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Kress M.,Innsbruck Medical University | Huttenhofer A.,Innsbruck Medical University | Landry M.,Institut Universitaire de France | Kuner R.,University of Heidelberg | And 13 more authors.
Frontiers in Molecular Neuroscience | Year: 2013

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals. © 2013 Kress, Hüttenhofer, Landry, Kuner, Favereaux, Greenberg, Bednarik, Heppenstall, Kronenberg, Malcangio, Rittner, Üçeyler, Trajanoski, Mouritzen, Birklein, Sommer and Soreq.

PubMed | the Heinrich Pette Institute, the European Molecular Biology Laboratory, the European XFEL GmbH, From the European Molecular Biology Laboratory and 3 more.
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2016

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.

News Article | February 3, 2016
Site: www.nature.com

Principal investigators (PIs) have a major role in advancing the careers of the junior scientists in their labs. They serve as bosses, role models, teachers and technical advisers. But often, those tasks prevent them from filling another essential need: mentor. “A PI by definition must be an authority figure,” says Kelly Edwards, associate dean for student and postdoctoral affairs at the University of Washington's graduate school in Seattle. “Someone who will be tough and strict.” But students and postdocs often need other kinds of support, she says: a counsellor, an advocate, a sympathetic ear, or even just “an aunt who will slip you $5 every once in a while”. These sorts of relationships can be hard to cultivate, especially for young scientists whose lives revolve around their labs. But making the effort to step away from the bench to socialize and network at conferences, seminars and university events can pay off later in the form of valuable career advice and guidance. Mentors can help junior researchers to make difficult career decisions, such as whether to pursue a non-academic career path or switch their research emphasis. And having mentors other than a PI is comparable to having an informal board of directors: the junior scientist benefits from the viewpoint and perspective of multiple people at various career stages. Some institutions have begun to encourage young scientists to build a mentor network by connecting them with alumni or hosting retreats that put them in direct contact with scientists from different professional backgrounds. These initiatives remove the need for junior researchers to build an entire mentor network on their own. The University of Washington's graduate school, for example, offers regular sessions to help students tap into the university's alumni group and other resources. It also requires that students' individual development plans,— which document young scientists' training and are required by many grants — include a section on mentor-team development. Part of the university's scheme involves telling both the PI and the student that mentors outside the department, or even the university, might be useful for goals such as exposure to industry or soft-skill development. Similarly, Indiana University–Purdue University Indianapolis hosts regular career-planning workshops in which professionals from the area meet with students, postdocs and faculty members. The university also offers training workshops for young faculty members. Katherine Shives, a microbiology graduate student at the University of Colorado Anschutz Medical Campus in Aurora, can attest to the usefulness of such events. It took her almost a year and four lab rotations to settle on her PI. But she needed just a few minutes at a university social function to find a key mentor. She started chatting with immunologist Kathryn Holmes, a professor emerita at the university, and felt an immediate rapport. “I felt very comfortable talking to her,” says Shives, who adds that Holmes has become an important mentor. “I spent a pretty decent amount of time in her office. She's given me excellent advice.” Holmes talked Shives through the stress of her first year in graduate school, and has given her the best advice she has received so far in her programme: “Even good change is stressful.” That phrase became Shives's mantra and has helped her to maintain her perspective during other difficult periods. Holmes also provided guidance as Shives rotated through labs while trying to choose her PI. When the options were carved down, she helped Shives to choose between an established PI with a large lab and a newly fledged PI with a smaller group. “She advised that established PIs have a solid track record of funding and mentoring students, and that with established labs you know where the PI is and what they are doing,” says Shives. “With younger PIs, there's no track record of funding and they may have never mentored a student, but there is also the potential of working with a rising star before they get big, and that being the only student means you can get more individual attention.” Ultimately, Shives chose the smaller lab, where she remains today. She has also stayed in contact with her undergraduate adviser and her PI from an internship during her final year at secondary school, who have also been valuable members of her team, in part because they are not nearby. “Having someone off-campus and removed from my programme was very valuable in the first year,” Shives says. “It helped me keep in perspective that it was not just me struggling, and that the difficulty was not a sign that I was incapable of the work.” A series of professional interactions through different stages of his education and career helped Tshaka Cunningham to construct his mentoring team. Now a scientific-programme manager at the US Department of Veterans Affairs in Washington DC, he had won a scholarship to attend Princeton University in New Jersey as an undergraduate. Donald Graham, then chairman of The Washington Post, was a member of the scholarship committee, and became part of Cunningham's mentor team after the student requested a personal meeting with him. Molecular biologist Arnold Levine advised Cunningham during his undergraduate programme. After Cunningham graduated, the two stayed in contact; Levine helped him with job interviews, and continued his mentoring role with Cunningham when the younger scientist joined Rockefeller University in New York and Levine became its president. And later, while Cunningham was working as an industry researcher in drug discovery, he connected with a company executive who remains one of his mentors. Cunningham says that he especially values the support and guidance he received from his mentors when he was conflicted about whether to accept his current job. His quandary was that he loved basic research, but wanted a more-even work–life balance. His mentors counselled him to evaluate what he most wanted, and the advice proved sound — eight years later, he remains pleased with his decision to choose the work–life balance. He warns, however, that although it is important for junior scientists to feel comfortable with their mentors, they should also look for people who will challenge them. “You want people always in your corner — but who can be brutally honest,” he says. “I seek someone who knows me well, and who has a good enough relationship with me that they don't worry about hurting my feelings.” His team helped him to weigh up the effort and sacrifice involved in an academic career, he says, and to understand that he could have professional success outside that — even though academia had been his dream. Once a junior scientist has built a network of mentors, the work is not over — he or she must continue to nurture the relationships. Cunningham stays in regular contact with his mentors, but is careful not to smother them. He sends short e-mails a couple of times a year asking how they are doing and what they are working on. Shives meets her mentors regularly for coffee or lunch, and makes a point of catching up with those who are based outside her area whenever they are in town. She makes sure to thank them all for whatever help and support they offer. Cunnningham points out that it is important to remember that a mentor is not a therapist who can devote endless amounts of time to a junior scientist's challenges. He asks for help only when he has a specific problem. That process helped him to remember both the power and limitations of mentors. “At the end of the day, a mentor can give you only a framework,” he says. Mentors outside the lab can help to relieve pressure on the PIs, because the relationships can meet needs that the PI might overlook or be unable to address. This is especially important when a student wants to explore skills or careers away from the bench or outside academia, says Edwards. “That can be a vulnerable point for the faculty member — 'This trainee doesn't want to be like me',” she says. Often, she adds, a senior faculty member simply does not have enough information about non-academic or non-research career pathways to properly guide a junior scientist into or through them. Mentors need not always be senior researchers: peers can successfully mentor one another. The European Molecular Biology Laboratory (EMBL) has fostered an initiative in which new students take courses together and learn from each other's skills. Such a structure can form a strong section of a professional network, says Regis Lengrand, head of administration at EMBL in Grenoble, France. Paul Sauer, a third-year PhD student studying cellular signalling at EMBL, has gleaned several benefits from the initiative. Taking the courses is an effective and easy way to build peer-mentor relationships — and to set the stage for later collaborations, he says. “Once your own project develops and you are looking for collaborators, or some advice from another field, you can turn to those peers,” he says. More-senior graduate students and postdocs at EMBL are tasked with teaching workshops, which puts them in touch with scientists at other sites. EMBL also hosts seminars given by external postdocs, companies and visiting faculty members, which provide opportunities for graduate students and postdocs to network and develop external mentor relationships. It also makes its alumni network available to students and postdocs. The US National Institutes of Health has created a similar model in its Broadening Experience in Scientific Training programme, which sponsors 'boot camps' for new graduate students that encourage them to form both peer and faculty mentor networks (see Nature 525, 147–148; 2015). A strong mentor group can give a junior scientist lifelong help with career progression — not just through graduate school and postdocs. “Having a diverse group of mentors, both inside and outside science, was really helpful for me as I discovered my own truth for the career path I wanted to pursue,” says Cunningham. Shives appreciates the continuing presence in her life of one of her off-campus mentors from early in her education. She still turns to him for emotional support. “You need someone to help you keep your feet on the ground,” she says.

Monteiro F.,Institute Biologia Experimental e Tecnologica | Monteiro F.,New University of Lisbon | Bernal V.,University of Murcia | Bernal V.,Repsol | And 7 more authors.
Journal of Biotechnology | Year: 2016

The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts. © 2016 Elsevier B.V.

Baer M.M.,University of Basel | Baer M.M.,University of Cologne | Palm W.,Max Planck Institute of Molecular Cell Biology and Genetics | Eaton S.,Max Planck Institute of Molecular Cell Biology and Genetics | And 3 more authors.
Journal of Cell Science | Year: 2012

The Drosophila tracheal system is a useful model for dissecting the molecular mechanisms controlling the assembly and expansion of tubular organs. We have identified microsomal triacylglycerol transfer protein (MTP) as a new player involved in the lumen expansion in unicellular tubes. MTP is an endoplasmic reticulum resident protein that can transfer triglycerides and phospholipids between membranes in vitro. MTP lipid transfer activity is crucial for the assembly and secretion of apoB family lipoproteins, which are carriers of lipids between different tissues. Here we describe an unexpected role of MTP in tracheal development, which we postulate to be independent of its known function in lipoprotein secretion. We propose that, in tracheal cells, MTP is involved in regulation of de novo apical membrane delivery to the existing lumen and thus promotes proper expansion of the larval tracheal system. © 2012. Published by The Company of Biologists Ltd.

The European Molecular Biology Laboratory, Fahy Gurteen Laboratories Ltd. and Ucl Business Plc | Date: 2012-12-20

The present invention describes methods for determining the risk that a breast precursor lesion will progress to invasive breast cancer and/or the risk of recurrent non-invasive disease in a patient, comprising detecting the presence and/or level of PAPPA and/or PAPPA functional activity in a breast tissue sample obtained from the patient, wherein if PAPPA is not present, or is present at a reduced amount compared to a control, there is the risk of progression to invasive cancer and/or the risk or recurrent disease. The present invention also enables the chemosensitisation of mitotically delayed breast cancer cells to anti-proliferative agents, preferably anti-mitotic agents, by restoring normal progression through mitosis. In this embodiment a first drug is applied to release breast cancer cells from the mitotic block and, sequentially, a second drug affecting proliferating cells is administered for cancer cell killing.

PubMed | University of Padua, University of Parma and the European Molecular Biology Laboratory
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2015

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR.

Crepin T.,French National Center for Scientific Research | Swale C.,French National Center for Scientific Research | Monod A.,French National Center for Scientific Research | Garzoni F.,French National Center for Scientific Research | And 6 more authors.
Current Opinion in Structural Biology | Year: 2015

Polyproteins are chains of covalently conjoined smaller proteins that occur in nature as versatile means to organize the proteome of viruses including HIV. During maturation, viral polyproteins are typically cleaved into the constituent proteins with different biological functions by highly specific proteases, and structural analyses at defined stages of this maturation process can provide clues for antiviral intervention strategies. Recombinant polyproteins that use similar mechanisms are emerging as powerful tools for producing hitherto inaccessible protein targets such as the influenza polymerase, for high-resolution structure determination by X-ray crystallography. Conversely, covalent linking of individual protein subunits into single polypeptide chains are exploited to overcome sample preparation bottlenecks. Moreover, synthetic polyproteins provide a promising tool to dissect dynamic folding of polypeptide chains into three-dimensional architectures in single-molecule structure analysis by atomic force microscopy (AFM). The recent use of natural and synthetic polyproteins in structural biology and major achievements are highlighted in this contribution. © 2015.

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