Gellan microspheres application for capture or purification of plasmid DNA vaccine

Cervical cancer is the 4th cause of death among women worldwide and is profoundly associated with HPV infection, due to apoptosis inhibition and uncontrolled cell proliferation caused by oncoproteins E6 and E7 action. At the moment, some prophylactic vaccines are available in the market, but that are only capable of preventing HPV infection. Thus, the development of effective treatment for HPV-infected individuals is fundamental. DNA vaccines emerged as a promising way to prevent and treat several diseases since it can stimulate both cellular and humoral immune responses. The biotechnological process for obtaining plasmid DNA (pDNA) includes several steps, which present an environmental impact and makes it quite expensive to the pharmaceutical industry. Therefore, it is crucial to explore new alternatives. In this work, copper-crosslinked gellan microspheres were produced through a water-in-oil emulsion in order to capture pDNA directly from the Escherichia coli (E. coli) lysate seeking a reduction in the recovery and clarification-associated costs. The lowest diameter of gellan microspheres was achieved with 1.41 % of an aqueous gellan gum solution, previously heated at 90ºC, and dripped through a syringe to the oil solution formerly heated at 100 ºC with constant stirring of 750 rpm at a flow rate of 75 µL/min. Afterwards batch method optimization, the gellan microspheres captured 15.61 % of pDNA with 2.42 % of purity by a strategy based on immobilized metal affinity chromatography (IMAC), by manipulating the pH and ionic strength of binding and elution buffers. Another strategy was developed in order to increase the pDNA capture by precipitating the E. coli lysate with ammonium sulfate. The elimination of major impurities improved the recovery percentage to 32.41 % and the purity degree to 12.43 %. Moreover, copper-crosslinked gellan microspheres were functionalized with polyethylenimine (PEI), in order to increase the pDNA capture by increasing the functional groups in the microspheres surface. This allowed an improvement in the recovery percentage to 88.09 %, but the same did not happen to the purity percentage, 3.18 %. Thus, if the central aim is total pDNA capture from crude lysates without resorting to salts or organic solvents, the strategy in which the microspheres were functionalized with PEI showed great potential. On the other hand, if the main objective is to capture pDNA with higher purity, it is recommended to perform a prior step to the capture with ammonium sulfate, where copper-crosslinked microspheres may be applied or also the ones functionalized with PEI. In conclusion, these simple, fast and low-cost strategies allow lysate clarification since an E. coli lysate usually has 1.07 % of pDNA.O vírus do papiloma humano (HPV) é um vírus sexualmente transmissível e a persistência da sua infeção é considerada a maior causa para o desenvolvimento do cancro do colo do útero. Este potencial oncogénico do HPV está diretamente relacionado com a expressão das oncoproteínas E6 e E7, visto que estas têm a capacidade de interferir na desregulação do ciclo celular, indução da apoptose, entre outros fenómenos biológicos. O cancro do colo do útero corresponde à 4º maior causa de morte nas mulheres a nível mundial. No entanto, com as evoluções alcançadas ao nível científico tem sido possível melhorar e desenvolver novas terapias à base de DNA para tratar vários problemas de saúde como cancro e doenças genéticas. Sendo uma delas, as vacinas de DNA, uma vez que estas têm a capacidade de despoletar todos os tipos de imunidade desejada, através da resposta celular e resposta humoral, evitando a evolução da doença, o que é uma vantagem em relação às vacinas convencionais. Os vetores de DNA plasmídico (pDNA) codificantes de determinados antigénios têm sido muito explorados como vacinas de DNA, uma vez que apresentam baixa toxicidade e são mais simples de desenvolver. O processo biotecnológico de preparação do biofármaco de pDNA compreende etapas sequenciais de produção, clarificação e purificação com objetivo de obter a isoforma superenrolada (sc) com o grau de pureza recomendado pelas agências reguladoras, já que é considerada a conformação de pDNA biologicamente ativa. Contudo, este processo é bastante dispendioso para a indústria farmacêutica e apresenta um impacto ambiental acentuado, devido ao uso de elevadas quantidades de solventes orgânicos (isopropanol) e sais caotrópicos (sulfato de amónio e sulfato de sódio). Desta forma, é fundamental desenvolver novas estratégias de captura ou purificação do pDNA sc de modo a simplificar a amostra e evitar o uso de determinados reagentes, tornando o processo mais “green” e económico. A goma gelana é um exopolissacárido microbiano aniónico que tem a capacidade de, em determinadas condições (presença de catiões, concentração de polímero, temperatura), alterar a sua estrutura conformacional e formar uma rede tridimensional dando origem a um gel termorreversível com diferentes características estruturais e propriedades, consoante o objetivo desejado. A gelana apresenta diversas aplicações na indústria alimentar (espessante, gelificante), farmacêutica (formulações oftálmicas), cosmética (loções, cremes), biotecnológica (substituinte do agar), e devido, a características como a sua biocompatibilidade, hidrofilicidade, porosidade e versatilidade tem vindo a ser explorada nos últimos anos como matriz cromatográfica. Recentemente foi explorada e otimizada por desenho experimental a formulação de microsferas de gelana através do método de emulsão água-em-óleo e reforço com iões divalentes para capturar proteínas em função da sua carga ou afinidade. Assim, o objetivo central deste trabalho foi formular as microsferas de gelana e desenvolver uma estratégia de captura de pDNA sc a partir de um lisado bruto de Escherichia coli (E. coli) aplicando o método de batch. Para tal, a amostra de lisado foi obtida através de uma fermentação em E. coli, com posterior lise alcalina. As microesferas de gelana foram preparadas pelo método de emulsão água–em–óleo, reforçadas com alguns iões divalentes, cobre, níquel, zinco, cobalto, e algumas formulações foram posteriormente funcionalizadas com o polímero de polietilenoimina (PEI). O menor diâmetro das formulações foi obtido com 1.41 % de uma solução aquosa de gelana, previamente aquecida a 90 ºC que foi gotejada através de uma seringa para uma solução de óleo aquecida anteriormente a 100 ºC sob constante agitação de 750 rpm a uma velocidade de 75 µL/min. Ambas as formulações foram caracterizadas relativamente ao diâmetro médio (microscopia de semiótica), à morfologia (SEM), à carga global (potencial zeta) e à composição elementar (EDX e FTIR). Dos resultados obtidos quanto à morfologia, ambas as topologias de microesferas apresentam uma forma esférica e consistente. A estabilidade das duas formulações foi avaliada pela medição do diâmetro médio ao longo de vinte dias e ambas se apresentam estáveis ao longo do período em análise. Em relação ao diâmetro médio, as microesferas funcionalizadas com PEI apresentam um menor diâmetro que as microesferas reforçadas com cobre, o que pode ser devido à formação de uma ligação de coordenação entre o PEI e o cobre tornando a sua estrutura mais compacta. Esta ligação pode ter também um efeito na composição elementar pois, as microesferas reforçadas com cobre apresentam cerca de 9 % de cobre e as que foram funcionalizadas com PEI, apresentam 17 % de aminas e apenas 0.62 % de cobre. As microesferas reforçadas com cobre apresentam uma carga superficial de cerca de – 5 mV e as que que foram funcionalizadas com PEI, apresentam uma carga superficial de cerca de + 5 mV. As estratégias de captura desenvolvidas tiveram por base a interação entre os iões metálicos reticulados nas microesferas e o pDNA presente no lisado celular, uma vez que a carga negativa do polímero de gelana repele os ácidos nucleicos. Após inúmeros estudos de otimização, as melhores condições de ligação do pDNA sc às microesferas de gelana reticuladas com cobre foram obtidas a pH 5.0 e a eluição com 200 mM NaCl em 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, permitindo assim recuperar 15.61 % de pDNA sc com 2.42 % de pureza. Adicionalmente, outra estratégia desenvolvida para melhorar a captura de pDNA consistiu na precipitação do lisado de E. coli com 2,5 M de sulfato de amónio. Ao eliminar a maior parte das impurezas (RNA e proteínas) a quantidade de pDNA sc capturado aumentou para 32.41 % com um grau de pureza de 12.43 %. Por fim, as microesferas de gelana foram funcionalizadas com PEI, com objetivo de melhorar a captura de pDNA, tendo em conta o aumento de grupos funcionalizados para interagirem com DNA na superfície das microesferas. Nas microsferas funcionalizadas com PEI, o passo de ligação foi realizado com um tampão de 10 mM MES a pH 5.0 e a eluição com 200 mM NaCl em 10 mM Tris-HCl, 1 mM EDTA, pH 10.5. Nestas condições, foi possível a captura total do pDNA presente na amostra de lisado e uma recuperação/eluição 88.09 % de pDNA sc. Contudo, também ocorreu a retenção de grande parte do RNA da amostra que resultou num decréscimo do grau de pureza para 3.18 %. Assim sendo, se o objetivo principal for a captura total de pDNA de lisados brutos sem recorrer a sais ou solventes orgânicos, a estratégia que apresenta grande potencial é aquela em que as microesferas foram funcionalizadas com PEI. Por outro lado, se o objetivo principal for a captura de pDNA com um grau de pureza mais elevado, é recomendado realizar um passo prévio de tratamento com sulfato de amónio antes do passo de captura, sendo que neste se pode aplicar as microesferas reticuladas com cobre ou também as funcionalizadas com PEI. Concluindo, estas estratégias simples e de baixo custo permitiram a clarificação do lisado sem ser necessário recorrer a solventes orgânicos como o isopropanol, visto que o lisado de E. coli normalmente apresenta cerca de 1.07 % de pDN

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio

Mammals in Portugal: a data set of terrestrial, volant, and marine mammal occurrences in Portugal

Mammals are threatened worldwide, with ~26% of all species being included in the IUCN threatened categories. This overall pattern is primarily associated with habitat loss or degradation, and human persecution for terrestrial mammals, and pollution, open net fishing, climate change, and prey depletion for marine mammals. Mammals play a key role in maintaining ecosystems functionality and resilience, and therefore information on their distribution is crucial to delineate and support conservation actions. MAMMALS IN PORTUGAL is a publicly available data set compiling unpublished georeferenced occurrence records of 92 terrestrial, volant, and marine mammals in mainland Portugal and archipelagos of the Azores and Madeira that includes 105,026 data entries between 1873 and 2021 (72% of the data occurring in 2000 and 2021). The methods used to collect the data were: live observations/captures (43%), sign surveys (35%), camera trapping (16%), bioacoustics surveys (4%) and radiotracking, and inquiries that represent less than 1% of the records. The data set includes 13 types of records: (1) burrows | soil mounds | tunnel, (2) capture, (3) colony, (4) dead animal | hair | skulls | jaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8), observation in shelters, (9) photo trapping | video, (10) predators diet | pellets | pine cones/nuts, (11) scat | track | ditch, (12) telemetry and (13) vocalization | echolocation. The spatial uncertainty of most records ranges between 0 and 100 m (76%). Rodentia (n =31,573) has the highest number of records followed by Chiroptera (n = 18,857), Carnivora (n = 18,594), Lagomorpha (n = 17,496), Cetartiodactyla (n = 11,568) and Eulipotyphla (n = 7008). The data set includes records of species classified by the IUCN as threatened (e.g., Oryctolagus cuniculus [n = 12,159], Monachus monachus [n = 1,512], and Lynx pardinus [n = 197]). We believe that this data set may stimulate the publication of other European countries data sets that would certainly contribute to ecology and conservation-related research, and therefore assisting on the development of more accurate and tailored conservation management strategies for each species. There are no copyright restrictions; please cite this data paper when the data are used in publications

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora

Make EU trade with Brazil sustainable

Brazil, home to one of the planet's last great forests, is currently in trade negotiations with its second largest trading partner, the European Union (EU). We urge the EU to seize this critical opportunity to ensure that Brazil protects human rights and the environment