Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.     

Epigenetic biomarkers of T-cells in human glioma

Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10⁻¹⁶), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10⁻¹¹). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10⁻⁹) as well as Tregs (p = 5.2 × 10⁻¹¹) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10⁻⁷), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks.     

Overwintering physiology and microhabitat use of Phyllocnistis populiella (Lepidoptera: Gracilliariidae) in interior Alaska

We investigated the overwintering physiology and behavior of Phyllocnistis populiella Chambers, the aspen leaf miner, which has caused severe and widespread damage to aspen in Alaska over the past 10 yr. Active P. populiella moths caught in spring and summer supercooled to an average temperature of -16°C, whereas dormant moths excavated from hibernacula in the leaf litter during fall and winter supercooled to an average of -32°C. None of the moths survived freezing in the laboratory. Counts of overwintering moths in leaf litter across microhabitats in interior Alaska demonstrated that moths occurred at significantly higher density beneath white spruce trees than beneath the aspen host, several other hardwood species, or in open areas among trees. During winter, the temperature 1-2 cm below the surface of the leaf litter beneath white spruce trees was on average 7-9°C colder than beneath aspen trees, and we estimate that during at least one period of the winter the temperature under some white spruce trees may have been cold enough to cause mortality. However, the leaf litter under white spruce trees was significantly drier than the litter from other microhabitats, which may assist P. populiella moths to avoid inoculative freezing because of physical contact with ice. We conclude that in interior Alaska, P. populiella overwinter in a supercooled state within leaf litter mainly under nonhost trees, and may prefer relatively dry microhabitats over moister ones at the expense of lower environmental temperature.     

Nothing but the truth

Many scientists blame the media for sensationalising scientific findings, but new research suggests that things can go awry at all levels, from the scientific report to the press officer to the journalist.Everything gives you cancer, at least if you believe what you read in the news or see on TV. Fortunately, everything also cures cancer, from red wine to silver nanoparticles. Of course the truth lies somewhere in between, and scientists might point out that these claims are at worst dangerous sensationalism and at best misjudged journalism. These kinds of media story, which inflate the risks and benefits of research, have led to a mistrust of the press among some scientists. But are journalists solely at fault when science reporting goes wrong, as many scientists believe [1]? New research suggests it is time to lay to rest the myth that the press alone is to blame. The truth is far more nuanced and science reporting can go wrong at many stages, from the researchers to the press officers to the diverse producers of news.Many science communication researchers suggest that science in the media is not as distorted as scientists believe, although they do admit that science reporting tends to under-represent risks and over-emphasize benefits [2]. “I think there is a lot less of this [misreported science] than some scientists presume. I actually think that there is a bit of laziness in the narrative around science and the media,” said Fiona Fox, Director of the UK Science Media Centre (London, UK), an independent press office that serves as a liaison between scientists and journalists. “My bottom line is that, certainly in the UK, a vast majority of journalists report science accurately in a measured way, and it''s certainly not a terrible story. Having said that, lots of things do go wrong for a number of reasons.”Fox said that the centre sees everything from fantastic press releases to those that completely misrepresent and sensationalize scientific findings. They have applauded news stories that beautifully reported the caveats and limitations of a particular scientific study, but they have also cringed as a radio talk show pitted a massive and influential body of research against a single non-scientist sceptic.“You ask, is it the press releases, is it the universities, is it the journalists? The truth is that it''s all three,” Fox said. “But even admitting that is admitting more complexity. So anyone who says that scientists and university press officers deliver perfectly accurate science and the media misrepresent it […] that really is not the whole story.”Scientists and scientific institutions today invest more time and effort into communicating with the media than they did a decade ago, especially given the modern emphasis on communicating scientific results to the public [3]. Today, there are considerable pressures on scientists to reach out and even ‘sell their work'' to public relations officers and journalists. “For every story that a journalist has hyped and sensationalized, there will be another example of that coming directly from a press release that we [scientists] hyped and sensationalized,” Fox said. “And for every time that that was a science press officer, there will also be a science press officer who will tell you, ‘I did a much more nuanced press release, but the academic wanted me to over claim for it''.”Although science public relations has helped to put scientific issues on the public agenda, there are also dangers inherent in the process of translation from original research to press release to media story. Previous research in the area of science communication has focused on conflicting scientific and media values, and the effects of science media on audiences. However, studies have raised awareness of the role of press releases in distorting information from the lab bench to published news [4].In a 2011 study of genetic research claims made in press releases and mainstream print media, science communication researcher Jean Brechman, who works at the US advertising and marketing research firm Gallup & Robinson, found evidence that scientific knowledge gets distorted as it is “filtered and translated for mass communication” with “slippages and inconsistencies” occurring along the way, such that the end message does not accurately represent the original science [4]. Although Brechman and colleagues found a concerning point of distortion in the transition between press release and news article, they also observed a misrepresentation of the original science in a significant portion of the press releases themselves.In a previous study, Brechman and his colleagues had also concluded that “errors commonly attributed to science journalists, such as lack of qualifying details and use of oversimplified language, originate in press releases.” Even more worrisome, as Fox told a Nature commentary author in 2009, public relations departments are increasingly filling the need of the media for quick content [5].Fox believes that a common characteristic of misrepresented science in press releases and the media is the over-claiming of preliminary studies. As such, the growing prevalence of rapid, short-format publications that publicize early results might be exacerbating the problem. Research has also revealed that over-emphasis on the beneficial effects of experimental medical treatments seen in press releases and news coverage, often called ‘spin'', can stem from bias in the abstract of the original scientific article itself [6]. Such findings warrant a closer examination of the language used in scientific articles and abstracts, as the wording and ‘spin'' of conclusions drawn by researchers in their peer-reviewed publications might have significant impacts on subsequent media coverage.Of course, some stories about scientific discoveries are just not easy to tell owing to their complexity. They are “messy, complicated, open to interpretation and ripe for misreporting,” as Fox wrote in a post on her blog On Science and the Media (fionafox.blogspot.com). They do not fit the single-page blog post or the short press release. Some scientific experiments and the peer-reviewed articles and media stories that flow from them are inherently full of caveats, contexts and conflicting results and cannot be communicated in a short format [7].In a 2012 issue of Perspectives on Psychological Science, Marco Bertamini at the University of Liverpool (UK) and Marcus R. Munafo at the University of Bristol (UK) suggested that a shift toward “bite-size” publications in areas of science such as psychology might be promoting more single-study models of research, fewer efforts to replicate initial findings, curtailed detailing of previous relevant work and bias toward “false alarm” or false-positive results [7]. The authors pointed out that larger, multi-experiment studies are typically published in longer papers with larger sample sizes and tend to be more accurate. They also suggested that this culture of brief, single-study reports based on small data sets will lead to the contamination of the scientific literature with false-positive findings. Unfortunately, false science far more easily enters the literature than leaves it [8].One famous example is that of Andrew Wakefield, whose 1998 publication in The Lancet claimed to link autism with the combined measles, mumps and rubella (MMR) vaccination. It took years of work by many scientists, and the aid of an exposé by British investigative reporter Brian Deer, to finally force retraction of the paper. However, significant damage had already been done and many parents continue to avoid immunizing their children out of fear. Deer claims that scientific journals were a large part of the problem: “[D]uring the many years in which I investigated the MMR vaccine controversy, the worst and most inexcusable reporting on the subject, apart from the original Wakefield claims in the Lancet, was published in Nature and republished in Scientific American,” he said. “There is an enormous amount of hypocrisy among those who accuse the media of misreporting science.”What factors are promoting this shift to bite-size science? One is certainly the increasing pressure and competition to publish many papers in high-impact journals, which prefer short articles with new, ground-breaking findings.“Bibliometrics is playing a larger role in academia in deciding who gets a job and who gets promoted,” Bertamini said. “In general, if things are measured by citations, there is pressure to publish as much and as often as possible, and also to focus on what is surprising; thus, we can see how this may lead to an inflation in the number of papers but also an increase in publication bias.”Bertamini points to the real possibility that measured effects emerging from a group of small samples can be much larger than the real effect in the total population. “This variability is bad enough, but it is even worse when you consider that what is more likely to be written up and accepted for publication are exactly the larger differences,” he explained.Alongside the endless pressure to publish, the nature of the peer-reviewed publication process itself prioritizes exciting and statistically impressive results. Fluke scientific discoveries and surprising results are often considered newsworthy, even if they end up being false-positives. The bite-size article aggravates this problem in what Bertamini fears is a growing similarity between academic writing and media reporting: “The general media, including blogs and newspapers, will of course focus on what is curious, funny, controversial, and so on. Academic papers must not do the same, and the quality control system is there to prevent that.”The real danger is that, with more than one million scientific papers published every year, journalists can tend to rely on only a few influential journals such as Science and Nature for science news [3]. Although the influence and reliability of these prestigious journals is well established, the risk that journalists and other media producers might be propagating the exciting yet preliminary results published in their pages is undeniable.Fox has personal experience of the consequences of hype surrounding surprising but preliminary science. Her sister has chronic fatigue syndrome (CFS), a debilitating medical condition with no known test or cure. When Science published an article in 2009 linking CFS with a viral agent, Fox was naturally both curious and sceptical [9]. “I thought even if I knew that this was an incredibly significant finding, the fact that nobody had ever found a biological link before also meant that it would have to be replicated before patients could get excited,” Fox explained. “And of course what happened was all the UK journalists were desperate to splash it on the front page because it was so surprising and so significant and could completely revolutionize the approach to CFS, the treatment and potential cure.”Fox observed that while some journalists placed the caveats of the study deep within their stories, others left them out completely. “I gather in the USA it was massive, it was front page news and patients were going online to try and find a test for this particular virus. But in the end, nobody could replicate it, literally nobody. A Dutch group tried, Imperial College London, lots of groups, but nobody could replicate it. And in the end, the paper has been withdrawn from Science.”For Fox, the fact that the paper was withdrawn, incidentally due to a finding of contamination in the samples, was less interesting than the way that the paper was reported by journalists. “We would want any journal press officer to literally in the first paragraph be highlighting the fact that this was such a surprising result that it shouldn''t be splashed on the front page,” she said. Of course to the journalist, waiting for the study to be replicated is anathema in a culture that values exciting and new findings. “To the scientific community, the fact that it is surprising and new means that we should calm down and wait until it is proved,” Fox warned.So, the media must also take its share of the blame when it comes to distorting science news. Indeed, research analysing science coverage in the media has shown that stories tend to exaggerate preliminary findings, use sensationalist terms, avoid complex issues, fail to mention financial conflicts of interest, ignore statistical limitations and transform inherent uncertainties into controversy [3,10].One concerning development within journalism is the ‘balanced treatment'' of controversial science, also called ‘false balance'' by many science communicators. This balanced treatment has helped supporters of pseudoscientific notions gain equal ground with scientific experts in media stories on issues such as climate change and biotechnology [11].“Almost every time the issue of creationism or intelligent design comes up, many newspapers and other media feel that they need to present ‘both sides'', even though one is clearly nonsensical, and indeed harmful to public education,” commented Massimo Pigliucci, author of Nonsense on Stilts: How to Tell Science from Bunk [12].Fox also criticizes false balance on issues such as global climate change. “On that one you can''t blame the scientific community, you can''t blame science press officers,” she said. “That is a real clashing of values. One of the values that most journalists have bred into them is about balance and impartiality, balancing the views of one person with an opponent when it''s controversial. So on issues like climate change, where there is a big controversy, their instinct as a journalist will be to make sure that if they have a climate scientist on the radio or on TV or quoted in the newspaper, they pick up the phone and make sure that they have a climate skeptic.” However, balanced viewpoints should not threaten years of rigorous scientific research embodied in a peer-reviewed publication. “We are not saying generally that we [scientists] want special treatment from journalists,” Fox said, “but we are saying that this whole principle of balance, which applies quite well in politics, doesn''t cross over to science…”Bertamini believes the situation could be made worse if publication standards are relaxed in favour of promoting a more public and open review process. “If today you were to research the issue of human contribution to global warming you would find a consensus in the scientific literature. Yet you would find no such consensus in the general media. In part this is due to the existence of powerful and well-funded lobbies that fill the media with unfounded skepticism. Now imagine if these lobbies had more access to publish their views in the scientific literature, maybe in the form of post publication feedback. This would be a dangerous consequence of blurring the line that separates scientific writing and the broader media.”In an age in which the way science is presented in the news can have significant impacts for audiences, especially when it comes to health news, what can science communicators and journalists do to keep audiences reading without having to distort, hype, trivialize, dramatize or otherwise misrepresent science?Pigliucci believes that many different sources—press releases, blogs, newspapers and investigative science journalism pieces—can cross-check reported science and challenge its accuracy, if necessary. “There are examples of bloggers pointing out technical problems with published scientific papers,” Pigliucci said. “Unfortunately, as we all know, the game can be played the other way around too, with plenty of bloggers, ‘twitterers'' and others actually obfuscating and muddling things even more.” Pigliucci hopes to see a cultural change take place in science reporting, one that emphasizes “more reflective shouting, less shouting of talking points,” he said.Fox believes that journalists still need to cover scientific developments more responsibly, especially given that scientists are increasingly reaching out to press officers and the public. Journalists can inform, intrigue and entertain whilst maintaining accurate representations of the original science, but need to understand that preliminary results must be replicated and validated before being splashed on the front page. They should also strive to interview experts who do not have financial ties or competing interests in the research, and they should put scientific stories in the context of a broader process of nonlinear discovery. According to Pigliucci, journalists can and should be educating themselves on the research process and the science of logical conclusion-making, giving themselves the tools to provide critical and investigative coverage when needed. At the same time, scientists should undertake proper media training so that they are comfortable communicating their work to journalists or press officers.“I don''t think there is any fundamental flaw in how we communicate science, but there is a systemic flaw in the sense that we simply do not educate people about logical fallacies and cognitive biases,” Pigliucci said, advising that scientists and communicators alike should be intimately familiar with the subjects of philosophy and psychology. “As for bunk science, it has always been with us, and it probably always will be, because human beings are naturally prone to all sorts of biases and fallacious reasoning. As Carl Sagan once put it, science (and reason) is like a candle in the dark. It needs constant protection and a lot of thankless work to keep it alive.”     

Refractoriness of sural nerve in humans

High-frequency stimulation of peripheral nerve bundles is frequently used in clinical tests and physiologic experiments to study presynaptic and postsynaptic effects. To understand the postsynaptic effects, it is important to ensure that each pulse in the train is equally effective in stimulating the presynaptic nerve bundle; however, the optimal interpulse interval (IPI) and the stimulus intensity at which each pulse is equally effective in stimulating the same number of axons are not known. The magnitude of the compound action potential produced by each pulse in a train was tested on the sural nerve of 4 healthy human subjects. The stimulus train (2-4 pulses) was applied to the sural nerve at the lateral malleolus, and neural responses were recorded from just below the knee. With 2-pulse trains, families of curves between IPIs (1-6 ms) and normalized amplitudes of the second response were plotted for different stimulus intensities. Visual inspection of the data showed that the curves fell into 2 groups: with stimulus intensities <2.5x perception threshold (Th), the test response appeared partially at longer IPIs, whereas with stimulus intensities >=3x Th, partial recovery of the test response was earlier. The interval for complete recovery was statistically the same for low- and high-intensity stimulation. With more than 2 pulses in a stimulus train (IPI = 5 ms), the amplitude of the compound action potential (CAP) was not affected significantly. These results are important in understanding both the presynaptic and postsynaptic responses when presynaptic axon bundles are stimulated at high frequencies.     

Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral disease in North America (NA) and is a potential biological weapon. However, genetically and antigenically distinct strains from South America (SA) have seldom been associated with human disease or mortality despite serological evidence of infection. Because mice and other small rodents do not respond differently to the NA versus SA viruses like humans, we tested common marmosets (Callithrix jacchus) by using intranasal infection and monitoring for weight loss, fever, anorexia, depression, and neurologic signs. The NA EEEV-infected animals either died or were euthanized on day 4 or 5 after infection due to anorexia and neurologic signs, but the SA EEEV-infected animals remained healthy and survived. The SA EEEV-infected animals developed peak viremia titers of 2.8 to 3.1 log(10) PFU/ml on day 2 or 4 after infection, but there was no detectable viremia in the NA EEEV-infected animals. In contrast, virus was detected in the brain, liver, and muscle of the NA EEEV-infected animals at the time of euthanasia or death. Similar to the brain lesions described for human EEE, the NA EEEV-infected animals developed meningoencephalitis in the cerebral cortex with some perivascular hemorrhages. The findings of this study identify the common marmoset as a useful model of human EEE for testing antiviral drugs and vaccine candidates and highlight their potential for corroborating epidemiological evidence that some, if not all, SA EEEV strains are attenuated for humans.     

The reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl in three different sediment cultures: evidence for the involvement of phylogenetically similar Dehalococcoides-like bacterial populations

Anaerobic cultures capable of reductively dechlorinating 2,3,4,5-tetrachlorobiphenyl (CB) were enriched from three different sediments, one estuarine, one marine and one riverine. Two different electron donors were used in enrichments with the estuarine sediment (elemental iron or a mixture of fatty acids). The removal of doubly flanked meta and para chlorines to form 2,3,5-CB and 2,4,5-CB was observed in all cultures. Bacterial community analysis of PCR-amplified 16S rRNA gene fragments revealed different communities in these cultures, with the exception of one common population that showed a high phylogentic relatedness to Dehalococcoides species. No Dehalococcoides-like populations were ever detected in control cultures to which no PCBs were added. In addition, the dynamics of this Dehalococcoides-like population were strongly correlated with dechlorination. Subcultures of the estuarine sediment culture demonstrated that the Dehalococcoides-like population disappeared when dechlorination was inhibited with 2-bromoethanesulfonate or when 2,3,4,5-CB had been consumed. These results provide evidence that Dehalococcoides-like populations were involved in the removal of doubly flanked chlorines from 2,3,4,5-CB. Furthermore, the successful enrichment of these populations from geographically distant and geochemically distinct environments indicates the widespread presence of these PCB-dechlorinating, Dehalococcoides-like organisms.     

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation

When amino acids vary during evolution, the outcome can be functionally neutral or biologically‐important. We previously found that substituting a subset of nonconserved positions, “rheostat” positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether “phylogenetic” patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10–15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non‐neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously‐identified hLPYK rheostat, “toggle” (most substitution abolished function), and “neutral” (all substitutions were like wild‐type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12–13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co‐evolutionary or eigenvector centrality measures of evolutionary change.