An antibiotic effect minus resistance

After 70 years, antibiotics are still the primary treatment for halting the spread of bacterial infections. But the prevalence of antibiotic resistance is now outpacing the rate of new drug discovery and approval.

A microbiologist at the University of Wisconsin-Milwaukee (UWM) has discovered a different approach: Instead of killing the bacteria, why not disarm them, quashing disease without the worry of antibiotic resistance?

Ching-Hong Yang, associate professor of biological sciences, has developed a compound that shuts off the "valve" in a pathogen's DNA that allows it to invade and infect.

The research is so promising that two private companies are testing it with an eye toward commercialization.

"We analyzed the genomic defense pathways in plants to identify all the precursors to infection," says Yang. "Then we used the information to discover a group of novel small molecules that interrupt one channel in the intricate pathway system."

Yang and collaborator Xin Chen, a professor of chemistry at Changzhou University in China, have tested the compound on two virulent bacteria that affect plants and one that attacks humans. They found it effective against all three and believe the compound can be applied to treatments for plants, animals and people.

The work was published online this month in the journal Antimicrobial Agents and Chemotherapy.

Urgent concerns about antibiotics

The economic costs and health threats of antibiotic resistance have become so serious that the World Health Organization (WHO) this year dedicated World Health Day to call global attention to the issue.

Antibiotics are routinely sprayed on crops and widely used in factory farming of animals, which causes resistance to develop quickly. That antibiotic resistance is then transferred to humans who eat the food containing antibiotic-resistant bacteria.

Among the bacteria tested by the researchers is Pseudomonas aeruginosa, which is resistant to a broad range of antibiotics. It causes infections in people with compromised immune systems, such as HIV and cancer patients. It's also responsible for lung infections in patients with cystic fibrosis, and hospital-related infections such as urinary tract infections, pneumonia and infections from burns.

The fatality rate from these is about 50 percent. Hospital-acquired urinary tract infections by P. aeruginosa alone cost more than $3.5 billion a year in the U.S.

Road to the market

The research has attracted interest from two companies. Creative Antibiotics, a Swedish pharmaceutical company, is testing the compound and derivatives for human therapeutic uses and Wilbur-Ellis Agribusiness Division, based in Washington and California, is examining them for agricultural uses.

Despite the constant threat of disease in agriculture, says John Frieden, a biologist and R&D manager with Wilbur-Ellis, the industry has not had access to any new antibiotics in many years. U.S. regulatory agencies do not allow agribusiness to use antibiotics that are also used for human health – even if they would be effective.

"The thing that caught my attention," Frieden says, "was that this was not an antibiotic, but it accomplishes the same thing as an antibiotic."

Although he says it is too soon to tell if a product could spring from the research, the approach is "incredibly unique. I've never seen anything that is even close to a commercial application like this. It could be very big."

The researchers have filed two patents on the work through the UWM Research Foundation (UWMRF), and Yang is partially funded through two UWMRF Bradley Catalyst Grants and a UWM Research Growth Initiative (RGI) grant.

Virulence factors

The compounds Yang and Chen have developed are unique because they take aim at one component of a cluster that makes pathogenic bacteria harmful.

One of those components, the type III secretion system (T3SS), gives pathogens their ability to invade a cell, letting in a host of proteins that enhance the bacterium's ability to cause disease.

"These bacteria are very smart," says Yang. "They grow a narrow appendage that acts as a 'needle,' injecting the virulence factors, such as toxins, into the host cell. The host cell cannot recognize the pathogen's 'needle,' so its defense mechanism is not triggered."

Yang and Chen's compounds block the production of T3SS. Although they have tested the compounds on only three pathogens, they have reason to believe the compounds will be effective against far more.

"T3SS exists in many different kinds of disease-causing bacteria," says Yang, "so the compounds can target multiple pathogens. That's the beauty of it."

http://www.eurekalert.org/pub_releases/2011-10/uow--aae102811.php

fatty acid binding protein (FABP4)

A large pad of fat cells that extends from the stomach and covers the intestines provides nutrients that promote the spread and growth of ovarian cancer, reports a research team based at the University of Chicago in the journal Nature Medicine, published online October 30th, 2011.

Ovarian cancer, the fifth leading cause of cancer deaths in women, tends to spread within the abdominal cavity as opposed to distant organs. In 80 percent of women, by the time ovarian cancer is diagnosed, it has spread to the pad of fat cells, called the omentum. Often, cancer growth in the omentum exceeds the growth of the original ovarian cancer.

"This fatty tissue, which is extraordinarily rich in energy-dense lipids, acts as a launching pad and energy source for the likely lethal spread of ovarian cancer," said study author Ernst Lengyel, MD, PhD, professor of obstetrics and gynecology at the University of Chicago. "The cells that make up the omentum contain the biological equivalent of jet fuel. They feed the cancer cells, enabling them to multiply rapidly. Gaining a better understanding of this process could help us learn how to disrupt it."

The researchers performed a series of experiments to identify the role of these fat cells as major mediators of ovarian cancer metastasis. The first step was to understand the biological signals that attract ovarian cancer cells to the omentum and use it for rapid growth.

The spread of ovarian cancer cells to the omentum can happen quickly. Ovarian cancer cells injected into the abdomen of healthy mice find their way to the omentum within 20 minutes. The researchers found that protein signals emitted by the omentum can attract the tumor cells. Inhibitors which disturbed these signals reduced this attraction by at least 50 percent.

Once ovarian cancer cells reach the omentum, they quickly develop the tools to devour the sustenance provided by this fatty tissue, reprogramming their metabolism to thrive on lipids acquired from fat cells. Ovarian cancer can rapidly convert the entire omentum, a soft fat pad, into a solid mass of cancer cells.

"This mechanism may not be limited to ovarian cancer cells," the authors note. Fat metabolism may also contribute to cancer development in other environments where fat cells are abundant, such as breast cancer.

A protein known as fatty acid binding protein (FABP4), a fat carrier, may be crucial to this process and could be a target for treatment.

When the researchers compared primary ovarian cancer tissue with ovarian cancer tissue which had spread to the omentum, they found that tumor cells next to omental fat cells produced high levels of FABP4. Cancer cells distant from the fat cells did not produce FABP4.

When they inhibited FABP4, the transfer of nutrients from fat cells to cancer cells was drastically reduced. Inhibition of FABP4 also reduced tumor growth and the ability of tumors to generate new blood vessels.

"Therefore," the authors wrote, "FABP4 emerges as an excellent target in the treatment of intra-abdominally disseminating tumors, which preferentially metastasize to adipose tissue such as ovarian, gastric, and colon cancers."

http://www.eurekalert.org/pub_releases/2011-10/uocm-fci102611.php

new DNA letter in brain suggest distinct function

In 2009, the DNA alphabet expanded. Scientists discovered that an extra letter or "sixth nucleotide" was surprisingly abundant in DNA from stem cells and brain cells.

Now, researchers at Emory University School of Medicine have mapped the patterns formed by that letter in the brains of mice, observing how its pattern of distribution in the genome changes during development and aging.

Those patterns, stable or dynamic depending on the gene, suggest that 5-hydroxymethylcytosine (5-hmC) has its own distinct functions, which still need to be fully brought to light.

"Our data tells us that 5-hmC is not just an intermediate state," says senior author Peng Jin, PhD, associate professor of human genetics at Emory University School of Medicine. "It looks like it has specific functions in stem cells and brain. 5-hmC may poise a gene to be turned on after being repressed."

The results were published online Sunday by the journal Nature Neuroscience. The paper is the first report on how the patterns of 5-hmC's distribution change in mouse brain during development, and also contains data on 5-hmC in DNA samples from human brain.

Postdoctoral fellow Keith Szulwach and instructor Xuekun Li are co-first authors, and collaborators from the University of Chicago and the University of Wisconsin-Madison contributed to the paper.

5-hydroxymethylcytosine (5-hmC) is an epigenetic modification of cytosine, one of the four bases or "letters" making up DNA. Epigenetic modifications are changes in the way genes are turned on or off, but are not part of the underlying DNA sequence. 5-hmC resembles 5-methylcytosine (5-mC), another modified DNA base that scientists have been studying for decades. Until recently, chemical techniques did not allow scientists to tell the difference between them.

In contrast to 5-mC, 5-hmC appears to be enriched on active genes, especially in brain cells. 5-mC is generally found on genes that are turned off or on repetitive "junk" regions of the genome. When stem cells change into the cells that make up blood, muscle or brain, 5-mC helps shut off genes that aren't supposed to be turned on. Changes in 5-mC's distribution also underpin a healthy cell's transformation into a cancer cell.

It looks like 5-hmC can only appear on DNA where 5-mC already was present. This could be a clue that 5-hmC could be a transitory sign that the cell is going to remove a 5-mC mark. Jin says the patterns his team sees tell a different story, at least for some genes. On those genes, the level of 5-hmC is stably maintained and increases with age.

The Emory team used a method for chemically labeling 5-hmC they developed in cooperation with scientists at the University of Chicago. They find that 5-hmC is ten times more abundant in brain than in stem cells, and it is found more in the body of some genes, compared to stem cells.

In addition, the researchers found a relative lack of 5-hmC on X chromosomes in both males and females. That result is a surprise, Jin says, because it was already known that the X chromosome is subject to a special form of regulation in females only. Males have one X chromosome and females have two, and in female cells one of the X chromosomes is inactivated.

Jin's team is beginning to map how 5-hmC changes in neurological disorders, including Rett syndrome and autism, and refining techniques for detecting 5-hmC in DNA at high resolution.

http://www.eurekalert.org/pub_releases/2011-10/eu-pon102711.php

stem cell key to lung regeneration

Scientists at A*STAR'S Genome Institute of Singapore (GIS) and Institute of Molecular Biology (IMB), have made a breakthrough discovery in the understanding of lung regeneration. Their research showed for the first time that distal airway stem cells (DASCs), a specific type of stem cells in the lungs, are involved in forming new alveoli to replace and repair damaged lung tissue, providing a firm foundation for understanding lung regeneration.

Lung damage is caused by a wide range of lung diseases including influenza infections and chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). Influenza infection induces acute respiratory distress syndrome (ARDS) which affects more than 150,000 patients a year in the US, with a death rate of up to 50 percent. COPD is the fifth biggest killer worldwide.

The team took a novel approach in tackling the question of lung regeneration. They cloned adult stem cells taken from three different parts of the lungs - nasal epithelial stem cells (NESCs), tracheal airway stem cells (TASCs) and distal airway stem cells (DASCs). Despite the three types of cells being nearly 99 percent genetically identical, the team made the surprising observation that only DASCs formed alveoli when cloned in vitro.

"We are the first researchers to demonstrate that adult stem cells are intrinsically committed and will only differentiate into the specific cell type they originated from. In this case, only DASCs formed alveoli because alveolar cells are found in the distal airways, not in the nasal epithelial or tracheal airway", said Dr Wa Xian, Principal Investigator at IMB. "This is a big advancement in the understanding of adult stem cells that will encourage further research into their potential for regenerative medicine."

Using a mouse model of influenza, the team showed that after infection, DASCs rapidly grow and migrate to influenza-damaged lung areas where they form "pods". These "pods" mature to new alveoli which replace the alveoli that were destroyed by the infection, leading to lung regeneration.

"We have harvested these "pods" to provide insight into genes and secreted factors that likely represent key components in tissue regeneration.

These secreted factors might be used as biological drugs (biologics) to enhance regeneration of the lung and airways," said Dr Frank McKeon, Senior Group Leader of the Stem Cell and Developmental Biology at GIS.

The research was jointly led by Dr Frank McKeon from GIS and Dr Wa Xian from IMB in collaboration with scientists at the National University of Singapore (NUS), and clinicians at the Harvard Medical School and the Brigham and Women's Hospital in Boston.

Prof Birgitte Lane, Executive Director of IMB, said, "This groundbreaking work is a fine example of collaborative research, which has brought us new insight into lung epithelial stem cells. This will have breakthrough consequences in many areas." Dr Edison Liu, Executive Director of GIS, added, "We will continue to seek impactful collaborations and build upon this research area where there is a need for novel therapies, which will offer hope for patients suffering from respiratory diseases."

http://www.eurekalert.org/pub_releases/2011-10/afst-asf102711.php

Toughest Exam Question: What Is the Best Way to Study?

Here's a pop quiz: What foods are best to eat before a high-stakes test? When is the best time to review the toughest material? A growing body of research on the best study techniques offers some answers.

Chiefly, testing yourself repeatedly before an exam teaches the brain to retrieve and apply knowledge from memory. The method is more effective than re-reading a textbook, says Jeffrey Karpicke, an assistant professor of psychological sciences at Purdue University. If you are facing a test on the digestive system, he says, practice explaining how it works from start to finish, rather than studying a list of its parts.

In his junior year of high school in Cary, N.C., Keenan Harrell bought test-prep books and subjected himself to a "relentless and repetitive" series of nearly 30 practice SAT college-entrance exams. "I just took it over and over again, until it became almost aggravating," he says.

Practice paid off. Mr. Harrell, now 19, was accepted at University of North Carolina-Chapel Hill, a college he's dreamed of attending since the third grade. He scored 1800 (out of 2400) on the SAT, up 50% from 1200 on the PSAT, a preliminary test during his sophomore year.

Taking pretests "felt like hard work," Mr. Harrell says, but seeing steady increases in his scores boosted his confidence. Practice tests also help with test-taking skills, such as pacing, says Paul Weeks, vice president of educational services for the ACT, which creates and administers college-entrance exams.


Getty Images
Repeated practice tests help master test format and pacing.

Sleep also plays a role in test performance, but in two unexpected ways. Review the toughest material right before going to bed the night before the test. That approach makes it easier to recall the material later, says Dan Taylor, director of a sleep-and-health-research lab at the University of North Texas in Denton. And don't wake up earlier than usual to study; this could interfere with the rapid-eye-movement sleep that aids memory, he says.

A common study habit—the all-nighter—is a bad idea. Although 60% of college students stay up all night at some point in school, the practice is linked to lower grades, says Pamela Thacher, an associate professor of psychology at St. Lawrence University in Canton, N.Y., based on a 2008 study of 120 students. It also impairs reasoning and memory for as long as four days.

Being Confident

Write down fears and anxieties before the test to free working memory and prevent distractions during the test.

To combat self-doubts (such as 'I'm bad in math'), remind yourself of proven personal traits and strengths that can propel you to success.

Practice in advance facing all the pressures you will face on exam day, such as driving to the testing center or visiting an unfamiliar testing room.

Test yourself by recalling broad concepts rather than trying to memorize facts or re-reading textbooks.

Before the test, envision yourself answering questions calmly and with confidence.

Everybody knows you should eat breakfast the day of a big test. High-carb, high-fiber, slow-digesting foods like oatmeal are best, research shows. But what you eat a week in advance matters, too. When 16 college students were tested on attention and thinking speed, then fed a five-day high-fat, low-carb diet heavy on meat, eggs, cheese and cream and tested again, their performance declined. The students who ate a balanced diet that included fruit and vegetables, however, held steady, says Cameron Holloway, a senior clinical researcher at the University of Oxford. The brain requires a constant supply of energy and "has only a limited backup battery," he says.

While many teens insist they study better while listening to music or texting their friends, research shows the opposite: Information reviewed amid distractions is less likely to be recalled later, says Nicole Dudukovic, assistant professor of psychology at Trinity College, Hartford, Conn.

In her research, college students categorized and made judgments about pictures of more than 100 items. Then, they were tested on a new mix of pictures and asked to recall which ones they had already seen and how they had categorized them; half the time, they were also asked to listen and respond to a set of rhythmic sounds. When the students were tested later, they were more likely to remember correctly what they had studied without distractions.

"Students do have this belief that they can do it all and they aren't really being distracted" by music or sounds from a noisy cafe, Dr. Dudukovic says. But while the sounds may "make them feel more relaxed," she says, they won't help them ace the midterm.


Bryan Almanza says he did poorly on the PSAT as a high-school sophomore because he didn't know how to prepare. He got too little sleep the night before and ate only a bowl of cereal for breakfast. On the test, some hard physics questions made him nervous and distracted, says Mr. Almanza, 18, a senior at Campbell High in Smyrna, Ga. "I'm going to fail," he remembers thinking at the time. A test-prep program at his school taught him to get plenty of sleep, eat a good breakfast and pace himself on the test. By staying calm, optimistic and focused, he raised his score significantly on the SAT.

Tips on Conquering Test-Day Jitters

Even when students are fully prepared, anxiety can be another burden on test day.

An estimated 35% of students are so nervous before high-stakes tests that it impairs their performance, says Richard Driscoll, a Knoxville, Tenn., clinical psychologist who has researched text anxiety.

To help ease fears, Julie Hartline, lead counselor at Campbell High School in Smyrna, Ga., helped start a three-week program last year to teach juniors anxiety-reduction techniques.

One calming tactic that has been shown to improve scores is to teach yourself in advance to think differently about the test, Dr. Driscoll says. Envision yourself in a situation you find challenging and invigorating; a soccer player might imagine scoring a goal, or a mountain climber might envision herself topping a ridge, he says. Then switch your mental image to the testing room and imagine yourself feeling the same way. With practice, you'll be able to summon up more confidence on test day.

Also, reducing "novelty and stress on the day of the exam" can prevent choking under pressure, says Sian Beilock, a researcher and author on cognitive performance. If you are taking the exam in an unfamiliar place, visit the room in advance.

If you are still feeling anxious, set aside 10 minutes beforehand to write down your worries, says Dr. Beilock, an associate professor of psychology at the University of Chicago. She and a fellow researcher tested 106 ninth-graders for anxiety before their first high-pressure exam, then asked half of them to spend 10 minutes writing down their thoughts right before the test. The anxious kids who did the writing exercise performed as well on the
test as the students who had been calm all along. But anxious students who didn't do the writing performed more poorly. Expressing one's worries in writing, Dr. Beilock says, unburdens the brain.

http://online.wsj.com/public/search?article-doc-type=%7BWork+%26+Family%7D&HEADER_TEXT=work+%26+family

Structure of human mitochondrial RNA polymerase

Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts.

Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA5.

TFAM is an abundant protein that binds and bends promoter DNA 15–40 base pairs upstream of the transcription start site, and stimulates the recruitment of mtRNAP and TFB2M to the promoter.

TFB2M assists mtRNAP in promoter melting and reaches the active site of mtRNAP to interact with the first base pair of the RNA–DNA hybrid10.

The X-ray structure of human mtRNAP at 2.5 Å resolution, which reveals a T7-like catalytic carboxy-terminal domain, an amino-terminal domain that remotely resembles the T7 promoter-binding domain, a novel pentatricopeptide repeat domain, and a flexible N-terminal extension.

The pentatricopeptide repeat domain sequesters an AT-rich recognition loop, which binds promoter DNA in T7 RNAP, probably explaining the need for TFAM during promoter binding.

Consistent with this, substitution of a conserved arginine residue in the AT-rich recognition loop, or release of this loop by deletion of the N-terminal part of mtRNAP, had no effect on transcription.

The fingers domain and the intercalating hairpin, which melts DNA in phage RNAPs, are repositioned, explaining the need for TFB2M during promoter melting.

The results provide a new venue for the mechanistic analysis of mitochondrial transcription. They also indicate how an early phage-like mtRNAP lost functions in promoter binding and melting, which were provided by initiation factors in trans during evolution, to enable mitochondrial gene regulation and the adaptation of mitochondrial function to changes in the environment.

http://www.nature.com/nature/journal/v478/n7368/full/nature10435.html

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO–AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.

http://www.nature.com/nature/journal/v478/n7368/full/nature10491.html

Gene Mutation Link to Inflammation Identified

A loss-of-function mutation in a gene known as ADAM17 is likely to be the cause of neonatal inflammatory skin and bowel lesions in two children born to consanguineous parents, researchers reported.

This gene, located on chromosome 2, encodes a protein that cleaves membrane-bound tumor necrosis factor (TNF)-α, converting it to soluble TNF-α, according to David P. Kelsell, PhD, of the London School of Medicine and Dentistry, and colleagues.

The two out of three affected siblings, one boy and one girl, both developed severe erythema and pustular rashes within two days of birth, and later showed abnormalities of the hair and nails.

They also were susceptible to skin infections throughout childhood.
In addition, within a week of birth, both children developed bloody diarrhea suggestive of malabsorption, which in the girl was associated with failure to thrive.

She died at age 12 from myocarditis associated with parvovirus B19 infection.

Subsequent evaluation of her affected brother revealed the presence of mild left ventricular abnormalities.

To identify the genetic basis of this disorder, Kelsell and colleagues obtained biopsy specimens from the skin and intestines of the family members and age-matched controls.

Genetic analysis identified a deletion in exon 5 of ADAM17 in the two affected children that was not present in the unaffected sibling. Both affected children were homozygous for the mutation.

Histochemical analysis then revealed a lack of expression of ADAM17 in biopsy specimens and peripheral blood mononuclear cells in the affected children, which was not the case in unaffected family members or controls.

Because of the inflammatory nature of the disorder, the researchers also examined cytokine production in peripheral blood mononuclear cells.

Stimulation with lipopolysaccharide or anti-CD3 and anti-CD28 antibodies led to robust production of TNF-α in samples from controls and the mother, but only weak production in samples from the affected boy.

Other cytokines, including interleukin-1β, interleukin-6, and interferon-γ, were secreted in all samples, including those from the affected boy.

A knockout mouse model lacking ADAM17 exhibits a severe phenotype with a lack of development of epithelial cells in various organs including the intestine, and few mice carrying the mutation survive long after birth.

The mutant mice typically also have abnormalities of the skin and hair.

The researchers noted that there were phenotypic similarities between the knockout mice and the affected children, but there also were differences.

Although the children manifested skin and gut abnormalities soon after birth, intestinal biopsies done later in childhood found few abnormalities.

This, according to the researchers, suggests "the presence in humans of compensatory mechanisms" for epithelial repair even when ADAM17 is lacking.

"The cause of the gut problems in both children has never been satisfactorily resolved and requires more investigation," they noted.

A possible explanation for the attenuated phenotype in humans compared with the mice is that the expression of ADAM10 was retained, and there is some overlap in the activity of that enzyme and ADAM17, such as in targeting desmoglein 2 in the skin and hair follicles.

Desmoglein 2 is known to be expressed in myocytes in the heart, which may help explain the cardiac abnormalities in the affected children, they suggested.

Moreover, the lack of TNF-α, which has cardioprotective properties as well as immune-regulating activity, may have played a role in the affected girl's death at age 12.

The inadequate levels of TNF-α also may have contributed to the children's susceptibility to opportunistic skin infections. "Given the redundancy in the immune system, however, other pathways were probably operating in the patient's skin and gut to limit infection and inflammation," the researchers hypothesized.

ADAM17 has previously been considered a potential pharmacologic target in diseases characterized by chronic inflammation and overproduction of TNF-α, but the severe phenotype seen in the knockout mice suggested that blocking this target could be lethal in humans as well.

These researchers' findings suggest that such a concern may be unwarranted, and they noted that reconsideration of targeting ADAM17 might be useful for patients with conditions such as rheumatoid arthritis and psoriasis, although the possibility for adverse cardiac effects exists.

Source reference:
Blaydon D, et al "Inflammatory skin and bowel disease linked to ADAM17 deletion" N Engl J Med 2011; 365: 1502-1508.

Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection

Antiviral Research In Press, Accepted Manuscript - Note to users doi:10.1016/j.antiviral.2011.10.005 | How to Cite or Link Using DOI   Permissions & Reprints Short communication Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection Miguel Rodríguez-Pulidoa, b, Francisco Sobrinob, Belén Borregoc, Margarita Sáizb, ,  Purchase a Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Bellaterra, 08193 Barcelona, Spain b Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain c CISA-INIA, Valdeolmos, 28130 Madrid, Spain Received 20 June 2011; revised 3 October 2011; Accepted 5 October 2011. Available online 12 October 2011. Abstract We have recently described the ability of in vitro-transcribed RNAs, mimicking structural domains in the 5´ and 3´ non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome, to trigger the innate immune response in porcine cultured cells and mice. In this work, the antiviral effect exerted in vivo by these small synthetic non-infectious RNA molecules was analyzed extensively. The susceptibility of transfected newborn Swiss mice to FMDV challenge was tested using a wide range of viral doses. The level of protection depended on the specific RNA inoculated and was dose-dependent. The RNA giving the best protection was the internal ribosome entry site (IRES), followed by the transcripts corresponding to the S fragment. The time course of resistance to FMDV of the RNA-transfected mice was studied. Our results show the efficacy of these RNAs to prevent viral infection as well as to contain ongoing FMDV infection in certain time intervals. Protection proved to be independent of the serotype of FMDV used for challenge. These results support the potential use of the FMDV NCR transcripts as both prophylactic and therapeutic molecules for new FMDV control strategies. Highlights ► FMDV NCRs have prophylactic and therapeutic activity against viral infection ► FMDV IRES induces heterotypic protection against virus challenge in suckling mice ► FMDV 5´ and 3´ NCR transcripts induce dose-dependent protection against FMDV Keywords: Foot-and-mouth disease; viral non-coding regions; RNA antiviral activity; anti-FMDV strategies; innate immunity

Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells

Brain, Behavior, and Immunity In Press, Accepted Manuscript - Note to users doi:10.1016/j.bbi.2011.10.001 | How to Cite or Link Using DOI   Permissions & Reprints Short Communicaton Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells Adam C. Silvera, , , Alvaro Arjonaa, Michael E. Hughesb, Michael N. Nitabachb, Erol Fikriga, c Purchase a Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA b Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA c Howard Hughes Medical Institute, Chevy Chase, MD Received 4 August 2011; revised 28 September 2011; Accepted 5 October 2011. Available online 13 October 2011. Abstract In mammals, circadian and daily rhythms influence nearly all aspects of physiology, ranging from behavior to gene expression. Functional molecular clocks have been described in the murine spleen and splenic NK cells. The aim of our study was to investigate the existence of molecular clock mechanisms in other immune cells. Therefore, we measured the circadian changes in gene expression of clock genes (Per1, Per2, Bmal1, and Clock) and clock-controlled transcription factors (Rev-erbα and Dbp) in splenic enriched macrophages, dendritic cells, and B cells in both mice entrained to a light-dark cycle and under constant environmental conditions. Our study reveals the existence of functional molecular clock mechanisms in splenic macrophages, dendritic cells, and B cells. Highlights ► Mouse splenic macrophages, dendritic cells, and B cells possess functional circadian molecular clocks. Keywords: Circadian Rhythm; B Cells; Macrophages; Dendritic Cells; Gene Expression Corresponding author. Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, P.O Box 208022, New Haven, Connecticut 06520-8022, USA. Tel.: (203) 785 4140; fax: (203) 785-6815.

Real-time PCR (qPCR) assays cannot distinguish between DNA from dead and live cells

Highlights ► Real-time PCR (qPCR) assays cannot distinguish between DNA from dead and live cells ► DNA binding molecules allow specific detection of viable cells ► PMA-qPCR was able to specifically detect viable cells in pure cultures and food samples ► Power ultrasound has been successfully applied for microbial decontamination ► PMA-qPCR results correlate precisely with plate counts of ultrasound treated water samples. Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of E. coli O157:H7 in fresh-cut vegetable wash water P. Elizaquívela, G. Sánchezb, M.V. Selmac, R. Aznara, b, ,  doi:10.1016/j.fm.2011.10.008 | How to Cite or Link Using DOI   Permissions & Reprints Purchase a Department of Microbiology and Ecology, University of Valencia, Burjassot, Valencia, Spain b Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC). Burjassot, Valencia, Spain c Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain Received 18 April 2011; revised 23 September 2011; Accepted 5 October 2011. Available online 13 October 2011. Abstract The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a propidium monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with E. coli O157:H7 (106 CFU/mL) and treated by means of a continuous ultrasonic irradiation with a power density of 0.280 kW/L. Quantification data obtained by PMA-qPCR and plate counts were statistically similar during the viability reduction of 99.996% which corresponds to 4.4 log reductions. Further reductions of E. coli O157:H7 were not detected by the PMA-qPCR method due to the limit of detection of this technique (20 CFU/mL). Inactivation data obtained by both techniques successfully fitted a linear model, giving no significant differences in kinetic parameters. These results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria.

Synergy between intraepithelial lymphocytes and lamina propria T cells drives intestinal inflammation during infection

C E Egan, K J Maurer, S B Cohen, M Mack, K W Simpson and E Y Denkers Abstract Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7–10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and subepithelial bacterial translocation. As such, these features share similarity to Crohn's disease. Recently, we uncovered a role for intraepithelial lymphocytes (IELs) in mediating pathology after Toxoplasma infection. We show here that αβ and not γδ T-cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1−/− mice, we demonstrate that IELs do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria (LP). Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IELs and LP lymphocytes. Our data provide valuable new insights into the mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease. http://www.nature.com/mi/journal/v4/n6/full/mi201131a.html?WT.ec_id=MI-201111

Enzyme 'switch' clue to infertility and miscarriage

Scientists have identified a "fertility switch" protein which appears to increase infertility if levels are too high and fuel miscarriage if too low.

An Imperial College London team took samples from the womb lining of more than 100 women.

Writing in Nature Medicine they said women with unexplained infertility had high levels of the enzyme SGK1, while those who miscarried had low levels.

One fertility expert said the research offered new avenues for research.

About one in six women have difficulty getting pregnant, and one in 100 women trying to conceive experience recurrent miscarriages, defined as the loss of three or more consecutive pregnancies.

The Imperial team also carried out mouse studies which found levels of SGK1 in the womb lining decline during the window of time during which they can fall pregnant.

When extra copies of the SGK1 gene were implanted into the womb lining, these mice were unable to get pregnant.

The researchers say this suggests a fall in SGK1 levels is essential for making the uterus receptive to embryos.

However, if low levels of SGK1 persist into pregnancy, this appears to cause different problems.

When the researchers blocked the SGK1 gene, mice had no problem getting pregnant but they had smaller litters and showed signs of bleeding, suggesting a lack of SGK1 made miscarriage more likely.

'Focus for research'

Prof Jan Brosens, who led the research at Imperial's Institute of Reproductive and Developmental Biology, said: "Our experiments on mice suggest that a temporary loss of SGK1 during the fertile window is essential for pregnancy, but human tissue samples show that they remain high in some women who have trouble getting pregnant.

"I can envisage that in the future, we might treat the womb lining by flushing it with drugs that block SGK1 before women undergo IVF."

After an embryo is implanted, the lining of the uterus develops into a specialised structure called the decidua.

The team say lab tests show low levels of the enzyme may impair the ability of cells in the decidua to protect themselves against oxidative stress, a condition in which there is an excess of reactive chemicals inside cells.

Dr Madhuri Salker, who also worked on the study, said: "We found that low levels of SGK1 make the womb lining vulnerable to cellular stress, which might explain why low SGK1 was more common in women who have had recurrent miscarriage.

"In the future, we might take biopsies of the womb lining to identify abnormalities that might give them a higher risk of pregnancy complications, so that we can start treating them before they get pregnant."

Prof Richard Fleming, of the Glasgow Centre for Reproductive Medicine, said the research was "encouraging".

"To have something as clear as this, with a specific enzyme, is great. It is giving us something to focus on."

But, Prof Fleming, who is also a member of the British Fertility Society, warned it would be some time before the discovery translated into day-to-day practice.

"It's all very well to measure something that is missing - whether or not you can correct it is the next step.

"But at least we know somewhere that's directly involved, and can explore that.

http://www.bbc.co.uk/news/health-15305064

Vitamin D Needed to Fight Off TB

By John Gever, Senior Editor, MedPage Today
Published: October 12, 2011
Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston and
Dorothy Caputo, MA, RN, BC-ADM, CDE, Nurse Planner


Effective immune responses to tuberculosis infection are critically dependent on vitamin D availability, researchers said.

In a healthy response to Mycobacterium tuberculosis invasion, macrophages mount a frontal assault on the pathogens -- but this failed to happen in vitro when human macrophages were incubated in serum with low levels of vitamin D, according to Robert Modlin, PhD, of the University of California Los Angeles, and colleagues.

On the other hand, when the serum was supplemented with 25-hydroxyvitamin D3 (25-OH-D, the active metabolite of vitamin D), antimicrobial activity in the macrophages was restored, the researchers reported online in Science Translational Medicine.

"The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection," Modlin and colleagues wrote.

Pathogens such as M. tuberculosis that take up lodging inside human host cells can only be fought off with T cell-mediated adaptive immune responses, the researchers explained.

Mounting this attack is a multi-step process: T cells that recognize infected host cells are generated, which then secrete interferon-gamma. This, in turn, mobilizes macrophages that carry out the dirty work of killing the infected cells and the pathogens within them.

This process has been understood for some time and has prompted investigations of interferon-gamma as a means of boosting the immune response in tuberculosis -- futilely, it turned out.

"Multiple studies of interferon-gamma treatment of human macrophages have consistently failed to demonstrate antimicrobial activity against intracellular M. tuberculosis," Modlin and colleagues wrote.

But subsequent research showed that innate immune responses, mediated by Toll-like receptors, could also stimulate macrophages to attack cells infected with M. tuberculosis as long as vitamin D was available.

These findings suggested to Modlin and colleagues that this process may also influence T-cell-mediated responses to tuberculosis infection.

In their key experiment, the researchers cultured human macrophages in serum from Caucasian donors who had mean 25-OH-D levels of 113 nM (SD 11) and from African American donors with mean levels of 56 nM (SD 2).

When then treated with interferon-gamma, the macrophages incubated in the white donors' serum produced cathelicidin and expressed other genes characteristic of antimicrobial activity. This activity was lacking in the cells incubated in the black donors' serum.

Moreover, when the serum from African Americans was supplemented with additional 25-OH-D, antimicrobial activity was then apparent in the macrophages.

In other experiments, Modlin and colleagues found similar results in monocytes, which also showed substantial antimicrobial peptide expression when incubated in the vitamin D-sufficient serum but not when grown in the deficient serum.

The researchers also infected human monocytes and macrophages with virulent M. tuberculosis and then dosed them with interferon-gamma in the two types of serum. The pathogen's growth was inhibited in the presence of the white donors' serum but not with the black donors' serum.

The difference between sera from whites and African Americans is clinically significant but not surprising. Dark skin reduces the ability to make vitamin D from sun exposure; furthermore, "dark-skinned populations ... are known to have increased susceptibility to tuberculosis and other infectious diseases," Modlin and colleagues wrote.

They noted as well that, in earlier studies, low vitamin D and African ancestry were both linked to impairments in the innate immune system.

"Vitamin D deficiency may compromise both the innate and the acquired antimicrobial host defense pathways against tuberculosis infection and likely other infections known to be greater in blacks," the researchers wrote.

In discussing their results, Modlin and colleagues stressed that the role of vitamin D in immune responsiveness must be investigated in human cells and tissues, not in mouse models.

"The interferon-gamma-induced antimicrobial pathway described here is vitamin D-dependent in humans but not in the mouse," they wrote. In humans, for example, several vitamin D response elements have been identified in the mechanisms leading to antimicrobial peptide expression that are lacking in mice.

"The evolution of distinct antimicrobial mechanisms makes sense teleologically as well because mice are nocturnal animals and humans are not, and the amount of vitamin D increases with sun exposure," they added

Fabri M, et al "Vitamin D is required for IFN-gamma–mediated antimicrobial activity of human macrophages" Sci Transl Med 2011; DOI: 10.1126/scitranslmed.3003045.

Communicating RNA

Commenting on communicator RNA

S Pascolo¹

¹Department of Oncology, University Hospital of Zurich, Haeldeliweg 4, 8044 Zurich, Switzerland.

Correspondence: Dr S Pascolo, E-mail: steve.pascolo@ifiz.uni-tuebingen.de

Harking back to the ‘RNA world’ that is considered to be the beginning of life about 4.2 billion years ago,^1, 2 RNA (ribonucleic acid) molecules recapitulate all biological activities necessary for life: containment of genetic information (for example, messenger RNA (mRNA)), regulation of gene expression (small-interfering RNA and micro RNA), scaffolding of tri-dimensional structures (for example, transfer RNA), enzymatic activities (for example, ribosomic RNA), storage of energy (for example, adenine and guanine in their triphosphate form) and protection of an organism's integrity by stimulating host defence mechanisms (immunostimulating RNA).

Another fundamental feature of pluricellular living is communication between cells. The presence of abundant and highly efficient RNases in the intercellular space and body fluids has led scientists to consider improbable the existence of functional extracellular naked RNA in organisms. This notion should, however, be challenged in the light of several pieces of experimental data, including those presented by Diken et al.³ in the July 2011 issue of Gene Therapy. The authors document that naked mRNA injected into the lymph nodes of mice is taken up by phagocytic cells through macropinocytosis. This can be recapitulated in vitrousing human and mice phagocytes, such as dendritic cells or macrophages. Similar phenomena were documented more than 20 years ago when Wolff et al.⁴ reported that intradermal injection of naked mRNA in mice resulted in local protein expression and when Gilboa and colleagues⁵ reported that co-incubation (so called ‘passive pulsing’) of mRNA with human dendritic cells resulted in the presentation of major histocompatibility complex (MHC)-associated peptides derived from the antigen encoded by the mRNA. We further documented that resident cells in mouse and human dermis take up locally injected naked mRNA in a saturable and calcium-dependant way.⁶ In these previous works, it could be shown that the uptake process is active: RNA molecules do not simply diffuse through membranes but are phagocytosed and transported to the cytosol in an (as yet) unknown way. Thus, mRNA molecules prejudiced as very labile in the RNase-contaminated extracellular milieu are surprisingly functional after penetrating local cells adjacent to the site of their delivery.

To date, no experimental data are available to explain the capacity of exogenous RNA to survive and then penetrate cells before being degraded by RNases. Underlying this unexpected observation could be the pyrimidine-specificity of extracellular RNases,⁷ which may lead to relative stability of purine-rich RNA and of RNA molecules with pyrimidine bases protected within three-dimensional structures. Alternatively, cationic proteins such as anti-viral peptides (for example, LL37) eventually present in the intercellular milieu could complex and stabilise the injected mRNA before it is degraded by RNases.⁸

Up to now, cross presentation has been thought to rely on the uptake of exogenous antigens in the form of protein. Although this format may be appropriate for MHC class II antigen presentation, it is not optimal for the presentation of therapeutically relevant endogenous MHC class I-associated peptides.⁹ Indeed, the set of MHC class I-associated peptides made from endogenous, that is intracellularly translated proteins can be distinct (though overlapping) from the set made from exogenous proteins.¹⁰ As shown in the study by Diken et al.,³ phagocytes such as antigen-presenting cells (APCs) are particularly efficacious in taking up RNA from the extracellular space. This would allow APCs to cross present through the MHC class I pathway exogenous antigens expressed after uptake of extracellular mRNA, either injected or released from dying cells. Phagocytosis by APCs of mRNA released by neighbouring cells that die because of infection or endogenous dysfunction (chromosomal damage for example), may be of importance for the MHC class I presentation of antigens derived from viruses that cannot infect APCs¹¹ and tumor-specific antigens, respectively. No data are yet available to quantify the relative importance of protein uptake versus mRNA uptake for immune (cross-) presentation.

Furthermore, it can be envisaged that the spontaneous uptake of recombinant naked mRNA by phagocytes in vitro as well as by phagocytes (for example, lymph node-resident dendritic cells³) and other cells (for example, skin fibroblasts⁶) in vivo at a site of injection, reflects an ancestral biological mechanism that uses naked RNA as a communicator between cells. As RNA is chemically very stable compared with double-stranded DNA or proteins at acidic pH for example, it could have been the ideal communicator between neighbouring or even distant cells at early times of evolution. Beyond immune cross-presentation as mentioned above, it can be speculated that specific (selective or induced) or non-specific (cell death) release of naked RNA (micro RNA or mRNA for example) by cells and uptake by neighbouring cells could be a very controlled and precisely coded pathway for intercellular communication. Although the capacity of sorted RNA contained in exosomes to serve as a communicator between neighbouring as well as distant cells and tissues is presently attracting much attention,^12, 13 our knowledge about the existence and physiological relevance of naked RNA as a local communicator RNA is in its infancy.

After uptake by macropinocytosis as shown by Diken et al.,³ translocation of large RNA molecules from endosomes to the cytosol has to be achieved. Disruption of the endosome's membranes, encapsulation into exosome structures followed by release and re-uptake or selective export of RNA across the endosomal membrane could be proposed as mechanisms. Further work is required to determine the mechanisms actually involved.

The unexpected capacity of injected naked mRNA to be internalised and efficiently translated by local cells including APCs and then to prime specific immune responses, has started the hunt for what may well turn out to be to a wealth of biologically important roles for naked communicator RNA (CoRNA).

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Conflict of interest

The author declares no conflict of interest.

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References

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Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model

D Ladage, I C Turnbull, K Ishikawa, Y Takewa, K Rapti, C Morel, I Karakikes, L Hadri, J Müller-Ehmsen, K D Costa, R J Hajjar and Y Kawase

Abstract

Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.

http://www.nature.com/gt/journal/v18/n10/full/gt201152a.html?WT.ec_id=GT-201110