The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Organizing a PhD symposium—an inside view

Planning a symposium organized by PhD students is a challenging prospect. Insight from the organizers of three such symposia sheds light on the highs and lows of the experience.When we took on the responsibility for our respective annual PhD symposia (Sidebar A), none of us had any idea how much we would have to learn about organization, management and logistics; how many e-mails would be sent; how many deadlines missed. In the end, however, organizing a PhD symposium was in many ways the most instructive part of our first year as PhD students. We had the opportunity to meet and speak with brilliant scientists and we learnt how to coordinate, plan and execute different tasks efficiently with a good team spirit. Both the contacts we made and the skills we acquired should prove useful in our future careers. If you have the opportunity to get involved in organizing a symposium, we hope our experience will help you in making a start.

Sidebar A | The conferences we organized

The 3rd PhD Symposium in Computational Biology and Innovation hosted at the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland (5–7 December 2012).The 14th European Molecular Biology Laboratory (EMBL) PhD Symposium hosted at EMBL campus in Heidelberg, Germany (24–27 October 2012).The 8th FinBioNet PhD Symposium: Revolutionary bioscience: from advanced technologies to personalized medicine hosted at the University of Tampere, Finland (2–3 October 2012).Perhaps more than any other kind of meeting, a PhD symposium is a great opportunity for early-stage researchers to be exposed to a broad range of science and to meet fellow PhD students and potential collaborators in a friendly atmosphere. By organizing one, you will contribute both to your own career and to those of everyone involved. Many university graduate programmes and societies encourage the organization of such events.If you have the opportunity to get involved in organizing a symposium, we hope our experience will help you in making a startThe amount of work involved, however, is huge and requires the coordination and cooperation of a committee. Your first step, then, is to recruit that committee. An announcement to pique the interest of local PhD students is a good idea to get started, followed by the cooption of members from different areas, groups and institutes to improve diversity and broaden the committee''s knowledge. It also helps to divide the committee into different teams, and to elect a chair, vice-chair and team leaders to keep things on track. Defining clear responsibilities and setting deadlines is vital, but keep in mind that you will need to be flexible, as committee members might find they have more or less time than they planned. In addition to research, both internships and placements are common during the early phases of a PhD and you might unexpectedly lose core committee members along the way. In our case, losing a committee chair meant that the vice-chair had to take over most of the coordination and invest more time during his or her absence.In general, keep in mind that the people you are working with are not full-time employees. It is unlikely that anyone on the organizing committee has done something similar to this before, and so time must be set aside to bring people on board and get them up to speed. Some of your committee members will prove capable of working independently and will require little management; others might be overwhelmed by the demands of their research and will require assistance and micromanagement.In addition to research, internships and placements are common during the early phases of a PhD and you might unexpectedly lose core committee members…You therefore need to monitor people and be prepared for setbacks. As you will collectively bear responsibility for the symposium, work left undone will often fall to other committee members or to you if you are the chair or vice-chair. Just how much slack you have to pick up will depend on your management skills. As long as you have a plan and a clear overview of what needs to be done, management should be straightforward, if not always easy. A well-managed symposium will be a pleasure to organize. A poorly managed one will become a stressful and unpleasant experience.Dividing the labour correctly is the first crucial step. This should happen as early as possible. Sidebar B lists important categories of tasks that must be managed. Large areas—finance, participants and speakers—will probably need sub-committees of their own, while smaller areas such as website management and design might need only one or two people. A large symposium might require a team of 12 people for speakers alone, with one assigned to each speaker. Ideally you will have previous symposia hosted at your institution to use as a blueprint. If not, contact another institute and ask them for an outline. Above all, each task must have a committee member clearly responsible for it, as ambiguous instructions and diffusion of responsibility will result in inaction. Predicting how much work will be involved is difficult, and it is all too easy to under-estimate, leaving you understaffed on the day of the event. Too many staff is far better than too few, and you will find that the ability to be flexible and reassign people to tricky areas or problems is vital to your success.

Sidebar B | Important committee tasks

Speakers—contacting speakers, soliciting abstracts and direct assistance to speakersParticipants—selecting plenary speakers and editing abstracts for the abstract bookFinance—fundraising and book-keepingPR—marketing through e-mails and posters, and securing sponsorsPosters—organizing posters on the day and judging any competitionsCatering—providing food and refreshmentsDesign—designing abstract booklets, posters and logosTransport and accommodation—arranging transport and accommodation for speakers (and participants)Website management—ensuring that the website is updated regularly with information about speakers, sessions and travelWithout the right content—speakers, topics and networking opportunities—no symposium will succeed, whether it is organized by PhD students or seasoned professionals. With a committee formed, the most important task is to decide on a scientific theme and a title for the symposium. It is always good to seek suggestions from your peers and mentors, especially because this will raise awareness that the symposium is going to take place. How you ultimately settle on a theme is down to you, but a voting system might be helpful. Remember, however, that the people organizing the symposium need to be confident in its direction and vision. Once a theme is chosen, the next step should be to set the number of sessions and agree on topics for each of these sessions (Sidebar C).

Sidebar C | Deciding on a topic and title for your symposium

• The subject of the symposium should be broader than most academic conferences.You want to avoid competing directly with specialized conferences, and instead appeal to a broad range of PhD students.
• Avoid being vague—at the same time, it is vital that a PhD student in any field will read your poster and think ‘this conference is for me''. Moreover, they will need to be able to convince their group leader of it as well.
• Be aware of competitors—the topics of the symposium should not overlap with symposia that are going to be held around the same time.

Once you have settled on your theme (or themes) and set a date—keep in mind the dates of other symposiums and any public holidays—you need to begin recruiting speakers. Invitations can be sent out by e-mail and should be followed up after a week or so either by e-mail or, preferably, by telephone. Be audacious in inviting speakers—we were often surprised how willing top-tier speakers were to attend specifically because we were organizing a PhD symposium. There are two advantages for them in attending these kinds of events: the atmosphere tends to be more relaxed than other prestigious conferences, and senior attendees can interact with and influence the emerging generation of scientists. Therefore, be confident to select and invite high-profile speakers first. They will usually have personal assistants who respond to their e-mails and invitations and manage their busy schedules. Remember that you will probably receive several rejections, so take these in your stride. You can also ask the speakers to suggest the names of other suitable people to invite. Bear in mind that good speakers tend to run on extremely tight schedules, so they need to be contacted early. They should not be expected to stick to the first deadline given, or the second, or any other deadline. They will also need to be reminded gently every now and then to send their abstracts and other information. Do not be afraid to use connections you might have for ideas and information on speakers: professors, admins, old supervisors and industry contacts will all probably help in finding people and getting them to say yes.When choosing a keynote speaker, young principal investigators and accomplished post-doctoral fellows are also excellent choices. Young investigators have often achieved success by advocating new or controversial ideas and methodologies, and their presence at a symposium can enliven debate. They can also offer advice to students more immediately relevant to success in today''s scientific climate. In this vein, it is beneficial to have speakers from different stages of their scientific careers to provide a variety of perspectives in discussions.It is a good idea to have a backup plan with a small list of local and national speakers for each session in case a speaker cancels at the last minute. In all three of the symposia that the authors were variously involved in organizing (Sidebar A), there were last minute difficulties or cancellations. For example, last minute travel changes might be too expensive for your budget to cover, or planned travel might not work out as a result of weather conditions or illness.The risk with a back-up plan, of course, is that you might end up with too many speakers and find yourself in the embarrassing position of having invited someone and not actually needing them. There is no simple answer to this, but try to have a good personal relationship with your backup speakers and be upfront with them about the circumstances in which you are inviting them.Finally, PhD student symposia should be a platform for students to acquire knowledge and present their work, so make sure you include poster sessions and student presentations. They are also an important opportunity for your peers to impress future employers, make contacts and gain insight into the ‘hot'' research areas and future opportunities. Student talks can be chosen from among the submitted abstracts.With your topic chosen, teams defined, organizers recruited and speakers selected, the real work of planning sessions, sorting out speaker invites and travel, arranging catering and so on will begin (see the timeline depicted in Fig 1). It is important to start planning early—up to a year in advance—but as every good manager knows, long-term goals will be forgotten, so short-term goals are crucial to success. Three weeks is a good rule of thumb for the maximum length of time you can set for a deadline. The whole committee should have a shared checklist of everything that needs to be done, when and by whom. You should have a clear record of who has agreed to what responsibilities and everyone should know whose job it is to chase things up if deadlines are missed. This should be enough to ensure that the work gets done. If someone seems incapable or unwilling, then simply transfer his or her responsibility to someone else.Open in a separate windowFigure 1Timeline of symposium organization.Regular meetings will allow you to check in on each committee member and will facilitate communication more effectively than e-mail. Video-conferencing will need to be well-coordinated if it is to work. Distribute an agenda before each meeting and stick to it. Minutes should also be taken during each meeting and distributed afterwards by e-mail. Above all, make sure that the various teams are communicating—ask your committee members what they are doing and what information or help they need to get it done. Special care should be taken to ensure communication if committee members are geographically separated.Predicting how much work will be involved is difficult, and it is all too easy to under-estimate, leaving you understaffed on the day of the eventThe success of your symposium will be measured in a few ways. The most obvious and important will be the quality of the talks, the networking opportunities provided and whether or not the attendees had an enjoyable and interesting time. However, you will also be judged on your finances, so you need to have a clear view of how much you can spend. Handling such large amounts of money can be intimidating, so keeping good records and staying on top of things is the only way to feel comfortable about doing this and coming out the other side.The money you obtain from fundraising, sponsorship and grant applications cannot be accurately assessed early on, but you should account for it as best as you can. If you have the luxury of blueprints from previous symposia, get hold of their final budgets for guidelines on general expenses such as food, advertisement and printing (see Fig 2 for the example of our experience).Open in a separate windowFigure 2Financial breakdown of our symposia. Two different types of symposium are represented. The ‘Example income breakdown'' and ‘Example expenses breakdown 1'' are based on the example of the University College Dublin Symposium, the funding for which was external (grants, sponsorships and registration fees). ‘Example expenses breakdown 2'' is from the FinBioNet Symposium, which is supported by several doctoral programmes in Finland and, thus, grad students from participating programmes in Finland can attend free of charge. As such, it includes full payment of accommodation for speakers and attendees, and is a special case—a 50/50 split between flights and hotels is unusual for a symposium. An important message in the charts is that catering will probably take up half of the expenses of a symposium, and keynotes and speakers probably around a quarter.The attendance fees you will receive can deviate significantly from your projected numbers. This can be mitigated to some extent by early registration deadlines with discounts to encourage the majority of your attendees to register as soon as possible, as well as an easy and efficient payment system and interesting keynote speakers. Regular discussions of the budget are also crucial to keep on top of things.In terms of raising other money, consider submitting grant applications to as many societies, universities, trusts or foundations as possible. They will have a fixed timeframe in which you can apply for grants: for example, 3–6 months in advance for smaller grants and more than 6 months in advance for substantial sponsorship. It is important to have confirmations from invited speakers and estimates of attendance numbers before applying for grants.To save money, it is also beneficial to book the flights and hotel accommodation for invited speakers well in advance, rather than leaving it to them. The organizers of the PhD symposium held in 2012 at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, for example, were able to cut down the transport costs of their speakers by more than half compared with the previous year simply by booking in advance and using budget airlines. Flights within Europe with the wrong non-budget airlines can cost as much as three or four times more. If the invited speakers are left to choose their own flights, the budget for speaker travel might be bigger than expected.Commercial sponsorship will also help you balance your books. When approaching sponsors, aim for a mix of sponsorship levels and offers. Do not ignore the small sponsors, but make sure that you provide the right opportunities for the big ones to pay extra for certain privileges. A large sponsor could be the essential cash injection that your symposium needs to get a brilliant keynote speaker flown in from the USA, or food that actually tastes as good as it looks.If you are lucky, your predecessors will have been able to secure one or more big companies for sponsorship in previous years, so be sure to use the contacts that you already have. If the representatives liked your event, they will probably recommend it internally. Big companies also tend to give more non-money items. Offer them the opportunity to sponsor prizes or provide items for the conference bags. Even gaming-related companies will readily provide you with gadgets if you can manage to get them interested or tie one of their products to your symposium. Smaller companies will not have a big yearly budget to fund events, but if your symposium has a specific focus, contacting related companies close to your city might give you a greater chance of getting them interested.…the atmosphere tends to be more relaxed than other prestigious conferences, and senior attendees can interact with and influence the emerging generation of scientistsIn general, you need to keep your sponsors happy. They need to have a place to interact with your attendees, a booth for product advertising and a short presentation during the symposium. For smaller symposia, it might also be better to arrange specific sponsorship deals directly with a company, rather than relying on trying to block sell traditional tiered sponsorship—silver, gold and platinum levels. For the PhD Symposium in Computational Biology and Innovation in Dublin for example, Logitech agreed to donate three of their high quality wireless presenters that we could give away as prizes. Always keep in mind that global companies often have small departments that have useful gadgets which can be used as prizes or give-aways.Spreading the word is one of the most important steps in planning your symposium and should be started as early as possible. You should have a conference website that provides information and regular updates about your event. An eye-catching logo tied to the scientific topic also helps. Social networks—especially Facebook and Twitter—and e-mails make it extremely easy to reach interested researchers and PhD students.If your scientific field has an active international society, you should apply for an affiliation. The affiliation can be purely to help with promotion, but financial sponsorship is also possible in some cases and can open the door to more advanced grant applications that require an affiliation with a scientific society.Bear in mind that advertising your symposium too early can mean that interest might wane by the time that registration opens, whilst advertising too late might mean attendees have already made other plans. A good strategy is to spread the word once the date, venue and the first invited speakers have been confirmed. Follow this up by bursts of advertisements just before you open up registration and abstract submission. Most focused research communities have dedicated mailing lists—either regional or international—that will reach a substantial group of interested researchers. Contacting supervisors or programme directors in various universities who have students in a similar field is also a good idea. Ask them to forward your advertisement e-mail to mailing lists or PhD programmes they might know of. Generally, social or viral advertisement is free of charge and can reach far more people than any other medium.On the day itself, organization and a clear division of labour will be doubly important, so if you have already established good communication and working practices in the planning stages, this will pay dividends. If possible, have backup committee members ready to fill in for important tasks. Pay particular attention to speakers and sponsors: you have the reputation of your institute to consider. If possible, each speaker should have an assigned committee member. Make sure that each person knows exactly what he or she is supposed to be doing and leave enough time to account for delays. It is a good idea to do some practice runs before the event itself, as you will not have much time on the day to accommodate major changes. Things you might want to consider include: are there enough signs that direct you from the bus stop to the right building? Is it clear what opportunities there are for social events? What else will participants need? Hopefully you will already have considered these questions when people registered and will have captured much of the information on the registration forms. Similarly, you should have asked attendees to provide the kind of information you will need to know in advance about their dietary needs, disabled access needs and so on.PhD student symposia should be a platform for students […] so make sure you include poster sessions and student presentationsRemember that you are really doing this for the participants. Although the lectures and speakers are important, they are a part of the bigger picture of poster presentations, networking and PhD student talks, which are all equally important. The best conversation starters are social events, so take the keynote speakers out to a pub and invite everyone to join, or organize a dinner for everybody. Formal dinners are nice for large conferences, but a small PhD symposium greatly benefits from its informal environment. The first night, especially, can be a great chance to use for networking and social outings.Once the final talks are finished and the prizes are given, make sure you thank everyone: speakers, attendees, sponsors and supporters, and last but not least, yourselves, the organizing committee. Taking feedback from attendees is important, but it can be equally important to provide your own feedback for the group of PhD students who will organize the symposium next year. If you are part of a recurring event, you have a responsibility to them and should keep good records and be ready to advise them when they need it.Finally, we would recommend treating yourselves to committee t-shirts or hoodies so that you are easily identifiable to the multitude of people who will have questions for you or will need your help on the day. And so that you have a souvenir of all the hard work you put in. Good luck!     

ArchAlign: coordinate-free chromatin alignment reveals novel architectures

To facilitate identification and characterization of genomic functional elements, we have developed a chromatin architecture alignment algorithm (ArchAlign). ArchAlign identifies shared chromatin structural patterns from high-resolution chromatin structural datasets derived from next-generation sequencing or tiled microarray approaches for user defined regions of interest. We validated ArchAlign using well characterized functional elements, and used it to explore the chromatin structural architecture at CTCF binding sites in the human genome. ArchAlign is freely available at http://www.acsu.buffalo.edu/~mjbuck/ArchAlign.html.     

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit

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Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-gamma/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-alpha, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-gamma/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to "physiological" levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-alpha, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.     

19 A solution structure of the filarial nematode immunomodulatory protein,ES-62

ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19 A model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.     

PGF(2alpha)-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Lower esophageal sphincter (LES) tone depends on PGF(2alpha) and thromboxane A(2) acting on receptors linked to G(i3) and G(q) to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF(2alpha) signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(13) and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [(35)S]GTPgammaS-binding assay confirmed that G(13), RhoA, and ARF1 were activated by PGF(2alpha). Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(q) and G(i3), and a [(35)S]GTPgammaS-binding assay confirmed that G(q) and G(i3) were activated by PGF(2alpha). PGF(2alpha)-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF(2alpha) concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF(2alpha) activates a PLD- and protein kinase C (PKC)-dependent pathway through G(13), RhoA, and ARF1. In LES, PGF(2alpha) receptors are coupled to G(q) and G(i3), activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF(2alpha) activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.     

Inverse Rap1 and phospho-ERK expression discriminate the maintenance phase of tolerance and priming of antigen-specific CD4+ T cells in vitro and in vivo

T cell recognition of Ag can result in priming or tolerance depending on the context in which Ag is recognized. Previously, we have reported that these distinct functional outcomes are associated with marked differences in the amplitude, kinetics, and cellular localization of activated, pERK signals at the level of individual Ag-specific T cells in vitro. Here, we show that the GTPase Rap1, which can antagonize the generation of such pERK signals and has been reported to accumulate in tolerant cells, exhibits an inverse pattern of expression to pERK in individual Ag-specific primed and tolerized T cells. Although pERK is expressed by more primed than tolerized T cells when rechallenged with Ag in vitro, Rap1 is expressed by higher percentages of tolerant compared with primed Ag-specific T cells. Moreover, whereas pERK localizes to the TCR and lipid rafts in primed cells, but exhibits a diffuse cellular distribution in tolerized cells, Rap1 colocalizes with the TCR and lipid raft structures under conditions of tolerance, but not priming, in vitro. This inverse relationship between Rap1 and pERK expression is physiologically relevant, given that we observed the same patterns in Ag-specific T cells in situ, following induction of priming and tolerance in vivo. Together, these data suggest that the maintenance of tolerance of individual Ag-specific T cells may reflect the recruitment of up-regulated Rap1 to the immune synapse, potentially resulting in sequestration of Raf-1 and uncoupling of the TCR from the Ras-ERK-MAPK cascade.     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.