemented to assure efficiency. This paper presents the materials, guidelines for board construction, equipment, and preplanning and diving safety procedures necessary for the sam-board towing operation.

Utilização de imagem do sensor Aster para discriminação espectral de rochas carbonáticas aflorantes na região Noroeste do Estado do Rio de Janeiro

Este trabalho teve como objetivo geral avaliar o potencial das imagens do sensor ASTER, utilizando a região do infravermelho de ondas curtas (SWIR), para discriminação espectral de rochas carbonáticas aflorantes na região Noroeste do Estado do Rio de Janeiro, complementando produtos existentes de mapeamento geológico. As rochas carbonáticas servem de matéria-prima para produção de cimento, que atualmente apresenta forte demanda dado o crescimento de obras civis devido à expansão da infraestrutura do Estado do Rio de Janeiro. Este crescimento no consumo oferece desafios às companhias produtoras, tornando-se de vital importância a identificação de novas áreas para exploração de insumos para a indústria civil. Neste sentido, o carbonato tem sofrido grande pressão com relação a sua produção pois é a principal matéria-prima utilizada na fabricação do cimento. Imagens do sensor Aster vem sendo utilizadas na área da geologia com êxito, discriminando litologias e minerais como quartzo, óxido de ferro e calcita. Na região do intervalo de ondas entre 2,235-2,285 μm e 2,295-2,365 μm , as bandas 7 e 8 do sensor ASTER na região do SWIR, mostram-se adequadas para a identificação de minerais de calcita e dolomita. Como metodologia, foram aplicadas as técnicas de razões de bandas para separação de calcários e dolomitos e para a classificação espectral, foi utilizada a técnica SAM. Tornou-se como referência para a classificação espectral amostras de áreas de rochas carbonáticas aflorantes e espectros da biblioteca espectral da USGS. As classificações espectrais obtiveram resultados significativos na discriminação espectral das áreas carbonáticas, no entanto as técnicas de razões de bandas não obtiveram resultados suficientes para a discriminação de calcários e dolomitos. Para trabalhos futuros sugere-se a realização de trabalho de campo para a coleta de espectros, através da espectrorradiometria dos afloramentos dos carbonatos.

Avaliação citotóxica, mutagênica e genotóxica dos análogos do Megazol

A doença de Chagas é uma doença tropical infecciosa e negligenciada responsável por um grande número de pessoas infectadas e em risco de infecção, principalmente nas regiões pobres da América Latina. No momento, apenas duas drogas, Benzonidazol e Nifurtimox, estão disponíveis para o tratamento da doença de Chagas, mas são ineficazes por apresentarem baixa taxa de cura. O Megazol é um importante representante da classe dos nitroimidazóis e é uma alternativa promissora devido ao seu potencial tripanocida com um perfil superior de ação quando comparado ao tratamento disponível. No entanto, o Megazol não é utilizado clinicamente uma vez que possui atividade mutagênica e carcinogênica relatada. O Instituto de Tecnologia em Fármacos (Farmanguinhos) desenvolveu três análogos do Megazol: PTAL 05-02 (3-amino-5-(1-metil-5-nitro-1H-imidazol-2-il)-1H-1,2,4-triazol), PAMT 09 (2-amino-N-(1-metil-4-nitro-1H-imidazol-5-il)-5-(trifluorometil)-1H-1,2,4-triazol) e PTAL 04-09 (1-(1-metil-4-nitro-1H-imidazol-5-il)-1H-pirazol). O objetivo deste trabalho é apresentar novas moléculas análogas do Megazol com atividade tripanocida, desenvolvidas a partir de estratégias racionais de desenvolvimento de substâncias bioativas ao manter o perfil farmacodinâmico do Megazol enquanto tenta diminuir ou remover o efeito genotóxico. Testes genotóxicos na avaliação segura de novas substâncias bioativas foram utilizados, de acordo com as diretrizes da OECD. O teste da Salmonella/microssoma foi utilizado na avaliação mutagênica e citotóxica, utilizando linhagens de Salmonella enterica sorovar Typhimurium, deficientes e supercompetentes na síntese de enzimas nitroredutase e acetiltransferase. O análogo PAMT 09 não foi mutagênico em nenhuma concentração e linhagem utilizada. Os análogos PTAL 05-02 e PTAL 04-09 foram mutagênicos, na ausência de S9 mix, para a linhagem TA98/1,8-DNP6. Na avaliação de citotoxicidade, os três análogos foram citotóxicos, independente de metabolização exógena S9 mix. O teste do micronúcleo, utilizando células de macrófago de rato, foi realizado para a avaliação genotóxica dos análogos do Megazol. Os três análogos foram capazes de induzir a formação de micronúcleos e apresentaram efeito citotóxico.

Detection of molecular interactions with modified ferrocene self-assembled monolayers.

Ferrocene-terminated self-assembled monolayers (Fc-SAMs) are one of the most studied molecular aggregates on metal electrodes. They are easy to fabricate and provide a stable and reproducible system to investigate the effect of the microenvironment on the electron transfer parameters. We propose a novel application for Fc-SAMs, the detection of molecular interactions, based on the modification of the SAM with target-specific receptors. Mixed SAMs were fabricated by coimmobilization on Au electrodes of thiolated alkane chains with three different head groups: hydroxy terminating head group, ferrocene head group, and a functional head group such as biotin. Upon binding, the intrinsic electric charge of the target (e.g., streptavidin) modifies the electrostatic potential at the plane of electron transfer, causing a shift in the formal potential E degrees '. The SAMs were characterized by AC voltammetry. The detection mechanism is confirmed by measurements of formal potential as a function of electrolyte pH.

Benthic habitats of Buck Island Reef National Monument

NOAA’s Center for Coastal Monitoring and Assessment’s Biogeography Branch has mapped and characterized large portions of the coral reef ecosystems inside the U.S. coastal and territorial waters, including the U.S. Caribbean. The complementary protocols used in these efforts have enabled scientists and managers to quantitatively compare different marine ecosystems in tropical U.S. waters. The Biogeography Branch used these same general protocols to generate three seamless habitat maps of the Bank/Shelf (i.e., from 0 ≤50 meters) and the Bank/Shelf Escarpment (i.e., from 50 ≤1,000 meters and from 1,000 ≤ 1,830 meters) inside Buck Island Reef National Monument (BIRNM). While this mapping effort marks the fourth time that the shallow-water habitats of BIRNM have been mapped, it is the first time habitats deeper than 30 meters (m) have been characterized. Consequently, this habitat map provides information on the distribution of mesophotic and deep-water coral reef ecosystems and serves as a spatial baseline for monitoring change in the Monument. A benthic habitat map was developed for approximately 74.3 square kilometers or 98% of the BIRNM using a combination of semi-automated and manual classification methods. The remaining 2% was not mapped due to lack of imagery in the western part of the Monument at depths ranging from 1,000 to 1,400 meters. Habitats were interpreted from orthophotographs, LiDAR (Light Detection and Ranging) imagery and four different types of MBES (Multibeam Echosounder) imagery. Three minimum mapping units (MMUs) (100, 1,000 and 5,000 square meters) were used because of the wide range of depths present in the Monument. The majority of the area that was characterized was deeper than 30 m on the Bank/Shelf Escarpment. This escarpment area was dominated by uncolonized sand which transitioned to mud as depth increased. Bedrock was exposed in some areas of the escarpment, where steep slopes prevented sediment deposition. Mesophotic corals were seen in the underwater video, but were too sparsely distributed to be reliably mapped from the source imagery. Habitats on the Bank/Shelf were much more variable than those seen on the Bank/Shelf Escarpment. The majority of this shelf area was comprised of coral reef and hardbottom habitat dominated by various forms of turf, fleshy, coralline or filamentous algae. Even though algae was the dominant biological cover type, nearly a quarter (24.3%) of the Monument’s Bank/Shelf benthos hosted a cover of 10%-<50% live coral. In total, 198 unique combinations of habitat classes describing the geography, geology and biology of the sea-floor were identified from the three types of imagery listed above. No thematic accuracy assessment was conducted for areas deeper than about 50 meters, most of which was located in the Bank/Shelf Escarpment. The thematic accuracy of classes in waters shallower than approximately 50 meters ranged from 81.4% to 94.4%. These thematic accuracies are similar to those reported for other NOAA benthic habitat mapping efforts in St. John (>80%), the Main Eight Hawaiian Islands (>84.0%) and the Republic of Palau (>80.0%). These digital maps products can be used with confidence by scientists and resource managers for a multitude of different applications, including structuring monitoring programs, supporting management decisions, and establishing and managing marine conservation areas. The final deliverables for this project, including the benthic habitat maps, source imagery and in situ field data, are available to the public on a NOAA Biogeography Branch website (http://ccma.nos.noaa.gov/ecosystems/coralreef/stcroix.aspx) and through an interactive, web-based map application (http://ccma.nos.noaa.gov/explorer/biomapper/biomapper.html?id=BUIS). This report documents the process and methods used to create the shallow to deep-water benthic habitat maps for BIRNM. Chapter 1 provides a short introduction to BIRNM, including its history, marine life and ongoing research activities. Chapter 2 describes the benthic habitat classification scheme used to partition the different habitats into ecologically relevant groups. Chapter 3 explains the steps required to create a benthic habitat map using a combination of semi-automated and visual classification techniques. Chapter 4 details the steps used in the accuracy assessment and reports on the thematic accuracy of the final shallow-water map. Chapter 5 summarizes the type and abundance of each habitat class found inside BIRNM, how these habitats compare to past habitat maps and outlines how these new habitat maps may be used to inform future management activities.

Shallow-water benthic habitats of southwest Puerto Rico

This report describes the creation and assessment of benthic habitat maps for shallow-water (<30m) marine environments of the Guánica/Parguera and Finca Belvedere Natural Reserve in southwest Puerto Rico. The objective was to provide spatially-explicit information on the habitat types, biological cover and live coral cover of the region’s coral reef ecosystem. These fine-scale habitat maps, generated by interpretation of 2010 satellite imagery, provide an update to NOAA’s previous digital maps of the U.S. Caribbean (Kendall et al., 2001) for these areas. Updated shallow-water benthic habitat maps for the Guánica/Parguera region are timely in light of ongoing restoration efforts in the Guánica Bay watershed. The bay is served directly by one river, the Rio Loco, which flows intermittently and more frequently during the rainy season. The watershed has gone through a series of manipulations and alterations in past decades, mainly associated with agricultural practices, including irrigation systems, in the upper watershed. The Guánica Lagoon, previously situated to the north of the bay, was historically the largest freshwater lagoon in Puerto Rico and served as a natural filter and sediment sink prior to the discharge of the Rio Loco into the Bay. Following alterations by the Southwest Water Project in the 1950s, the Lagoon’s adjacent wetland system was ditched and drained; no longer filtering and trapping sediment from the Rio Loco. Land use in the Guánica Bay/Rio Loco watershed has also gone through several changes (CWP, 2008). Similar to much of Puerto Rico, the area was largely deforested for sugar cane cultivation in the 1800s, although reforestation of some areas occurred following the cessation of sugar cane production (Warne et al., 2005). The northern area of the watershed is generally mountainous and is characterized by a mix of forested and agricultural lands, particularly coffee plantations. Closer to the coast, the Lajas Valley Agricultural Reserve extends north of Guánica Bay to the southwest corner of the island. The land use practices and watershed changes outlined above have resulted in large amounts of sediment being distributed in the Rio Loco river valley (CWP, 2008). Storm events and seasonal flooding also transport large amounts of sediment to the coastal waters. The threats of upstream watershed practices to coral reefs and the nearshore marine environment have been gaining recognition. Guánica Bay, and the adjacent marine waters, has been identified as a “management priority area” by NOAA’s Coral Reef Conservation Program (CRCP, 2012). In a recent Guánica Bay watershed management plan, several critical issues were outlined in regards to land-based sources of pollution (LBSP; CWP, 2008). These include: upland erosion from coffee agriculture, filling of reservoirs with sediment, in-stream channel erosion, loss of historical Guánica lagoon, legacy contaminants and sewage treatment (CWP, 2008). The plan recommended several management actions that could be taken to reduce impacts of LBSP, which form the basis of Guánica watershed restoration efforts.

Small-boat surveys for coastal dolphins: line-transect surveys of Hector’s dolphins (Cephalorhynchus hectori)

Management of coastal species of small cetaceans is often impeded by a lack of robust estimates of their abundance. In the Austral summers of 1997−98, 1998−99, and 1999−2000 we conducted line-transect surveys of Hector’s dolphin (Cephalorhynchus hectori) abundance off the north, east, and south coasts of the South Island of New Zealand. Survey methods were modified for the use of a 15-m sailing catamaran, which was equipped with a collapsible sighting platform giving observers an eye-height of 6 m. Eighty-six percent of 2061 km of survey effort was allocated to inshore waters (4 nautical miles [nmi] or 7.4 km from shore), and the remainder to offshore waters (4−10 nmi or 7.4–18.5 km from shore). Transects were placed at 45° to the shore and spaced apart by 1, 2, 4, or 8 nmi according to pre-existing data on dolphin density. Survey effort within strata was uniform. Detection functions for sheltered waters and open coasts were fitted separately for each survey. The effect of attraction of dolphins to the survey vessel and the fraction of dolphins missed on the trackline were assessed with simultaneous boat and helicopter surveys in January 1999. Hector’s dolphin abundance in the coastal zone to 4 nmi offshore was calculated at 1880 individuals (CV=15.7%, log-normal 95% CI=1384−2554). These surveys are the first line-transect surveys for cetaceans in New Zealand’s coastal waters.