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Use of benzodiazepines and related drugs common around Alzheimer’s diagnosis

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Benzodiazepines and related drugs are initiated frequently in persons with Alzheimer’s disease already before the diagnosis, and their use becomes even more common after the diagnosis, shows a recent study from the University of Eastern Finland. Benzodiazepines and related drugs are used as a sleep medication and for anxiolytic purposes. These drugs were initiated more frequently in persons with Alzheimer’s disease than in persons not diagnosed with AD. Compared to persons not diagnosed with AD, it was three times more likely for persons with Alzheimer’s disease to initiate benzodiazepine use after the diagnosis, and benzodiazepines were most commonly initiated six months after the diagnosis.

The findings are based on data from the Finnish Medication Use and Alzheimer’s Disease Study, Medalz. Medalz comprises nationwide, extensive register-based data from the Finnish health care registers, and it includes all persons diagnosed with Alzheimer’s disease in Finland between 2005 and 2011. The study, analysed the initiation of benzodiazepines and related drugs in 51,981 persons diagnosed with AD. Their use of drugs was monitored for a period of five years, and the follow-up started already two years before the diagnosis. The findings were compared to persons not diagnosed with Alzheimer’s disease who were matched based on age and gender.

According to the Finnish Current Care Guidelines, benzodiazepines can be used on a short-term basis to treat behavioural problems associated with Alzheimer’s disease. However, data on the benefits of these drugs in the treatment of behavioural problems is insufficient, but it is known that these drugs increase the risk of falls and cause cognitive impairment.

One of the earlier studies on Medalz study found that in Finland, benzodiazepines are used for extensive periods in persons with Alzheimer’s disease. This, together with the current finding of frequent initiations of these drugs, paints a picture of a possible delay in AD diagnoses and concerning practice of symptom-based treatment before and around diagnosis.

University of Eastern Finland www.uef.fi/en/-/bentsodiatsepiinien-ja-niiden-kaltaisten-laakkeiden-kaytto-yleistyy-alzheimerin-taudin-toteamisen-aikoihin

Watching the brain in action

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Watching millions of neurons in the brain interacting with each other is the ultimate dream of neuroscientists! A new imaging method now makes it possible to observe the activation of large neural circuits, currently up to the size of a small-animal brain, in real time and three dimensions. Researchers at the Helmholtz Zentrum Munchen and the Technical University of Munich have recently reported on their new findings.
Nowadays it is well recognized that most brain functions may not be comprehended through inspection of single neurons. To advance meaningfully, neuroscientists need the ability to monitor the activity of millions of neurons, both individually and collectively. However, such observations were so far not possible due to the limited penetration depth of optical microscopy techniques into a living brain.
A team headed by Prof. Dr. Daniel Razansky, a group leader at the Institute of Biological and Molecular Imaging (IBMI), Helmholtz Zentrum Munchen, and Professor of Molecular Imaging Engineering at the Technical University of Munich, has now found a way to address this challenge. The new method is based on the so-called optoacoustics, which allows non-invasive interrogation of living tissues at centimetre scale depths.
‘We discovered that optoacoustics can be made sensitive to the differences in calcium ion concentrations resulting from neural activity and devised a rapid functional optoacoustic neuro-tomography (FONT) system that can simultaneously record signals from a very large number of neurons’, said Dr. Xose Luis Dean-Ben, first author of the study. Experiments performed by the scientists in brains of adult zebrafish (Danio rerio) expressing genetically encoded calcium indicator GCaMP5G demonstrated, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in opaque brains. The technique was also able to trace neural activity during unrestrained motion of the animals.
‘So far we demonstrated real-time analysis on whole brains of adult animals with roughly 2x3x4 millimetre dimensions (approximately 24 mm3),’ says the study’s leader Razansky. State-of-the-art optical microscopy methods are currently limited to volumes well below a cubic millimetre when it comes to imaging of fast neural activity, according to the researchers. In addition, their FONT method is already capable of visualizing volumes of more than 1000 cubic millimetres with temporal resolution of 10 milliseconds.
Large-scale observation of neural activity is the key to understanding how the brain works, both under normal and diseased conditions. ‘Thanks to our method, one can now capture fast activity of millions of neurons simultaneously. Parallel neural networks with the social media: in the past, we were able to read along when someone (in this case, a nerve cell) placed a message with a neighbour. Now we can also see how this message spreads like wildfire,’ explains Razansky. ‘This new imaging tool is expected not only to significantly promote our knowledge on brain function and its pathophysiology but also accelerate development of novel therapies targeting neurological and neuropsychiatric disorders,’ he concludes.

Technical University of Munich http://tinyurl.com/goxtrm5

Silver ions prove effective in preventing, killing MRSA while forming bone

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University of Missouri College of Engineering Dean and Bioengineering Professor Elizabeth Loboa and a team of colleagues recently discovered a way to slow and, in some cases, prevent the spread of MRSA while also regenerating new bone.

Methicillin resistant Staphylococcus aureus, or MRSA, infections are a critical problem in the medical world, including the area of regenerative medicine. This form of antibiotic-resistant staph infection can cause serious complications after typical invasive procedures and can be easily spread through skin-to-skin contact. MRSA is one of the foremost causes of osteomyelitis, a disease that inflames and destroys bone as well as surrounding soft tissue.

But University of Missouri College of Engineering Dean and Bioengineering Professor Elizabeth Loboa and a team of colleagues – Mahsa Mohiti-Asli and Casey Molina of the Joint Department of Biomedical Engineering at the University of North Carolina and North Carolina State University, Diteepeng Thamonwan of Silpakorn University in Thailand and Behnam Pourdeyhimi of NCSU – recently discovered a way to slow and, in some cases, prevent the spread of MRSA while also regenerating new bone.

Loboa and her colleagues discovered that by seeding the proper amount of silver into a biodegradable scaffold alongside bone-forming stem cells, they could still rapidly form bone while either inhibiting MRSA growth or killing the infection outright.

‘The silver ions go in and completely disrupt the MRSA cell machinery, and they can inhibit growth and kill the bacteria,’ Loboa said. ‘It’s a fine line. If you overuse too much of the silver, it’s bad for the mammalian cells. We want to make sure we don’t hurt our host cells but kill the bacterial cells.’

The threads of the bone-creating scaffold were coated with a silver ion-containing solution before testing. Silver has proven effective in undoing bacteria mechanically, making it harder for bacteria to develop immunity.

University of Missouri College of Engineering engineering.missouri.edu/2017/01/silver-ions-prove-effective-treating-killing-antibiotic-resistant-staph-infection/

Heater-cooler devices blamed for global mycobacterium chimaera outbreak

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A global outbreak of Mycobacterium chimaera, an invasive, slow-growing bacterium, is linked to heater-cooler devices (HCD) used in cardiac surgery, according to a study. This study adds interim guidance to recent field reports on the outbreak, providing precautionary recommendations to hospitals and health systems to reduce the risk of infections.

‘It is surprising that a global outbreak like this could go unnoticed for years. This dangerous infection has put many patients at risk all over the world,’ said Rami Sommerstein, MD, of Inselspital, Bern University Hospital in Switzerland, the lead author of the study. ‘Now that we know HCDs are the source, individual action from the different players (healthcare institutions, manufacturers, etc.) is needed to contain the ongoing patient risk. The most important action a hospital can take is to remove contaminated HCDs from the operating room and other critical areas. That is the only way to ensure that patients are protected from this infection moving forward.’

HCDs are stand-alone devices needed for heat exchange in heart-lung machines used in some 250,000 surgeries annually in the U.S., according to the Centers for Disease Control and Prevention. In response to an increasing number of infections, investigators looked into hospital water sources and found M. chimaera in HCD water circuits – specifically, in the LivaNova 3T HCD used in most hospitals around the world. They also found the bacteria in air samples during surgeries with LivaNova HCDs, suggesting transmission through air particles.

To prevent future cases of invasive M. chimaera infections, the researchers made the following recommendations for hospitals and health systems, as well as public health authorities, based on their personal experience with the outbreak:

Ensure strict separation of contaminated HCDs from air of critical medical areas
Educate clinicians on the risks for and dangers associated with M. chimaera
Screen patients who had open heart surgery, heart transplantation or those who were exposed to ventricular assist devices and demonstrate prolonged and unexplained fevers.
M. chimaera is a non-tuberculous mycobacterium that was previously known to cause lung infections. Invasive M. chimaera in cardiac surgery patients is particularly difficult to treat because it requires surgery and prolonged antibiotic therapy.

‘While our understanding of the causes and the extent of the M. chimaera outbreak is growing, several aspects of patient management, device handling and risk mitigation still require clarification,’ said Sommerstein.

Society for Healthcare Epidemiology of America www.shea-online.org/journal-news/press-room/press-release-archives/497-heater-cooler-devices-blamed-for-global-mycobacterium-chimaera-outbreak

Holographic imaging and deep learning diagnose malaria

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Duke researchers have devised a computerized method to autonomously and quickly diagnose malaria with clinically relevant accuracy — a crucial step to successfully treating the disease and halting its spread.
In 2015 alone, malaria infected 214 million people worldwide, killing an estimated 438,000.

Malaria’s symptoms can look like many other diseases, and there are simply not enough well-trained field workers and functioning microscopes to keep pace with the parasite. While rapid diagnostic tests do exist, it is expensive to continuously purchase new tests. These tests also cannot tell how severe the infection is by tallying the number of infected cells, which is important for managing a patient’s recovery.

In a new study, engineers from Duke University report a method that uses computer deep learning’ and light-based, holographic scans to spot malaria-infected cells from a simple, untouched blood sample without any help from a human. The innovation could form the basis of a fast, reliable test that could be given by most anyone, anywhere in the field, which would be invaluable in the $2.7 billion-per-year global fight against the disease.

‘With this technique, the path is there to be able to process thousands of cells per minute,’ said Adam Wax, professor of biomedical engineering at Duke. ‘That’s a huge improvement to the 40 minutes it currently takes a field technician to stain, prepare and read a slide to personally look for infection.’
The new technique is based on a technology called quantitative phase spectroscopy. As a laser sweeps through the visible spectrum of light, sensors capture how each discrete light frequency interacts with a sample of blood. The resulting data captures a holographic image that provides a wide array of valuable information that can indicate a malarial infection.

‘We identified 23 parameters that are statistically significant for spotting malaria,’ said Han Sang Park, a doctoral student in Wax’s laboratory and first author on the paper. For example, as the disease progresses, red blood cells decrease in volume, lose haemoglobin and deform as the parasite within grows larger. This affects features such as cell volume, perimeter, shape and centre of mass.

‘However, none of the parameters were reliable more than 90 percent of the time on their own, so we decided to use them all,’ said Park.
‘To be adopted, any new diagnostic device has to be just as reliable as a trained field worker with a microscope,’ said Wax. ‘Otherwise, even with a 90 percent success rate, you’d still miss more than 20 million cases a year.’

To get a more accurate reading, Wax and Park turned to deep learning — a method by which computers teach themselves how to distinguish between different objects. By feeding data on more than 1,000 healthy and diseased cells into a computer, the deep learning program determined which sets of measurements at which thresholds most clearly distinguished healthy from diseased cells.

When they put the resulting algorithm to the test with hundreds of cells, it was able to correctly spot malaria 97 to 100 percent of the time — a number the researchers believe will increase as more cells are used to train the program. Because the technique breaks data-rich holograms down to just 23 numbers, tests can be easily transmitted in bulk, which is important for locations that often do not have reliable, fast internet connections, and that, in turn, could eliminate the need for each location to have its own computer for processing.
Wax and Park are now looking to develop the technology into a diagnostic device through a startup company called M2 Photonics Innovations. They hope to show that a device based on this technology would be accurate and cost-efficient enough to be useful in the field. Wax has also received funding to begin exploring the use of the technique for spotting cancerous cells in blood samples.

Duke University pratt.duke.edu/about/news/spotting-malaria

Rapid test identifies disease pathogens

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Researchers at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart are developing a test which rapidly and cost-effectively identifies bacteria, fungi or viruses. It can be carried out directly in situ without laboratory equipment and specialist knowledge. ‘The ImmuStick can even detect pathogens outside the body – on medical devices or in hospital rooms for example. However, the technology would certainly also be of interest for testing human blood for germs or allergies’, says Dr. Anke Burger-Kentischer.
The method works as simply as a pregnancy test. The ImmuStick is a test strip onto which a few drops of fluid are applied. If the fluid contains pyrogens, fragments of pathogens, this is shown by a coloured strip in a viewing window. First of all, human immune receptors sensitive to certain pyrogens are applied to the surface of the stick. These are laboratory-produced immune receptors which are synthesized on the basis of the biological model. During production, at the docking point of the immune receptors to which the pyrogens normally bind, a type of placeholder is mounted which is marked with a dye. When drops of a fluid containing pyrogens are then applied to the test strip, the pyrogens rush to the docking point on the immune receptor. The placeholders marked with the dye migrate with the fluid through the test strip until they are visible in the viewing window. The colour signal thus indicates that pyrogens that have docked on the immune receptors are present.
The ImmuStick project was financed with money from the Discover programme. In this way the Fraunhofer-Gesellschaft is supporting projects for the duration of one year in order to demonstrate the feasibility of a technology. The ImmuStick has passed this test. ‘We were able to show that it works very well for the bacterial pyrogen LPS. Together with industrial partners, we now want to develop it into a product’, says project manager Burger-Kentischer. ‘We are currently testing further immune receptors that are specific for other pyrogens.’
Currently envisaged are applications in the food and pharmaceuticals sector or in medical technology, as a complete absence of germs or pyrogens is required there. In principle, the ImmuStick would also be of interest for blood analysis. Pyrogens in the blood often lead to blood poisoning, sepsis, from which many people still die today, especially weakened intensive care patients. ‘However, blood is a special challenge as it is complex and contains many constituent parts. But in the medium term we are aiming at blood analysis’, says Burger-Kentischer.
As pyrogens also include certain allergy trigger factors, an application here would also be conceivable. In the food and pharmaceutical industries, for example, it is important that products are free of allergens. With the ImmuStick these could be detected quickly, cost-effectively and simply. Costly and laborious laboratory tests would therefore no longer be needed or could be supplemented. At present the IGB researchers are seeking cooperation partners who want to further develop the ImmuStick to make it ready for the market.
Pyrogens become a problem when hygiene is of particular importance – in the food and pharmaceutical industries for example, or on intensive care wards in hospitals. Especially people with weakened immune systems can become severely ill. For this reason, tests are frequently carried out and the surfaces of machines or medical devices are tested for pyrogens using swabs. However, to date these tests have been costly and laborious as pyrogens can only be detected with laboratory equipment. A widely used standard test is the detection of LPS, a structure that is present in the membrane of certain bacteria. At present this test takes up around two hours. Other pyrogens can even only be detected in animal experiment.

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB http://tinyurl.com/jyrlqct

New test could improve diagnosis of tuberculosis in developing nations

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In developing nations, the current test to diagnose tuberculosis (TB) is error-prone, complicated and time-consuming. Furthermore, patients in these resource-limited areas can’t easily travel back to a clinic at a later date to get their results. To make diagnoses simpler, faster and more accurate, chemists have developed a quick and easy diagnostic tool. Field trials of the experimental new test began in June in South Africa, which has a high incidence of TB.
In wealthier countries, a patient suspected of having TB can be examined with a chest X-ray. Or a sample of the patient’s sputum, or saliva, can be sent to a lab for testing by techniques such as polymerase chain reaction (PCR).
But in developing nations with limited resources and spotty access to electricity, patients are often checked for TB with the Ziehl-Neelsen (ZN) test, which was developed in the 1880s. Technicians using this 11-step procedure put a saliva sample on a microscope slide, then dye it and rinse it multiple times. The process takes several hours. Even worse, ‘the ZN test is not very sensitive. It misses some cases of TB, and it gives a lot of false positives,’ says Carolyn R. Bertozzi, Ph.D. These limitations led Bertozzi and her team at Stanford University to develop a new test.
But that wasn’t the researchers’ initial goal when they began studying TB 16 years ago. At the time, they were investigating molecules, known as glycolipids, in the cell walls of the bacteria that cause the disease. Each glycolipid consists of the sugar trehalose linked to a lipid, or fat. The researchers discovered that if they provided slightly modified forms of trehalose to the bacteria, the microbes would metabolize the sugar molecules and integrate them into their glycolipids.
Other researchers showed that the bacteria can take up forms of trehalose in which each sugar is attached to a fluorescent dye molecule. A cell that picks up these sugars glows green. ‘We thought we could use this to detect the bacteria in sputum samples,’ Bertozzi says. Unfortunately, the other researchers’ dye also sticks to other components in saliva, making it tough to distinguish the bacteria.
Bertozzi’s team solved this problem by attaching trehalose to a ‘solvatochromic’ dye that doesn’t glow until it’s incorporated into the cell walls. As a result, there’s no background glow. In addition, the process couldn’t be easier: the technician takes a sputum sample, squirts a little of the dye mixture onto it, and then after an hour looks at it under a microscope to see if anything is glowing.
Even better, only live bacterial cells can metabolize the trehalose/dye molecules. The dyes in the ZN test, however, label both live and dead cells. That means the traditional test can’t determine whether the number of live cells is decreasing, so it can’t be used to tell whether a patient’s treatment is working. Because many strains of TB bacteria are now resistant to the standard treatments, ‘if the drugs aren’t working, you want to switch the patient to the next treatment as quickly as possible so you don’t contribute to drug resistance,’ Bertozzi explains.
The group is now working with a collaborator in South Africa to see how the new test performs in real-world conditions. In the meantime, Bertozzi’s team is studying other fluorescent dyes that could work even better in a TB test. They are also using their current trehalose/dye molecule to explore the molecular structure and physical properties of the cell wall of TB bacteria. That knowledge could shed light on the bacteria’s drug-resistance mechanism, as well as potential new ways to kill the cells.

American Chemical Society http://tinyurl.com/jp7yel4

Progesterone may protect women from worst effects of the flu

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In mouse studies, researchers from the Johns Hopkins Bloomberg School of Public Health have found that progesterone – a female sex hormone contained in most forms of hormone-based birth control – appears to stave off the worst effects of influenza infection and, in an unexpected finding, help damaged lung cells to heal more quickly.

The findings suggest that sex hormones have an effect far beyond the reproductive system and that progesterone may one day be a viable flu treatment for women

The World Health Organization reports that more than 100 million young adult women around the world are on progesterone-based contraception. And women of reproductive age are twice as likely as men to suffer from complications related to the influenza virus.

‘Despite the staggering number of women who take this kind of birth control, very few studies are out there that evaluate the impact of contraceptives on how the body responds to infections beyond sexually transmitted diseases,’ says study leader Sabra L. Klein, PhD, an associate professor in the Bloomberg School’s Department of Molecular Microbiology and Immunology. ‘Understanding the role that progesterone appears to play in repairing lung cells could really be important for women’s health. When women go on birth control, they don’t generally think about the health implications beyond stopping ovulation, and it’s important to consider them.’

The World Health Organization (WHO) has already listed hormonal contraceptives as an essential medication because of the profound benefits these compounds can have on women’s health by widening the interval between pregnancies, including decreased rates of maternal mortality and improved outcomes for babies and children.

For their research, Klein and her colleagues placed progesterone implants in female mice and left other mice, also female, without. The mice were then infected with influenza A virus. Both sets of mice became ill, but those with implants had less pulmonary inflammation and better lung function and saw the damage to their lung cells repaired more quickly.

The researchers found that progesterone was protective against the more serious effects of the flu by increasing the production of a protein called amphiregulin by the cells lining the lungs. When the researchers bred mice that were depleted of amphiregulin, the protective effects of progesterone disappeared as well. Klein says she was not surprised that progesterone lessened the inflammation and damage associated with the flu. What she didn’t expect was to find that progesterone also helped induce repair.

When female mice (and possibly humans) get sick with the flu, their natural levels of progesterone fall. Women on hormonal contraceptives – be it a birth control pill, intrauterine device (IUD) or injection – get a steadier level of progesterone, which overrides what the ovaries make naturally or what the virus takes away during infection.

Klein says there is no scientific data to date showing whether progesterone in humans has any relationship to flu severity, since no researchers have asked those questions. Building on this research, Klein says researchers at the Johns Hopkins Center of Excellence for Influenza Research and Surveillance doing flu surveillance have added questions about specific forms of birth control to their questionnaires so they can get a better idea of how this protective effect may work in humans.

Johns Hopkins Bloomberg School of Public Health www.jhsph.edu/news/news-releases/2016/female-sex-hormone-may-protect-women-from-worst-effects-of-the-flu.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feedpercent3A+JHSPHNews+percent28Public+Health+News+Headlines+from+Johns+Hopkinspercent29

Doctors use lung ultrasound to diagnose hidden disorders

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Researchers have discovered that ultrasound is a better diagnostic test for early diagnosis of pulmonary embolism and other disorders than current tests.
The study, by Dr. Peiman Nazerian, shows that transthoracic lung ultrasound can detect alternative diagnoses including pneumonia and pleural effusion in lungs more accurately than the commonly used Wells score, as well as detecting early signs of pulmonary embolism.
The Wells score is the most commonly used test to predict the clinical probability of a person developing a pulmonary embolism, or blood clot that travels to their lungs.
‘One of the largest criticisms of the widely used Wells score for estimating likelihood of potentially fatal blood clots in the lung [PE] is the vagary that surrounds the definition of its term, alternative diagnosis more likely than PE,” Jeffrey Kline, vice chair of research in the Department of Emergency Medicine and professor in the Department of Cellular and Integrative Physiology at Indiana University School of Medicine and study author, said in a press release.
‘Most clinicians who believe an alternative diagnosis is more likely than PE cannot name the diagnosis. Nazerian et al, show that lung ultrasound can quickly and non-invasively allow physicians to literally see the identity of something else wrong’ other than blood clots in the lung. This advantage can help them be more confident in deciding not to order expensive testing that causes large doses of radiation exposure to patients.’

UPI http://tinyurl.com/jzleagl

Health diagnosis through bio-signal measuring electrodes on IoT devices

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DGIST announced that Professor Kyung-in Jang’s research team from the Department of Robotics Engineering succeeded in developing bio-signal measuring electrodes that can be mounted on Internet of Things (IoT) devices through joint research with a research team led by professor John Rogers of the University of Illinois, USA.

The bio-signal measuring electrodes developed by the research team can be easily mounted on IoT devices for health diagnosis, thus they can measure bio-signals such as brain waves and electrocardiograms without additional analysis and measurement equipment while not interfering or restricting human activities.

Conventional hydro-gel based electrodes required external analysis and measurement devices to measure bio-signals due to their pulpy gel forms, which made their attachment to and detachment from IoT devices instable. In addition, since these electrodes were wet-bonded to the skin, there have been disadvantages that the characteristics of the electrodes deteriorated or their performance decreased when the electrodes were dried in the air over a long period.

In contrast, the electrodes developed by Professor Kyung-in Jang can be easily interlocked as if they are a part of IoT devices for health diagnosis. Also, since they are composed only of polymer and metal materials, they have the advantage of there being no possibility of drying in the air.

Long-term usability with wireless EMG recording. Data captured using a) a hydrogel electrode and b) soft, folded magnetic electrode. Each red triangle indicates a specific point of muscle contraction of the flexor carpi radialis of the forearm.

The bio-signal measurement electrodes developed by the research team consist of a composite material in which a magnetic material is folded with a soft and adhesive polymer, with a conductive electrode material wrapped around the composite material. The conductive electrode material electrically connects the bottom surface touching the skin and the top surface touching the electrode of the IoT device.

Electrodes with this structure reacting to the magnetic field can be easily attached and detached by using the attraction that occurs between the magnet and the electrode mounted on the IoT devices. Then, through the conductive electrode materials that connect the skin and the electrode part of the IoT device, the electric signals generated on the skin can be directly transmitted to the IoT device for health diagnosis.

The research team succeeded in storing and analyzing brain waves (electroencephalogram, EEG), electrocardiograms (ECG), eye movements (electrooculogram, EOG), and limb movements and muscle contractions (electromyogram, EMG) of the wearer for a long period through an experiment in which IoT devices with the electrodes are attached to various parts of the human body.

The bio-signal measurement electrodes can measure the bioelectric signal generated from the skin without loss or noise by using the IoT platform, thus they are expected to be applicable to the medical and healthcare fields since they cannot only measure the electrical signals of the body, but also analyze various forms of bio-signals such as body temperature change, skin change, and in-body ion concentration change.

DGIST en.dgist.ac.kr/site/dgist_eng/menu/508.do?siteId=dgist_eng&snapshotId=3&pageId=429&cmd=read&contentNo=33460

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