Células do sangue são transformadas em células do coração que batem

As células do coração, geradas a partir de células-tronco induzidas, batem de verdade, abrindo caminho para sua implantação no coração de pessoas que sofreram ataques cardíacos.

Cientistas da Universidade Johns Hopkins, nos Estados Unidos, desenvolveram um procedimento simplificado e mais barato que poderá ser usado por cientistas de todo o mundo para transformar células do sangue em células cardíacas.

O método é mais seguro porque não utiliza vírus nas etapas intermediárias, como normalmente se faz, e produz as células do coração que batem com quase 100 por cento de eficiência.

"Nós transformamos o processo de uma complexa receita de minestrone em uma simples sopa de missô," disse o Dr. Elias Zambidis, coordenador da pesquisa.

Células-tronco sem vírus

Muitos cientistas duvidavam da possibilidade de um método não-viral para induzir células de sangue a se transformarem em células cardíacas funcionais, que batem de verdade.

Apesar de terem conseguido justamente isso, os cientistas alertam que as células cardíacas ainda não estão prontas para testes em humanos.

Para obter células-tronco retiradas de uma fonte (como o sangue) e transformá-las em uma célula de outro tipo (como células do coração), os cientistas geralmente usam um vírus para entregar um pacote de genes nas células que, em primeiro lugar, faz com que as células originais se transformem em células-tronco.

No entanto, é comum que os vírus induzam mutações nos genes, gerando um câncer nas células recém-transformadas.

Para inserir os genes sem usar um vírus, a equipe de Zambidis voltou-se para os plasmídeos, anéis de DNA que se replicam rapidamente no interior das células e, eventualmente, se degradam.

Receita de célula-tronco

Além da complexidade de persuadir as células-tronco em outros tipos celulares, é necessário elaborar uma receita cara e complicada de fatores de crescimento, nutrientes e condições ambientais que banham as células-tronco durante a sua transformação.

A receita desse "caldo" difere de laboratório para laboratório e de uma linhagem celular para outra.

A equipe de Zambidis simplificou a receita e as condições ambientais necessárias para que as células que estão se transformando em células cardíacas.

Eles verificaram que sua receita funcionou de forma consistente para 11 diferentes linhagens de células-tronco - um verdadeiro arroz com feijão, fácil de preparar e que cabe em qualquer ocasião.

A técnica funcionou tanto para as mais controversas células-tronco embrionárias, como para as linhagens de células-tronco induzidas - sobretudo para as linhagens de células geradas partir das células-tronco adultas.

No futuro, os cientistas esperam utilizar essas células-tronco para implantá-las em pacientes que sofreram ataques cardíacos.

Capacidade multitarefa do cérebro diminui com a idade

Memória de trabalho

A capacidade de realizar mais de uma tarefa ao mesmo tempo cai à medida que o homem envelhece. Uma nova pesquisa aponta que o motivo pelo qual pessoas mais velhas têm mais dificuldade em alternar tarefas está nas redes neurais.

Lidar com múltiplas tarefas envolve a memória de curta duração, que define a capacidade de manter e manipular uma determinada informação em um período de tempo.

Essa memória de trabalho é a base de todas as operações mentais, de decorar um número de telefone a digitá-lo em um aparelho, de manter o ritmo de uma conversa a conduzir funções complexas como raciocinar ou aprender.

"Os resultados do estudo sugerem que o impacto negativo das múltiplas tarefas na memória de trabalho não é necessariamente um problema com a memória, mas deriva de uma interação entre atenção e memória", disse Adam Gazzaley, professor da Universidade da Califórnia em San Francisco, um dos autores do estudo que será publicado esta semana na revista Proceedings of the National Academy of Sciences.

Multitarefa em declínio

De acordo com o estudo, a dificuldade em realizar mais de uma tarefa em um mesmo período de tempo está no momento de alternar entre uma atividade e outra.

O problema fundamental não são as próprias tarefas ou as interrupções, mas as distrações. A pesquisa indica que a capacidade do cérebro em ignorar informações irrelevantes cai com a idade e que isso impacta na memória de trabalho.

O estudo reforça que as "coisas da idade", como costumam ser chamados episódios comuns de distração e esquecimento, têm um impacto maior em indivíduos mais velhos.

Os pesquisadores compararam a memória funcional de jovens saudáveis (com idade média de 24,5 anos) e de idosos também saudáveis (com média de 69,1 anos) em testes envolvendo diversas tarefas simultâneas.

Capacidade de concentração

Por meio de imagens de ressonância magnética, analisaram o fluxo sanguíneo nos cérebros dos participantes de modo a tentar identificar as atividades de circuitos e redes neurais.

Os participantes tinham que observar uma determinada cena e fixá-la por 14,4 segundos. Durante o período, entrava uma interrupção, na forma da imagem de um rosto, e os voluntários tinham que determinar o sexo e a idade estimada da pessoa. Em seguida, tinham que lembrar a cena original.

Os mais velhos mostraram maior dificuldade em fixar a imagem original. Os exames de ressonância mostraram que quando os participantes eram interrompidos, o processo de fixação da memória dava lugar ao próprio processamento da interrupção.

Os mais jovens conseguiam restabelecer a conexão com a rede da memória após a interrupção, desligando-se da imagem que apareceu no meio do teste. Já os mais velhos, na média, tiveram dificuldade tanto para se desligar da interrupção como para restabelecer a rede neural associada com a memória da cena original.

"O impacto das distrações e das interrupções revela a fragilidade da memória de trabalho. Esse é um fato importante a se considerar, uma vez que vivemos em um meio em que cada vez há mais interferências e exigências, como o aumento na quantidade de dispositivos que transportam informação", disse Gazzaley.

Lesser-Known Escherichia Coli Types Targeted in Food Safety Research

ScienceDaily (Apr. 12, 2011) — Almost everyone knows about Escherichia coli O157:H7, the culprit behind many headline-making outbreaks of foodborne illness in the United States. But the lesser-known relatives of this pathogenic microbe are increasingly of concern to food safety scientists.

ARS microbiologist Pina M. Fratamico and her collaborators have developed gene-based PCR (polymerase chain reaction) assays to help identify and detect six newly important Escherichia coli species that are close relatives of E. coli O157:H7 (shown here at about 16,000 times normal size). 

That's according to U.S. Department of Agriculture (USDA) microbiologist and research leader Pina M. Fratamico. Researchers such as Fratamico, along with food safety regulators, public health officials and food producers in the United States and abroad, want to know more about these less-studied pathogens.

In the past few years, a half-dozen of these emerging E. coli species, also called "serogroups," have come to be known among food safety specialists as "the Big Six," namely E. coli O26, O45, O103, O111, O121, and O145.

Fratamico and her colleagues are sorting out "who's who" among these related pathogens so that the microbes can be identified and detected quickly and reliably. The researchers are doing that by uncovering telltale clues in the microbes' genetic makeup.

Building upon this work, Fratamico and her Agricultural Research Service (ARS), university, and industry collaborators have developed gene-based PCR (polymerase chain reaction) assays for each of the Big Six. With further work, the assays might be presented as user-friendly test kits for use by regulatory agencies and others. Foodmakers, for example, might be able to use such kits for in-house quality control, while public health agencies might rely on them when processing specimens from patients hospitalized with foodborne illness.

Analyses of test results might help researchers determine whether certain strains of Big Six E. coli species cause more illness than E. coli O157:H7 does, and if so, why.

Fratamico works in the ARS Molecular Characterization of Foodborne Pathogens Research Unit at the agency's Eastern Regional Research Center in Wyndmoor, Pa. ARS is the chief intramural scientific research agency of USDA, and this work supports the USDA priority of enhancing food safety.

Fratamico has collaborated in this work with Chin-Yi Chen, Yanhong Liu, Terence P. Strobaugh, Jr., and Xianghe Yan at Wyndmoor; Connie E. Briggs, formerly with ARS; and others. Their findings appeared in Applied and Environmental Microbiology, the Canadian Journal of Microbiology, and other scientific journals.

Discovery of Two New Genes Provides Hope for Stemming Staph Infections

ScienceDaily (Apr. 12, 2011) — The discovery of two genes that encode copper- and sulfur-binding repressors in the hospital terror Staphylococcus aureus means two new potential avenues for controlling the increasingly drug-resistant bacterium, scientists say in the April 15, 2011 issue of theJournal of Biological Chemistry.

Staphylococcus aureus encodes a DNA binding copper-sensitive operon repressor (CsoR, bottom) and a CsoR-like sulfur transferase repressor (CstR, top), which are very similar to one another. Unlike CsoR, the repressor CstR does not form a stable complex with copper, Cu(I). Instead, operator binding is inhibited by attaching a second repressor to the first, possibly via a disulfide or even trisulfide bridge. 

"We need to come up with new targets for antibacterial agents," said Indiana University Bloomington biochemist David Giedroc, who led the project. "Staph is becoming more and more multi-drug resistant, and both of the systems we discovered are promising."

The work was a collaboration of members of Giedroc's laboratory, and that of Vanderbilt University School of Medicine infectious disease specialist Eric Skaar, and University of Georgia chemist Robert Scott.

MRSA, or multidrug-resistantStaphylococcus aureus, is the primary cause of nosocomial infections in the United States. About 350,000 infections were reported last year, about 20 percent of which resulted in fatalities, according to the Centers for Disease Control. One to two percent of the U.S. population has MRSA in their noses, a preferred colonization spot.

One of the repressors the scientists discovered, CsoR (Copper-sensitive operon Repressor), regulates the expression of copper resistance genes, and is related to a CsoR previously discovered by the Giedroc group in Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. When the bacterium is exposed to excess copper, the repressor binds copper (I) and falls away from the bacterial genome to which it is bound, making it possible for the copper resistance genes to be turned on. This makes sense, since in the presence of a lot of copper -- a metal commonly used to kill bacteria -- a bacterium is well served by expressing genes that help the bacterium sequester and export extra copper before the metal can do any real damage.

The other repressor, CstR (CsoR-like sulfurtransferase Repressor), which the scientists found can react with various forms of sulfur, appears to prevent the transcription of a series of sulfur assimilation genes based on their homology with similar genes in other bacterial species. One of the genes in this system encodes a well known enzyme, sulfurtransferase, which interconverts sulfite (SO3 2-) and thiosulfate, (S2O3 2-).

The scientists have yet to confirm the functions of the other genes controlled by CstR, but a new four-year, $1.1 million grant from the National Institutes of Health to principal investigator Giedroc will fund crucial investigations into Staph's utilization of sulfur, an important element that bacteria -- and all organisms for that matter -- use to make protein.

The two repressors -- and the gene systems they regulate -- are possible new drug targets for controlling Staph growth. A drug could hypothetically target either of the repressors, causing bacteria to become unresponsive to toxic copper levels or incapable of properly integrating sulfur into their cell physiologies, respectively.

"One thing you could do is prevent the repressors from coming off the DNA in the first place," Giedroc said "although I think that's probably a long shot. I think the repressors are one step removed from where you'd like to have the action. At this point I think the better targets are going to be the genes they are regulating."

Among those genes, Giedroc says he's hopeful one of the sulfur utilization genes controlled by CstR turns out to be an effective drug target. And he wouldn't be surprised if that was the case.

"The metabolic process by which sulfur is assimilated is a proven drug target in Mycobacterium tuberculosis," Giedroc said. "We see no reason why this can't be the case forStaphylococcus aureus. Finding out will be one of the goals of this new NIH-funded project."

Nicholas Grossoehme and Zhen Ma of IU Bloomington, Thomas Kehl-Fie and Keith Adams of Vanderbilt, and Darin Cowart of Georgia also contributed to the report. The project was funded by grants from the National Institutes of Health, the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense, and the American Heart Association.

Tissue Engineers Use New System to Measure Biomaterials, Structures

ScienceDaily (Apr. 12, 2011) — Tissue engineering makes biologists builders, but compared to their civil engineering counterparts, they don't know much about the properties of the materials and structures they use, namely living cells. To improve that knowledge, Brown University researchers have developed a simple and reliable system for measuring the power that cells employ to assemble into three-dimensional tissue.

Climbing higher, higher Cells forming themselves into a doughnut-shaped tissue appear to climb a hydrogel cone. Researchers can calculate the energy expended by the cells in overcoming gravity. 

The research appears online in theProceedings of the National Academy of Sciences.

In addition to helping engineers evaluate how quickly and stably different cell types will combine into desired structures, the power measurements could also improve scientists' understanding of natural tissue growth, such as in fetal development, and how cancerous cells sometimes break off from a tumor and travel in the body, said Jeffrey Morgan, the paper's senior author and associate professor of medical science in Brown's Department of Molecular Pharmacology, Physiology and Biotechnology.

"Cells are the ultimate building parts, and it's important to understand how they are held together, how they assemble together and the energies with which they do that, if you want to delve into the field of tissue engineering," said Morgan, who last year co-developed the first artificial human ovary. "Sometimes these complex processes go wrong, and that's where it's relevant to cancer in terms of cell-to-cell adhesion. But it also plays out very nicely in developmental biology where a very complex 3-D orchestration of cell movement and forces gives rise to new tissues and organs."

Climb the cone

In the system, the researchers deposited cells in very small wells made of a specially designed hydrogel. The wells each have a cone of different steepness rising in the middle, like Bundt cake pans do. The cells form a doughnut shape around the cone. The mutual attraction of the cells then causes the doughnut of living cells to slide up the cone while a video microscope watches. The observed rate at which this mass of cells overcomes the force of gravity to ascend the cone yields a valuable number for the overall power exerted by the cells.

"There's no need to calibrate this device, because gravity is consistent and reliable and there are no moving parts other than the living cells," Morgan said.

Such overall measures of energy, time, and power have been hard to obtain, said lead author and doctoral candidate Jacquelyn Youssef. Many scientists have studied distinct forces and energies within and among cells, such as the bonding strength between particular proteins, but such measures leave tissue engineers to estimate the total energy in a structure by adding up what's known about the cells, related proteins, and their many interactions.

"What we've developed looks at all these things in this one system together," Youssef said. "There's lots of moving parts."

At the same time as it offers an aggregate measure, the system allows for teasing out the relative contributions of those moving parts. In their experiments, the team, which also included Lambert Freund, professor emeritus of engineering at Brown, and recent Ph.D. graduate Asha Nurse, used a drug treatment to inhibit the contractions cells use to "grab" each other. They found that among human skin fibroblast cells, eliminating that particular action took away about half of the total power of the doughnut structure formation.

The researchers worked with two types of cells in the paper. In addition to human skin fibroblasts, which aggregated and ascended the cones in a couple of hours, they also tested liver cells, which took days to reach the same peaks.

Morgan said the system will work for many other cell types and even mixtures of cells as well, making it a promising instrument for assessing the structural characteristics of the variety of building materials that tissue engineers might choose to use in their structures. Bioengineers can also use it to measure the effect different chemicals or drugs might have on the rate or energy of tissue formation.

"What we're driving at is an understanding of how cells will spontaneously form these three-dimensional structures," Morgan said. "The rate at which they do that is important to understanding how to design something more complex."

Funding for the research came from the National Science Foundation and the National Institutes of Health.

Environmental E. Coli: New Way to Classify E. Coli Bacteria and Test for Fecal Contamination

ScienceDaily (Apr. 12, 2011) — The meaning of the standard fecal coliform test used to monitor water quality has been called into question by a new study that identified sources of Escherichia coli bacteria that might not indicate an environmental hazard.

Georgia Tech civil and environmental engineering assistant professor Kostas Konstantinidis (left) and biology graduate student Chengwei Luo examine environmental and clinical E. coli cells, which cannot be differentiated with microscopy techniques.

Fecal pollution of surface waters is measured by the concentration of E. coli bacteria in the water because E. coli is believed to live only in the intestines and waste of humans and other warm-blooded animals, and quickly die outside its host. The presence of E. coli in water also serves as a marker for other potentially more harmful organisms that may accompany it. Positive E. coli tests may lead to the summertime closing of beaches and other recreational bodies of water.

In this new study, researchers report identifying and sequencing the genomes of nine strains of E. colithat have adapted to living in the environment independent of warm-blooded hosts. These strains are indistinguishable from typical E. colibased on traditional tests and yield a positive fecal coliform result though researchers say they may not represent a true environmental hazard.

"The basis for E. coli's widespread use as a fecal pollution indicator is the traditional thinking that E. coli cannot survive for extended periods outside a host or waste, but this study indicates that's not true," said Kostas Konstantinidis, an assistant professor in the Georgia Tech School of Civil and Environmental Engineering. "These results suggest the need to develop a new culture-independent, genome-based coliform test so that the non-hazardous environmental types of E. coliare not counted as fecal contamination."

A paper describing the research was published April 11 in the early edition of the Proceedings of the National Academy of Sciences. The work was sponsored by the National Science Foundation and the National Institutes of Health.

Konstantinidis and Georgia Tech School of Biology graduate student Chengwei Luo compared the genomes of 25 different strains of E. coli and close relatives, which were sequenced by the Center for Microbial Ecology at Michigan State University, the Broad Institute in Massachusetts, or were publicly available in the National Center for Biotechnology Information database. Nine strains that were recovered primarily from environmental sources encoded all genes required for classification as E. coli.

"The orders-of-magnitude higher abundances of the group of organisms represented by these nine strains in environmental samples relative to those in human feces and the clinic indicate that they represent truly environmentally adapted organisms that are not associated primarily with mammal hosts," explained Konstantinidis, who also holds a joint appointment in the Georgia Tech School of Biology.

By comparing the full genomes of the samples, the Georgia Tech researchers identified 84 genes specific to or highly enriched in the genomes of the environmental E. coli and 120 genes specific to the strains commonly found in the gastrointestinal tract of healthy humans, which are called commensal E. coli. They also detected recent genetic exchange of core genes within the environmental E. coli and within the commensal strains, but not from commensal genomes to their environmental counterparts.

The environment-specific bacteria included genes important for resource acquisition and survival in the environment, such as the genes required to utilize energy sources and to break down dead cellular material. In contrast, the gastrointestinal E. coliincluded several genes involved in the transport and use of nutrients thought to be abundant in the gut.

"The genomic data suggest that the environmental E. coli are better at surviving in the external environment, but are less effective competitors in the gastrointestinal tract than commensal E. coli, which tells us that the environmental bacteria are highly unlikely to represent a risk to public health," explained Konstantinidis.

Collectively, this data also indicates that the environmental E. coli strains represent a distinct species from their commensalE. coli counterparts even though they are identified as E. colibased on the standard taxonomic methods. This work is consistent with a more stringent and ecologic definition for bacterial species than the current definition and suggests ways to start replacing traditional, culture-based approaches for defining diagnostic phenotypes of new species with genomic-based procedures.

The scientific, medical, regulatory and legal communities expect species to reasonably reflect the traits and habitat of an organism -- especially an organism like E. coli that has ramifications for diagnostic microbiology and for assessing fecal pollution of natural ecosystems. Efforts toward a more refined definition of this bacterial species are needed, according to Konstantinidis.

This study's findings provide a way to start redefining E. colispecies and testing for fecal contamination with procedures based on genomics and ecology.

"We are now working to develop a molecular assay that uses the gastrointestinal-specific genes as robust biomarkers to count commensal E. coli cells in environmental samples more accurately than current methods," added Konstantinidis.

This project is supported by a National Science Foundation (NSF) award to Georgia Tech and Michigan State University (Award No. DEB0516252) and a National Institutes of Health (NIH/NIAID) award to the Broad Institute (Award No. HHSN2722009000018C). The content is solely the responsibility of the principal investigators and does not necessarily represent the official views of NSF or NIH.

'Pacman Strategy' to Boost the Immune System to Fight Cancer

ScienceDaily (Apr. 12, 2011) — A molecule that lies dormant until it encounters a cancer cell, then suddenly activates and rouses the body's immune system to fight cancer cells directly, marks the latest step in scientists' efforts to tap the body's own resources to fight the disease.

A protease is designed to destroy the link between IL-2 and its inhibitor, freeing IL-2 near tumors. 

The developers of the technology at the University of Rochester Medical Center dub it the "Pacman strategy" because it hinges upon molecular machines produced in abundance by tumors to chew through and gobble up particular chains of molecules.

The key feature of the work is a new type of fusion molecule with three parts: a potent immune cell activator; a second molecule to keep that molecule quiescent until it's needed; and a link between the two that gives scientists control over how the two interact.

The overall fusion molecule acts like a tiny anti-cancer grenade: The portion designed to arouse the immune system to attack cancer is inactive until it's freed, an act that occurs when the link between it and its inhibitory counterpart is cleaved by specialized tumor proteins that chew up such molecules.

The work, led by graduate student John Puskas and Professor John Frelinger, Ph.D., was published online recently in the journal Immunology. Puskas, who is defended his doctoral thesis April 12, is first author of the paper.

In its experiments the team used Interleukin-2 or IL-2, a cytokine or chemical messenger that amplifies the effects of the immune system. IL-2 has been central to the burgeoning field known as cancer immunotherapy; it turns on T cells and natural killer cells that recognize and kill cancer cells. It's approved by the U.S. Food and Drug Administration for the treatment of melanoma and kidney cancer, but it can have serious side effects, limiting its use in patients dramatically. That's largely because it can harm healthy tissue when it's active throughout the body.

"One reason we chose IL-2 is that it's approved and used to treat patients today. If we're able to reduce the toxicity associated with it, perhaps it could be used more broadly," said Frelinger, professor of Microbiology and Immunology.

In experiments using the technology in the lab, the activity of IL-2 in the fusion protein was weak but became 10 to 50 times more biologically active after cleavage. Importantly, in experiments in mice with cancer, tumor growth was inhibited in mice where IL-2 was turned on using the technology compared to mice in which it was not. In many of the treated mice, tumor cells could not be detected after one week.

A key to the technology is the molecular link between IL-2 and its inhibitor. Puskas and Frelinger built that link out of a chain of amino acids -- building blocks of proteins. Such chains are broken or cleaved constantly in the body by enzymes known as proteases. In these experiments, when the link is broken, IL-2 breaks free from its inhibitor and is suddenly available to activate other immune cells.

Puskas and Frelinger created links that are cleaved by molecules found much more commonly in cancer cells than other cells. For instance, in one set of experiments, they created a link that is broken only by prostate specific antigen, a protease that is found in prostate cancer cells. They also created links that are cleaved by proteases known as MMP2 and MMP9 -- both examples of matrix metalloproteinases commonly overactive in many types of tumors.

The approach is designed to turn on the immune system powerfully right in the neighborhood of cancer cells, to destroy those cells, but to avoid a system-wide immune response that could cause severe side effects.

Frelinger points out that the new work is quite different from other experimental anti-cancer efforts that have involved fusion proteins. In other fusion protein approaches, the molecules are active throughout the body. In the new work, the cytokine is designed to be active only near tumor cells, an approach designed to reduce unwanted side effects.

"The beauty of this approach is that you can change any part of the molecule you want," said Frelinger, who also has an appointment in the University's James P. Wilmot Cancer Center. "If you want to target a specific type of cancer, you change the protease sequence to tailor it to particular types of tumors. If you want to change the part of the immune system activated, you change the cytokine.

"Our hope is that an approach like this might someday be coupled with other types of therapy, so that the body could initiate and maintain a vigorous immune response to kill tumors."

Other authors besides Puskas and Frelinger include graduate students Denise Skrombolas and Abigail Sedlacek, and faculty members Edith Lord, Ph.D., and Mark Sullivan, Ph.D. The work was supported by the National Institutes of Allergy and Infectious Diseases as well as by Steven and Alison Krausz and F.C. Blodgett.