2009 Society for Medical Anthropology Conference: The Intersection of Anthropology and Medicine as Portrayed in Paul Farmer’s Photo Album

Paul Farmer, physician, anthropologist, and author, spoke at the 2009 Society for Medical Anthropology Conference at Yale University in September.Medical anthropology is a very young field, only approximately 50 years old. The underpinnings of medical anthropology have been around for some time, but as a discipline, the burden to ensure that it continues to flourish and grow belongs to future generations of students and scholars. However, future generations of medical anthropologists cannot carry the field forward unless they examine the teachings of previous teachers and scholars. By narrating his own story, just as he so frequently narrates the intricacies of Haiti [1], Paul Farmer, physician, anthropologist, and author of Pathologies of Power: Health, Human Rights, and the New War on the Poor [2], displayed a parallel between the stories of his own past with that of medical anthropology.At the 2009 Society for Medical Anthropology Conference at Yale University in September, Farmer began his aptly titled presentation, Photo Album, with a discussion of his introduction to medical anthropology while an undergraduate at Duke. He stumbled upon medical anthropology quite by chance as an ambitious pre-med who was interested in taking every course that had the word “medical” in its title. He credited many people, including Patricia Pessar, Arthur Kleinman, and Linda Garro with aiding the development of his ideas and perception of the world and teaching him to use medical anthropology not only in passive observation, but in the active practice of medicine. You “don’t have to be a faculty member to teach,” stressed Farmer. Some of the most important lessons to learn come from the poor, to whom few listen.Farmer believes that listening can form the work we do. He honed his listening skills, which are used in anthropology in an ethnographic context, after his first night in an emergency room, when he saw that many minor cases were brought in solely because individuals had no other outlet for treatment. Being a good listener allowed Farmer to understand the full impact of a 1981 slavery case involving migrant workers in Florida. It was this skill of listening that enabled Farmer to understand and tell Haiti’s story, as well as understand the intricate web that exists between privilege and privation. Just as the line between medical anthropology and primary care is often blurred, the “bracing connection between privilege and privation” becomes even more apparent the longer one spends studying both extremes.This is a vantage point Farmer was particularly susceptible to, given his trips from Haiti to Harvard and back again. Listening to his patients in Haiti and the United States would allow Farmer to draw parallels of inequality and injustice that exist for the impoverished in both places. The only difference between the United States and Haiti is that eventually many impoverished individuals in the United States will wind up in somewhat adequate medical facilities. In the story of global economics, Farmer said, “Good things get stuck in customs and bad things get traded freely.” A practicing physician may easily note that inequalities between the rich and poor are not unique to the United States or to Haiti, but what, Farmer asks, can anthropologists say about this division?The cursory glance through Farmer’s photo album ended with a picture of friends whom he fondly termed “the structural violence mafia” and anthropological ideas regarding unequal access to health care. While at first, the portion of anthropology that dissects the structures of violence seems isolated from medical anthropology, those structures of violence institute the vast inequalities that cause medicine to seem inaccessible. Farmer also stressed that “how we think about social theory influences global health.” Work in Haiti taught Farmer firsthand about the phenomenon of blaming the victim [3]. To understand this entrenched system of structural violence fully, an intensive bio-social analysis must be undertaken. Structural violence results in a system in which the victims are blamed, empowering those who suppress the victim while inhibiting the victim’s access to health care. Pointing fingers at the vulnerable is illustrated by the fact that Haiti is often blamed for the introduction of AIDS into North America [4,5]. Farmer stressed not only the inherent trauma of structural violence, but Carolyn Nordstrom’s ideas on violence having a distinct tomorrow [6]. The perpetual cycle of structural violence enables this concept of violence having a clear future with the inherent cultural systems that allow for violence remaining stagnant while the individuals entrapped within the system change.Beyond this concept of structural violence is that of structural healing [3]. Though structural healing is a new phenomenon being examined by anthropologists, it provides a balance to structural violence with the idea being that there are certain societal standards that are either in place or can be introduced that allow for an alleviation of the suffering caused by structural violence. While Farmer’s discussion of the path that led him to his current position was inspirational in itself, the sharing of his story is of even more importance because he has been a teacher to so many. His story reinforces the idea that even though structural violence has a definite past and future, so do medical anthropology and the idea of structural healing. Thankfully, medical anthropology may be used as a relatively new force to combat structural violence. Farmer’s speech may have been unexpected in its autobiographical content, but perhaps the main point is that the intersection between medicine and anthropology can be seen not as a single point but a line that runs the full length of each of these disciplines. We all have a distinct responsibility to not only hear but to listen and learn, not to just passively observe, but actively understand. It is with this listening and acting, that future medical anthropologists can bridge the gap between social sciences and practical medicine.     

Gain weight by “going diet?” Artificial sweeteners and the neurobiology of sugar cravings: Neuroscience 2010

America’s obesity epidemic has gathered much media attention recently. A rise in the percent of the population who are obese coincides with an increase in the widespread use of non-caloric artificial sweeteners, such as aspartame (e.g., Diet Coke) and sucralose (e.g., Pepsi One), in food products (Figure 1). Both forward and reverse causalities have been proposed [1,2]. While people often choose “diet” or “light” products to lose weight, research studies suggest that artificial sweeteners may contribute to weight gain. In this mini-review, inspired by a discussion with Dr. Dana Small at Yale’s Neuroscience 2010 conference in April, I first examine the development of artificial sweeteners in a historic context. I then summarize the epidemiological and experimental evidence concerning their effects on weight. Finally, I attempt to explain those effects in light of the neurobiology of food reward.Open in a separate windowFigure 1Time line of artificial sweetener use and obesity trends in the United States. Blue line: changes in the percentage of the population who are obese (BMI >30) from 1961 to 2006. Source: National Health and Nutrition Examination Survey [57]. Orange line: changes in the percentage of the population who are regular artificial sweetener users from 1965 to 2004. Source: National Household Survey [2]. Purple line: changes in the number of new artificial sweetener containing food products introduced to the American market from 1999 to 2004. Source: Mintel Market Analysis [14]. Bars below the time axis indicates the type and availability of artificial sweeteners in the United States over time. Source: Kroger et al [9].     

2009 Society for Medical Anthropology Conference: Training,Communication, and Competence: The Making of Health Care Professionals

The role of medical anthropology in tackling the problems and challenges at the intersections of public health, medicine, and technology was addressed during the 2009 Society for Medical Anthropology Conference at Yale University in an interdisciplinary panel session entitled Training, Communication, and Competence: The Making of Health Care Professionals.The discipline of medical anthropology is not very formalized in the health setting. Although medical anthropologists work across a number of health organizations, including schools of public health, at the Centers for Disease Control (CDC), and at non-governmental organizations (NGOs), there is an emerging demand for an influential applied medical anthropology that contributes both pragmatically and theoretically to the health care field.The role of anthropology at the intersections of public health, medicine, and technology was addressed during the 2009 Society for Medical Anthropology Conference at Yale University in September. In a conference session entitled Training, Communication, and Competence: The Making of Health Care Professionals, health professional career issues, including training and education, medical entrepreneurship, and the maintenance of clinical relationships with patients were examined. The presentations encompassed macro approaches to institutional reform in training, education, and health care delivery, as well as micro studies of practitioner-patient interaction. Seemingly disparate methodological, disciplinary, and theoretical orientations were united to assess the increasing relevance of medically oriented anthropology in addressing the challenges of health care delivery, health education, and training.Margaret Bentley, a professor of public health at the University of North Carolina, Chapel Hill, spoke about the increasing “epidemic of global health” in universities, noting a doubling of global health majors within the past three years. Despite this expansion of the field, a common discipline of global health continues to be developed. In September, the Association of Schools of Public Health (ASPH) and the University of Minnesota hosted a Global Health Core Competency Development Consensus Conference with the initiative to explore “workforce needs, practice settings, and to identify core constructs, competency domains, and a preliminary global health competency model”¹. Given the current variability in training, Bentley believes medical anthropology is uniquely suited to inform training in global health because of its offerings in the way of interdisciplinary methods and team-based applied field experience.Anthropologists Carl Kendall of Tulane University and Laetitia Atlani of Université de Paris X Nanterre have seen medical anthropologists examine models of health strictly within a clinical experience. Understanding of the social determinants of epidemiology, methodological issues of population health, and survey research is crucial. However, training individuals through a more formalized program (currently in development in Europe) will allow anthropologists to better understand context, explain complex models, humanize aggregate statistics, and articulate methods of the multidimensional “social field” of health outside of the clinical experience.The social field of health, however, as Robert Like of the University of Medicine and Dentistry of New Jersey explained, shares an uncomfortable interface with clinical medicine. Recent efforts by the New Jersey Board of Examiners to incorporate cultural competency legislation have been robustly criticized. Evaluations of six-hour training sessions on cultural competency training have revealed health professionals’ frustration with the health care system’s inability to deal with “culturally different” individuals. In fact, the majority of health professionals who were required to complete the training believe cultural competency to be an area of study that is a “waste of time.”This opposition to cross-cultural education and the value of “cultural competence” training also has been a topic of great debate among anthropologists and health researchers. Despite the ubiquitous use of the term among research and health professionals, cultural competency is a term that cannot be defined precisely enough to operationalize.In “Anthropology in the Clinic: The Problem of Cultural Competency and How to Fix It,” Arthur Kleinman and Peter Benson asserted that the static notion of culture in the medical field “suggests that a culture can be reduced to a technical skill for which clinicians can be trained to develop expertise” [1]. T.S. Harvey, a linguistic and medical anthropologist at the University of California, Riverside, expounded on Kleinman’s opposition to competence as an acquired “technical skill” [1] and suggested reconceptualizing the approach to competence as communication. Although Kleinman’s explanatory models approach [2] provides a health care professional with what to ask the patient, Harvey pulls from Dell Hymes’ communicative competence [3] to understand how to ask it. Harvey recommended viewing competence as a “sociolinguistic acquisition … like a foreign language” where competencies are rule-governed and communication and speech events are formulaic.Harvey also noted that the “onus of cultural competency” is too often placed on the practitioner. Inevitably, there is an asymmetry in every clinical encounter, whereby the “would-be patient” is perpetually considered the “passive receptor.” Patients also share a stake in their health and, as such, should be taught communicative competence as well.Harvey also noted that the “onus of cultural competency” is too often placed on the practitioner. Inevitably, there is an asymmetry in every clinical encounter, whereby the “would-be patient” is perpetually considered the “passive receptor.” Patients also share a stake in their health and, as such, should be taught communicative competence as well.The role of the patient is made ever more complex by the power relationship that exists in the patient-provider context. Through ethnographic research, Sylvie Fainzang, director of research in the Inserm (Cermes), examines how doctors and patients lie. She argues that lying, in the context of secrecy, is an indication of a power relationship [4]. Fainzaing’s further research on the relationship between doctors and patients has yielded additional information on how patients learn about their diagnoses and how they will react to these diagnoses. Though a clinical encounter between a doctor and patient is expected to be one of informed consent, doctors often judge patients upon their ability to “intellectually understand” [4] and assess who is “psychologically ready” [4] to bear the information. This leads to manipulated, misinformed, and “resigned consent” [4]. This sort of social training of obligation of a subject to medical authority provides the patient with the choice either to conform or overthrow the rules as defined by society.Collectively, this interdisciplinary panel worked to inform the discussion on how medical anthropology can address training, communication, and competence at the intersections of medicine, public health, and education. By reviewing health professionals’ growing interest in public health, training in health education and competence, and the patient-provider relationship, medical anthropology can be seen as both relevant and necessary to addressing the challenges faced by the medical and health community today.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Creative destruction of the microtubule array

Comment on: Mukherjee S, et al. Cell Cycle 2012; 11:2359-66.Typical cells contain a dense array of microtubules that serves as a structural backbone and also provides a substrate against which molecular motor proteins generate force. Cells transitioning through the cell cycle or undergoing significant morphological changes must be able to tear apart the microtubule array and reconstruct it into new configurations, either partially or completely. The microtubule field was revolutionized in the 1980s with the introduction of the dynamic instability model,¹ now broadly recognized as a fundamental mechanism by which microtubule populations are reconfigured.² Dynamic instability involves the catastrophic disassembly of microtubules, generally from their plus ends, as well as the rapid reassembly of microtubules and selective stabilization of particular ones. Microtubules can be stabilized along their length by binding to various proteins and can be attached at their minus ends to structures such as the centrosome and “captured” at their plus ends by proteins in the cell’s cortex.² Given the contribution of these stabilizing and anchoring factors, additional mechanisms beyond dynamic instability are required to tear down previous microtubule structures so that new ones can be constructed. Borrowing from the field of economics, we refer to this as creative destruction.Various proteins such as stathmin³ and kinesin-13⁴ contribute to creative destruction by promoting loss of tubulin subunits from the ends of the microtubules. We find especially interesting a category of AAA enzymes called microtubule-severing proteins that use the energy of ATP hydrolysis to yank at tubulin subunits within the microtubule, thereby causing the lattice to break.⁵ If this occurs along the length of the microtubule, the microtubule will be severed into pieces. If this occurs at either of the two ends of the microtubule, the microtubule will lose subunits from that end. The first discovered and best-studied microtubule-severing proteins are katanin and spastin.Thanks to David Sharp and his colleagues at Albert Einstein College of Medicine, as well as other workers in the field, we now know that cells express at least five other AAA proteins with potential microtubule-severing properties, on the basis of sequence similarity to katanin and spastin in the AAA region.⁵ Two of these, called katanin-like-1 and katanin-like-2, are very similar to katanin. The three others are similar to one another, collectively termed fidgetins (fidgetin, fidgetin-like-1 and fidgetin-like-2). One possibility is that all seven of the microtubule-severing proteins are regulated similarly and are functionally redundant with one another. A more compelling possibility is that, while there is some functional redundancy, there is also a division of labor, with each severing protein displaying distinct properties and carrying out its own duties. Thus far, Sharp’s studies on mitosis support the latter scenario, with katanin, fidgetin and spastin having characteristic distributions within the spindle, resulting in unique phenotypes when depleted.⁶In a new article, Sharp’s group has confirmed that fidgetin has microtubule-severing properties. Interestingly, fidgetin depolymerizes microtubules preferentially from the minus end.⁷ In addition, the new work shows that in human U2OS cells, fidgetin targets to the centrosome, where most minus ends of microtubules are clustered, suggesting a scenario by which fidgetin suppresses microtubule growth from the centrosome as well as attachment to it. Consistent with this scenario, the authors show that experimental depletion of fidgetin reduces that speed of poleward tubulin flux as well as the speed of anaphase A chromatid-to-pole motion and also results in an increase in both the number and length of astral microtubules. Notably, this contrasts with katanin, which favors the plus ends of microtubules, for example, at the chromosome during cell division⁶ and at the leading edge of motile cells.⁸The authors close their article by pointing out that microtubule-severing is important beyond mitosis, for example, in the restructuring of the microtubule array in neurons and migrating cells, and we would point to plants as well.⁹ We previously described a mechanism called “cut and run,” wherein the severing of microtubules is important for motility within the microtubule array, as short microtubules are more mobile than long ones.⁹ Now, inspired by the work of Sharp and colleagues, we envision “creative destruction” as another way of understanding the crucial roles played by a diversity of microtubule-severing proteins in cells.     

KSR Int'l Co. v. Teleflex, Inc.: no obvious changes for the biotechnology market

With the advent of molecular biology, genomics, and proteomics, the intersection between science and law has become increasingly significant. In addition to the ethical and legal concerns surrounding the collection, storage, and use of genomic data, patent disputes for new biotechnologies are quickly becoming part of mainstream business discussions. Under current patent law, new technologies cannot be patented if they are “obvious” changes to an existing patent. The definition of “obvious,” therefore, has a huge impact on determining whether a patent is granted. For example, are modifications to microarray protocols, popular in diagnostic medicine, considered “obvious” improvements of previous products? Also, inventions that are readily apparent now may not have been obvious when discovered. Polymerase chain reaction, or PCR, is now a common component of every biologist’s toolbox and seems like an obvious invention, though it clearly was not in 1983. Thus, there is also a temporal component that complicates the interpretation of an invention’s obviousness. The following article discusses how a recent Supreme Court decision has altered the definition of “obviousness” in patent disputes. By examining how the obviousness standard has changed, the article illuminates how legal definitions that seem wholly unrelated to biology or medicine could still potentially have enormous effects on these fieldsJust what is obvious or not is a question that has provoked substantial litigation in the Federal Circuit, the appellate court with special jurisdiction over patent law disputes. Under U.S. patent law, an inventor may not obtain a patent, which protects his invention from infringement by others, if the differences between the subject matter sought to be patented and the prior art are such that “the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill” in the patent’s subject matter area [1]. However, what was “obvious” at the time of invention to a person of ordinary skill is hardly clear and is, in effect, a legal fiction designed to approximate objectivity. As illustrated by Chief Justice John Roberts of the Supreme Court in a moment of levity, “Who do you get to ... tell you something’s not obvious … the least insightful person you can find?” [2] Despite the apparent objectivity provided by a “person of ordinary skill” obviousness standard, the difficulty lies in that such a standard is still susceptible to multiple interpretations, depending on the point of view and knowledge ascribed to the “ordinary person.” As such, how obviousness is defined and interpreted by the courts will have important implications on biotechnology patents and the biotechnology business.The issue of obviousness arose in April 2007 when the Supreme Court handed down its decision in KSR Int’l Co. v. Teleflex, Inc. [3] The facts of the case were anything but glamorous; in the suit, Teleflex, a manufacturer of adjustable pedal systems for automobiles, sued KSR, its rival, for infringement of its patent, which “describe[d] a mechanism for combining an electronic sensor with an adjustable automobile pedal so that the pedal’s position can be transmitted to a computer that controls the throttle in the vehicle’s engine.” [4] Teleflex believed that KSR’s new pedal design was too similar to its own patented design and therefore infringed upon it [5]. In defense, KSR argued that Teleflex’s patent was merely the obvious combination of two pre-existing elements and, thus, the patent, upon which Teleflex’s infringement claim was based, was invalid.Patent law relies on the concept of obviousness to distinguish whether new inventions are worthy of being protected by a patent. If a new invention is too obvious, it is not granted a patent and is therefore not a legally protected property interest. However, if an invention is deemed not obvious and has met the other patentability requirements, a patent will be granted, thereby conferring exclusive use of the invention to the patent holder. This exclusive right prohibits others from making, using, selling, offering to sell, or importing into the United States the patented invention [6]. Essentially, the definition of obviousness sets the balance between rewarding new inventions with exclusive property rights and respecting old inventions by not treating minor variations of existing patents as new patents. In this manner, the law seeks to provide economic incentives for the creation of new inventions by ensuring that the property right conferred by the patent will be protected against insignificant variations. The importance of where the line for obviousness is drawn and how clearly it is drawn is especially important in the biotechnology industry. Studies have shown that the development of a new pharmaceutical therapy can take up to 14 years with costs exceeding $800 million [7]. Such an enormous investment of time and money would not be practical if it did not predictably result in a legally enforceable property right.The standard for what constitutes a patentable discovery has evolved over the last 150 years. In 1851, the Supreme Court held in Hotchkiss v. Greenwood that a patentable discovery required a level of ingenuity above that possessed by an ordinary person [8]. Lower courts treated the Hotchkiss standard as a subjective standard, whereby courts sought to determine “what constitute[d] an invention” [9] and a “flash of creative genius” [10]. However, the attempts at imposing the Hotchkiss standard proved unworkable, and in 1952, Congress overrode the case law with the Patent Act, “mandat[ing] that patentability be governed by an objective nonobviousness standard.” [11] This new statutory standard moved the courts away from subjective determinations and toward a more workable, objective obviousness standard.While the Patent Act laid the foundation for the current obviousness standard, the Supreme Court in Graham v. John Deere Co. interpreted the statutory language in an attempt to provide greater clarity as to what exactly “obvious” meant [12]. The Supreme Court determined that the objective analysis would require “the scope and content of the prior art ... to be determined; differences between the prior art and the claims at issue ... to be ascertained; and the level of ordinary skill in the pertinent art resolved.” [13] In addition to analysis under this three-part framework, the Supreme Court called for several secondary considerations to be weighed, including “commercial success, long felt but unresolved needs, [and the] failure of others [to solve the problem addressed].” [13]Unsurprisingly, lower courts were unsatisfied with the Supreme Court’s attempts to clarify the obviousness standard and sought to provide “more uniformity and consistency” to their evaluation of obviousness than the Supreme Court’s jumble of factors provided [14]. In search of consistency, the Federal Circuit created the “teaching, suggestion, or motivation” test (TSM test) “under which a patent is only proved obvious if ‘some motivation or suggestion to combine prior art teachings’ can be found in the prior art, the nature of the problem, or the knowledge of a person having ordinary skill in the art.” [14] Through implementation of the TSM test, the Federal Circuit sought to maintain the flexibility envisioned by the Supreme Court in Graham, while at the same time providing more certainty and predictability to obviousness determinations.The issue before the Supreme Court in KSR Int’l Co. v. Teleflex, Inc. was whether the Federal Circuit’s elaboration on the statutory language of the Patent Act, the TSM test, was consistent with the terms of the Patent Act itself and the Supreme Court’s own analysis in Graham. The Supreme Court determined that while the TSM test was, on its terms, consistent with the framework set out in Graham, the rigid manner in which the Federal Circuit had taken to applying that standard was inconsistent with the flexible approach established by Graham [15]. More generally, it appears the Supreme Court was mainly interested in restoring a more rounded, thorough inquiry to the evaluation of obviousness: “Graham set forth a broad inquiry and invited courts, where appropriate, to look at any secondary considerations that would prove instructive.” [16] As stated by the Supreme Court, “[r]igid preventative rules that deny factfinders recourse to common sense, however, are neither necessary under our case law nor consistent with it.” [17] As such, the Supreme Court reversed the findings of the Federal Circuit, which had found the Teleflex patent valid, and remanded the case back to the lower court with directions to analyze, without rigid adherence to the TSM test, whether the Teleflex patent was obvious [18].The Supreme Court’s ruling in KSR Int’l Co. v. Teleflex, Inc. that the Federal Circuit apply its TSM test less rigidly may have implications for those seeking biotechnology patents in the future. As discussed above, the large investments necessary to develop a marketable biotechnology product demand that entrepreneurs making those investments be reasonably assured that they can predict any future legal hurdles in patenting their invention and in ultimately protecting their patent. As explained by the Biotechnology Industry Organization in its amicus curiae brief in KSR Int’l Co. v. Teleflex, Inc., “[i]nvestment thus is predicated on an expected return on investment in the form of products or services that are protected by patents whose validity can be fairly determined.” [19] Therefore, the Supreme Court’s insistence that the Federal Circuit no longer rigidly rely on the TSM test could increase uncertainty in the grant of future patents. However, the Supreme Court’s refusal to completely dismiss the TSM test, while in fact endorsing its continued use, albeit on a less rigid basis, has to be viewed as a profound victory for an industry with a significant stake in maintaining the status quo. Moreover, it is unclear how much the Supreme Court’s holding in KSR Int’l Co. v. Teleflex, Inc. will truly change the legal analysis of the lower courts, given the evidence that lower courts already were independently shifting away from rigid adherence to the TSM test before the Supreme Court’s ruling [20].More importantly, several aspects of the Supreme Court’s reasoning in KSR Int’l Co. v. Teleflex, Inc. seem to directly address relevant concerns of the biotechnology market in favorable ways. First, the Supreme Court made clear that though a product is the result of a combination of elements that were “obvious to try,” it is not necessarily “obvious” under the Patent Act. Retaining the possibility that “obvious to try” inventions still may be patentable is extremely important to the biotechnology industry in particular because “many patentable inventions in biotechnology spring from known components and methodologies found in [the] prior art.” [21] Rather than foreclosing all “obvious to try” inventions as being obvious, and therefore not patentable, the Supreme Court instead explained that where there is “a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions,” it is more likely that a person of ordinary skill would find it obvious to pursue “known options.” [22] Thus, the proper inquiry, as stated by the Supreme Court, is “whether the improvement is more than the predictable use of prior art elements according to their established functions.” [23] While this reasoning may prevent some “obvious to try” inventions from being patented, it is unlikely to have a substantial effect on inventions in the biotechnology market because “most advances in biotechnology are only won through great effort and expense, and with only a low probability of success in achieving the claimed invention at the outset.” [24] In other words, it would be hard to characterize the use of prior art in the biotechnology context as predictable based on the inherent unpredictability of obtaining favorable results. As such, most biotechnology inventions would presumably fall outside the Supreme Court’s “obvious to try” reasoning due to the very nature of the industry, meaning they would remain patentable under the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision.Second, the Supreme Court recognized the “distortion caused by hindsight bias” and the importance of avoiding “arguments reliant upon ex post reasoning,” though it lessened the Federal Circuit’s rigid protection against hindsight bias [24]. Hindsight bias requires that obviousness be viewed at the time the invention was made, because what may seem revolutionary at the time of invention may, upon the passage of time, seem “obvious.” Cognizance of hindsight bias is crucial for biotechnology patents because “there often is a long ‘passage of time between patent application filing and litigation with biotechnology inventions [that] can exacerbate the problem’ of hindsight bias.” [25] The problem is further exacerbated by the “significantly longer durations of commercial utility” biotechnology inventions enjoy as compared to those in other fields [25]. The more time between the filing of a patent and the subsequent litigation over its validity, the greater the risk that “reliable accounts of [the] context” in which the discovery is made will no longer exist [26]. As such, inventions that were not obvious when they were created will be inescapably colored by the passage of time and by new knowledge and discoveries; the likelihood of this occurrence is higher the further removed the litigation is from the patent filing date. Once again, however, it seems clear that despite the Supreme Court’s abandonment of the TSM test’s rigidity, strong protections against hindsight bias still were emphasized in the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision. In fact, lower courts applying KSR Int’l Co. v. Teleflex, Inc. acknowledge they are “cautious” to avoid “using hindsight” in biotechnology obviousness determinations [27].Finally, the Supreme Court seems to believe that the imposition of a more flexible approach will be more likely to benefit markets not directly at issue in KSR Int’l Co. v. Teleflex, Inc. The Supreme Court asserted, “[t]he diversity of inventive pursuits and of modern technology counsels against limiting the analysis” to the rigid TSM test of the Federal Circuit [28]. This language suggests that the Supreme Court expects lower courts to take into consideration the special considerations facing unique markets, such as the biotechnology market. As such, the specific concerns of the biotechnology market discussed above may receive more attention under the flexible framework asserted by the Supreme Court in KSR Int’l Co. v. Teleflex, Inc.Leading up to the oral argument in KSR Int’l Co. v. Teleflex, Inc., there was widespread speculation that the case could result in a watershed moment, significantly altering the definition of obviousness in patent law. For many, including those in the biotechnology industry, there was ample reason to be concerned. Any change in the definition of obviousness would effectively shift property rights from new patent holders to old, or vice versa. However, the Supreme Court acted with restraint. While the decision purports to make substantial changes by doing away with the Federal Circuit’s TSM test, the opinion seems more like a mild-mannered rebuke of lower courts that had become too complacent in the implementation of their beloved test. If anything, the Supreme Court’s insistence on a more flexible formula is simply a call for lower courts to employ common sense, in addition to considering the factors from Graham and the TSM test. Accordingly, the Supreme Court’s opinion in KSR Int’l Co. v. Teleflex, Inc. is unlikely to have a pronounced effect on the biotechnology market, despite the widespread concern generated before the actual decision was handed down.     

PrP assemblies: Spotting the responsible regions in prion propagation

The “protein only” hypothesis states that the key phenomenon in prion pathogenesis is the conversion of the host protein (PrP^C) into a β-sheet enriched polymeric and pathogenic conformer (PrP^Sc). However the region of PrP bearing the information for structural transfer is still controversial. In a recent report, we highlighted the role of the C terminal part i.e., the helixes H2 and H3, using mutation approaches on recombinant PrP. The H2H3 was shown to be the minimal region necessary to reproduce the oligomerization pattern of the full-length protein. The oligomers produced from isolated H2H3 domain presented the same structural characteristics as the oligomers formed from the full-length PrP. Combining other groups'' results, this paper further discusses the relative, direct or indirect role of different PrP regions in assembly. The H2H3 region represents the core of PrP oligomers and fibrils, whereas the N terminus could explain divergences among different aggregates. Finally this review evocates the possibility to separate the domain involved in prion information transference (i.e., prion replication) from the domain bearing the cytotoxicity properties.Key words: prion, H2H3, amyloid, domain of replication, unfolding, strain, polymer, fibersTransmissible spongiform encephalopathies (TSE), fatal neurodegenerative diseases affecting humans and other mammalians, induce in most cases loss of motor control and dementia. PrP is a protein physiologically present in parts of the animal kingdom (in mammals, birds, reptiles and fishes). According to the “protein-only” hypothesis,^1,2 the key phenomenon in the pathogenesis is the conversion of the α-helix rich host-encoded PrP form (PrP^C) into a pathogenic conformer (PrP^Sc) characterized by a higher content in β-sheet and a polymeric state. The conversion to an enriched β-sheet structure is supposed to be due to the modification—induced only by a PrP^Sc-like state acting as a template- of PrP^C into the PrP^Sc conformer. This hypothesis was first proposed by Griffith in 1967³ and revisited by Lansbury et al. in 1993.⁴ The prion hypothesis has now found increasing support from experimental evidence based on the synthetic production of β-sheeted recombinant PrP which shows pathogenic properties in a wide variety of physico-chemical conditions.^5–7 However, the molecular basis of prion conversion remains unclear, especially the various structural landscape of the PrP^Sc, which is the basis of the strain phenomenon.⁸To understand the mechanisms of transfer of the structural information, two mains issues have to be addressed: (1) we need to understand which region(s) of the protein act as template for conversion and (2) what is the “pathogenic” state of this domain. In this review, we shall assume that the region bearing the infectious information for replication and the region responsible for polymerization are identical. However, the link between the propensity of a domain to form aggregates and the ability to contain the necessary information for prion replication is far from being trivial. Generally the formation of amyloid assemblies results from the aggregation of disordered peptides or in some cases from disordered regions of a folded protein.⁸ If we consider that prion replication is only supported by the globular part of PrP⁹ the currently available model involves the folded domain. Since all structural transitions need at least a partial unfolding and refolding process, pre-required structural events should be considered prior to the conversion process.     

Rapid stomatal closure triggered by a short ozone pulse is followed by reopening to overshooting values

Stomatal pores, surrounded by the pairs of guard cells, regulate plant gas exchange. Correct stomatal regulation is crucial for plant survival under various stress conditions. We have recently utilized the air pollutant ozone (O₃) to study stomatal signaling and showed that application of O₃ induces rapid decrease in stomatal conductance. Here we have addressed the recovery of stomatal conductance and show that after exposures of plants to high O₃ pulses stomatal conductance recovered faster, reaching higher, “overshooting” values than were the pre-exposure values. We propose the hypothetical mechanism for this phenomenon and discuss it in the frames of current stomatal signaling models.Key words: ozone, stomata, signaling, Arabidopsis, overshooting, guard cells, stressRapid progress in understanding structural and molecular mechanisms of the core abscisic acid (ABA) signaling pathway and subsequent stomatal closure (reviewed in ref. ¹) has been achieved by using a variety of mostly in vitro technologies and approaches. Data on early induction of stomatal response by a brief ABA pulse in vivo is almost absent, largely due to difficulties in rapid removal of ABA from intact guard cells. Application of O₃, an air pollutant efficiently utilized to study stomatal signaling,^2–4 lacks this disadvantage and allows monitoring stomatal responses to brief, clean-cut, strictly dosed pulses of this powerful oxidant in planta. Application of O₃ for 1 min to intact Arabidopsis rosette triggered a Rapid Transient Decrease (RTD) in stomatal conductance which, after lasting for 8–10 min, was followed by a 3–4 times slower recovery.³ The entire RTD, lasting for up to 40–50 min, is a conserved response in plants; to date it is found to be present in about 90 Arabidopsis ecotypes/mutants³ and also in tobacco and birch (unpublished results). Absence of RTD in protein phosphatase ABI1 and ABI2 mutants (abi1-1 and abi2-1) which are unable to form complex with PYR/PYL ABA receptors, in protein kinase OST1 and in guard cell plasma membrane anion channel SLAC1 mutants, indicates that O₃-triggered signal propagates through the same phosphatase/kinase pair as does the signal triggered by ABA.³ Results of mostly proteomic, pharmacological and electrophysiological studies allow to suggest that the most likely reason for the rapid stomatal closure during RTD is the ABI1, ABI2 and OST1 mediated alterations in a battery of plasma membrane ion channels, including the outward-rectifying anion channel SLAC1 and the depolarization-activated K⁺ channel GORK1 which after their sequential activation result in efflux of osmotica, turgor loss and stomatal closure.Physiological background of the recovery during RTD which takes place also under continuous exposure to ozone² is less understood. To study this process further we exposed whole rosettes of intact 22–25 day old Arabidopsis plants to different O₃ concentrations for 3 min as described earlier³ and observed that after exposures to high concentration O₃ pulses stomatal conductance recovered faster and reached higher values than were the preexposure values. We term this phenomenon “overshooting”.Ozone concentration of 70 nl l⁻¹ did not induce RTD (Fig. 1A). At higher concentrations O₃ induced intense decrease in stomatal conductance within 4–6 min after application. This was followed by rapid stoppage of the closure, a brief transition period and a sluggish, almost linear recovery where the pre-exposure value of stomatal conductance was reached about 30 min after the onset of O₃ (Fig. 1A). The rates and extents of the O₃-induced stomatal closure, as well as rates of reopening were concentration dependent. Continuation of the linear increase in stomatal conductance after reaching the pre-exposure value resulted in almost two-fold higher values at 50 min after the onset of 385 nl l⁻¹ of O₃. Overshootings were dependent on ozone concentration (Fig. 1B) and on the extent of the initial decrease in stomatal conductance (Fig. 1C). Both dependencies were exponential indicating a presence of threshold at 150–200 nl l⁻¹ of O₃ and at 20% of initial O₃-induced decrease in stomatal conductance, respectively.Open in a separate windowFigure 1Ozone-triggered rapid decrease in stomatal conductance is followed by recovery to higher “overshooting” values. (A) Typical asymmetric time patterns of stomatal conductance after exposure of 22–25 day old Arabidopsis plant leaf rosettes to different concentrations of ozone as described in Kollist et al.² In (B and C) O₃-induced “overshooting” is plotted against O₃ concentration and O₃-induced decrease in stomatal conductance, respectively.What could be the reason and mechanistic explanation for described O₃-induced “overshooting” in stomatal conductance? The protein kinase OST1 is required for induction of rapid closure phase of the O₃-triggered RTD.³ Besides phosphorylating SLAC1,^3,5 OST1 has been shown to phosphorylate also the inward-rectifying K⁺ channel KAT1 resulting in its inhibition.⁶ Inhibition of K⁺ uptake, which allows faster membrane depolarization and stomatal closure, has been shown to occur under various stresses.⁷ Presumably, H⁺-ATPase activity and proton pumping, tightly coupled to K⁺ uptake via channel energization⁸ are also suppressed by O₃. It has been shown that in depolarized guard cell, plasma membrane proton pumping may precede volume and turgor increase.⁹ We speculate that in the O₃-triggered, SLAC1- and GORK-mediated stomatal closure, when ion efflux and turgor loss proceed at high rates, reactivation of H⁺-ATPase and proton pumping and associated recovery of K⁺ uptake are induced to avoid guard cell plasmolysis.¹⁰ Guard cells begin to regain turgor and stomata reopen. At the same time outward-rectifying ion channels are transiently locked (inactivated) as stomata become completely insensitive to repeated O₃-pulses during recovery phase.³ This interpretation is supported by our observation that the recovery in stomatal opening is heavily suppressed in kincless mutant³ where the inward rectifying K⁺ current is abolished.¹¹ In addition, peak densities of inward K⁺ currents (2–4 µA/cm² membrane⁹) are shown to be much lower than those for outward anion and K⁺ currents (17–20 µA/cm).^2,8,12,13 This could be a reason why stomatal reopening is much slower than the initial O₃-induced closure. Our findings (Fig. 1) suggest that the faster and deeper the O₃-triggered turgor loss, the faster and extensive is its recovery. The “overshootings” suggest plasma membrane hyperpolarization and predict a viable oscillation-like stomatal behavior where the system tends to restore the initial equilibrium. Longer experiments are needed to address whether such an oscillating response exists in Arabidopsis elicited by O₃.Taken together, our data suggest the presence of a “security” mechanism in plant guard cells which avoids the excessive dehydration and precipitous turgor loss by reswitching the reaccumulation of osmotica ultimately leading to stomatal opening. Molecular mechanism(s) linking feedback from low turgor to activation of plasma membrane proton pumping and subsequent ion uptake are obscure. Irrespective of mechanism(s), our data indicate that stomata tend to recover from stress the faster the stronger has been the perturbation at its onset. Undoubtedly, rapid O₃-induced transient decrease in stomatal conductance is one of countless expressions of the Le Chatelier''s principle having numerous wordings like: “any change in status quo prompts an opposing reaction in the responding system,” or paraphrased on the basis of our results—the stronger the stimulus (O₃ concentration) the stronger the response (“overshooting”).     

Reviewing once more the c-myc and Ras collaboration: Converging at the cyclin D1-CDK4 complex and challenging basic concepts of cancer biology

The c-myc is a proto-oncogene that manifests aberrant expression at high frequencies in most types of human cancer. C-myc gene amplifications are often observed in various cancers as well. Ample studies have also proved that c-myc has a potent oncogenicity, which can be further enhanced by collaborations with other oncogenes such as Bcl-2 and activated Ras. Studies on the collaborations of c-myc with Ras or other genes in oncogenicity have established several basic concepts and have disclosed their underlying mechanisms of tumor biology, including “immortalization” and “transformation”. In many cases, these collaborations may converge at the cyclin D1-CDK4 complex. In the meantime, however, many results from studies on the c-myc, Ras and cyclin D1-CDK4 also challenge these basic concepts of tumor biology and suggest to us that the immortalized status of cells should be emphasized. Stricter criteria and definitions for a malignantly transformed status and a benign status of cells in culture also need to be established to facilitate our study of the mechanisms for tumor formation and to better link up in vitro data with animal results and eventually with human cancer pathology.Key words: c-Myc, Cyclin D1, transformation, immortalization, oncogeneC-myc is the first proto-oncogene discovered and is known to participate in many cellular functions,¹ including maintenance of stem cell properties.² Most types of human cancer manifest aberrant expression of c-myc at high frequencies, and gene amplification occurs in many cases of various cancers as well. Ample studies have demonstrated that c-myc has a potent oncogenicity, which can be further enhanced by collaborations with other oncogenes such as a Ras mutant or with many extracellular growth stimuli that activate Ras, such as epidermal growth factor (EGF) or transforming growth factor α (TGFα). Studies on the collaborations of c-myc with Ras and other genes have provided us with mechanistic details behind several basic concepts of cancer biology, including the “two-hit principle”,³ “immortalization” and “transformation”. In the meantime, however, many results from these studies also challenge these basic concepts and thus confuse us. We now discuss the data on the collaborations of c-myc with Ras and other genes and present a perspective that these collaborations may converge at the cyclin D1-CDK4 complex. We also appeal to emphasize the importance of an immortalized status of cells and to establish stricter criteria to better define a transformed and benign statuses, so as to better connect in vitro results with animal data and with human cancer pathology.     

Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins—the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.Key words: integrin, fibronectin, migration, cytoskeleton, dynamicsCells attach to the extracellular matrix (ECM) that surrounds them in specialized structures termed “cell-matrix adhesions.” These come in different flavors including “focal complexes” (small adhesions found in membrane protrusions of spreading and migrating cells), “focal adhesions” (larger adhesions connected by F-actin stress fibers that are derived from focal complexes in response to tension), “fibrillar adhesions” (elongated adhesions associated with fibronectin matrix assembly), and proteolytically active adhesions termed “podosomes” or “invadopodia” found in osteoclasts, macrophages and certain cancer cells. Common to all these structures is the local connection between ECM proteins outside- and the actin cytoskeleton within the cell through integrin transmembrane receptors. The intracellular linkage to filamentous actin is indirect through proteins that concentrate in cell-matrix adhesions such as talin, vinculin, tensin, parvins and others.¹Cell migration is essential for embryonic development and a number of processes in the adult, including immune cell homing, wound healing, angiogenesis and cancer metastasis. In moving cells, cell-matrix adhesion turnover is spatiotemporally controlled.² New adhesions are made in the front and disassembled in the rear of cells that move along a gradient of motogenic factors or ECM proteins. This balance between formation and breakdown of cell-matrix adhesions is important for optimal cell migration. Several mechanisms regulate the turnover of cell-matrix adhesions. Proteolytic cleavage of talin has been identified as an important step in cell-matrix adhesion disassembly³ and FAK and Src family kinases are required for cell-matrix adhesion turnover and efficient cell migration.^4,5 Besides regulating phospho-tyrosine-mediated protein-protein interactions within cell-matrix adhesions, the FAK/Src complex mediates signaling downstream of integrins to Rho GTPases, thus controlling cytoskeletal organization.^6,7 The transition from a stationary to a motile state could involve (local) activation of such mechanisms.Interestingly, conditions of increased cell migration (development, wound healing, angiogenesis, cancer metastasis) are accompanied by shifts in integrin expression with certain integrins being lost and others gained. Most ECM proteins can be recognized by various different integrins. For instance, the ECM protein, fibronectin (Fn) can be recognized by nine different types of integrins and most of these bind to the Arg-Gly-Asp (RGD) motif in the central cell-binding domain. Thus, cell-matrix adhesions formed on Fn contain a mixture of different integrins and shifts in expression from one class of Fn-binding integrins to another will alter the receptor composition of such adhesions. This may provide an alternative means to shift from stationary to motile.Indeed, we have found that the type of integrins used for binding to Fn strongly affects cell migration. We made use of cells deficient in certain Fn-binding integrins and either restored their expression or compensated for their absence by overexpression of alternative Fn-binding integrins. This allowed us to compare in a single cellular background cell-matrix adhesions containing α5β1 to those containing αvβ3. Despite the fact that these integrins support similar levels of adhesion to Fn, only α5β1 was found to promote a contractile, fibroblastic morphology with centripetal orientation of cell-matrix adhesions⁸ (Fig. 1). Moreover, RhoA activity is high in the presence of α5β1 and these cells move in a random fashion with a speed of around 25 mm/h. By contrast, in cells using αvβ3 instead, adhesions distribute across the ventral surface, RhoA activity is low, and these cells move with similar speed but in a highly persistent fashion.^8,9 Finally, photobleaching experiments using GFP-vinculin and GFP-paxillin demonstrated that cell-matrix adhesions containing α5β1 are highly dynamic whereas adhesions containing αvβ3 are more static.⁹Open in a separate windowFigure 1Immunofluorescence images. GE11 cells, epithelial β1 knockout cells derived from mouse embryos chimeric for the integrin β1 subunit endogenously express various av integrins, including low levels of αvβ3 and αvβ5. Ectopic expression of β1 leads to expression of α5β1 and induced α5β1-mediated adhesion to Fn (left image) whereas ectopic expression of β3 (in the β1 null background) leads to strong expression of αvβ3 and induced αvβ3-mediated adhesion to Fn (right image). Adhesions containing either α5β1 or αvβ3 show distinct distribution and dynamics (paxillin; green) and cause different F-actin organization (phalloidin; red). Cartoons: Differences in cell-matrix adhesion dynamics may be explained by differential binding of soluble Fn molecules (blue) or different molecular determinants of the interaction with immobilized Fn (red). See text for details.It has been observed that α5β1 and αvβ3 use different recycling routes. Interfering with Rab4-mediated recycling of αvβ3 causes increased Rab11-mediated recycling of α5β1 to the cell surface. In agreement with our findings, the shift to α5β1 leads to increased Rho-ROCK activity and reduced persistence of migration.¹⁰ One possible explanation for the different types of migration promoted by these two Fn-binding integrins might involve different signaling and/or adaptor proteins interacting with specific amino acids in their cytoplasmic tails. However, this appears not to be the case: α5β1 in which the cytoplasmic tails of α5 or β1 are replaced by those of αv or β3, respectively, behaves identical to wild type α5β1: it promotes a fibroblast-like morphology with centripetal orientation of cell-matrix adhesions and it drives a non-persistent mode of migration.^8,11 Together, these findings point to differences between α5β1 and αvβ3 integrins in the mechanics of their interaction with Fn, which apparently modulates intracellular signaling pathways in control of cell-matrix adhesion dynamics and cell migration.How might this work? It turns out that although α5β1 and αvβ3 similarly support cell adhesion to immobilized (stretched) Fn, only α5β1 efficiently binds soluble, folded (“inactive”) Fn.¹¹ We have proposed that such interactions with soluble Fn molecules (possibly secreted by the cell itself) may weaken the interaction with the immobilized ligand thereby causing enhanced cell-matrix adhesion dynamics in the presence of α5β1,¹¹ (Fig. 1). Preferential binding of soluble Fn by α5β1 could be explained by differences in accessibility of the RGD binding pocket between α5β1 (more exposed) and αvβ3 (more hidden) as suggested by others.¹² If this is the case, immobilization (“stretching”) of Fn apparently leads to reorientation of the RGD motif in such a way that it is easily accessed by both integrins.The issue is considerably complicated by the fact that other recognition motifs are present in the Fn central cell-binding domain. In addition to the RGD sequence in the tenth Fn type 3 repeat (IIIFn10), binding of α5β1, but not αvβ3, also depends on the PHSRN “synergy” sequence in IIIFn9.^13–15 The relative contribution of these motifs is controversial and there is structural data pointing either towards a model in which IIIFn9 interacts with α5β1 or towards a model in which IIIFn9 exerts long-range electrostatic steering resulting in a higher affinity interaction without contacting the integrin.^16,17 Cell adhesion studies have suggested that an interaction of α5β1 with the synergy region stabilizes the binding to RGD.^14,18 Such a two-step interaction may facilitate binding to full length, folded Fn for instance by altering the tilt angle between IIIFn9 and IIIFn10 leading to optimal exposure of the RGD loop, perhaps explaining why αvβ3 (which may not interact with the synergy site) poorly binds soluble Fn.Others have shown that the RGD motif alone is sufficient for mechanical coupling of αvβ3 to Fn whereas the synergy region is required to provide mechanical strength to the α5β1-Fn bond.¹⁹ It appears that the interaction of α5β1 with Fn is particularly dynamic with various conformations of α5β1 interacting with different Fn binding surfaces, including the RGD and synergy sequences as well as other regions in IIIFn9. Thus, besides the above model based on differential binding to soluble Fn molecules, differences in the complexity and dynamics of interactions with immobilized Fn that determine functional binding strength could also underlie the different dynamics of cell-matrix adhesions containing either α5β1 or αvβ3 (Fig. 1).Precisely how mechanical differences in receptor-ligand interactions result in such remarkably distinct cellular responses is poorly understood. In addition to effects on cell-matrix adhesion dynamics and cytoskeletal organization it is also associated with different activities of Rho GTPases, indicating that mechanical differences between these two integrins must translate into differential activation of intracellular signaling pathways.^8,9,11 Possibly, different adhesion dynamics due to distinct mechanisms of receptor-ligand interaction result in different patterns of F-actin organization, which, in turn, affects the formation of signaling platforms. It is also possible that differences in the extent of integrin clustering have an impact on the conformation of one or more cytoplasmic components of the cell-matrix adhesions containing either α5β1 or αvβ3. This could lead to hiding or exposing binding sites for signaling molecules (e.g., upstream regulators of Rho GTPases) or substrates. Whatever the mechanism involved, altering the integrin composition of cell-matrix adhesions through shifts in integrin expression as observed during development, angiogenesis, wound healing and cancer progression may be a driving force in the enhanced cell migration that characterizes those processes.     

2009 Society for Medical Anthropology Conference: Global Public Health through the Eyes of Medical Anthropologist Didier Fassin

At the 2009 Society for Medical Anthropology Conference at Yale University, anthropologist Didier Fassin discussed social inequality and the politicization of health in the context of global public health.U.S. Rep. Joe Wilson shouted, “You lie!” during President Obama’s denial that the proposed health care reform bill would cover illegal immigrants, and anthropologist Didier Fassin used that antagonistic stance toward what the 1978 Declaration of Alma-Ata [1] had called a fundamental human right to best illustrate the issues of social inequality and the politicization of health.Global public health was one focus of the 2009 Society for Medical Anthropology Conference at Yale University in September. Since its inception in 1948, the World Health Organization (WHO) has striven to provide health assistance to the world population, especially those in developing countries. But Fassin, professor of social science at the Institute for Advanced Study at Princeton, professor of sociology at the Université de Paris, Nord, and director of studies in political and moral anthropology at the Ecole des Hautes Études en Sciences Sociales, argued that the concept of global health, albeit well-meaning, is problematic. Its utopian nature is clearly apparent in the rhetoric of politicians, he said, adding that health as a gift of nature, a common good, and the core of the WHO, quickly becomes an object of politics and the coverage in times of sickness of a select few is akin to entitlement and privilege.The present age of globalization certainly makes health threats such as epidemics a threat to all, and nations are in it together to take preventive measures or put up a concerted fight. However, threats like bioterrorism or predicted consequences of global warming such as population migration may be viewed, particularly by Western countries, as security issues that menace national interests and state sovereignties. The consequence being that new policies are implemented that may directly or indirectly affect the rest of the world population.And then there is the issue of humanitarian intervention, which Fassin refers to as “politics of life” [2]. How can we view humanitarianism with the eye of a cynic when it is, in essence, the effort to demonstrate the very best of our nature? Yet the transformation of some humanitarian interventions into military operations and the decision to intervene (Iraq, Kosovo, Bosnia) or not (Rwanda, Ethiopia, Cambodia), politicize this notion. Additionally, Fassin believes that the key nation-states integrate their own cultural and political biases during interventions in troubled regions.Nowhere is this subjectivity more apparent than in the image of suffering as depicted by psychologists and psychiatrists working for non-government organizations (NGOs). NGOs compile testimonies of traumatized people in war and conflict zones, but their subjective narratives enmeshed in the diagnosis reports are increasingly supplanting faithful witness accounts. Fassin sees this trauma as “political expression of the world” [3]. The experts, in trying to raise awareness on issues that need immediate attention, may dramatize certain situations or get emotionally involved during their missions and take sides. They become the new voice of the conflict and their efforts may throw the victims into a state of confusion.It’s no surprise then that some nations view with distrust Western practices and their portrayal of aggressors and victims [4]. In 2000, Thabo Mbeki, then president of South Africa, convened an advisory panel that aimed to collect scientific data to prove that HIV does not cause AIDS. In return, he received the Durban Declaration with the signatures of more than 5,000 scientists and doctors who unilaterally declared the opposite to be scientifically true.Fassin brings up the abovementioned issues in order to shift attention to the difficulties that face our common efforts for better health services. It is truly challenging for Western leaders to mend the rift between their political agendas and accessible health for all, and as long as that continues to be the case, health care will elude millions.     

Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge,Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.Enhanced biological phosphorus removal (EBPR) has been applied in many wastewater treatment plants to reduce the phosphorus that causes eutrophication in surface waters. EBPR employs polyphosphate-accumulating organisms (PAOs), which are enriched through alternating aerobic-anaerobic cycles (34). Since PAOs are essential for an understanding of EBPR, many candidates have been proposed as potential PAOs, such as Acinetobacter spp. (11), Tetrasphaera spp. (31), Microlunatus phosphovorus (36), Lampropedia spp. (40), and Gram-positive Actinobacteria (24). However, those organisms do not exhibit all of the characteristics of the EBPR biochemistry model. Recently developed culture-independent approaches such as PCR-clone libraries, fluorescence in situ hybridization (FISH), and microautoradiography (MAR) have highlighted an uncultured Rhodocyclus-related bacterium, “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), as one of the most important PAO candidates (2, 5, 16, 22, 23, 27, 28, 47).Numerous studies have sought to investigate uncultured “Ca. Accumulibacter” and have shown the presence of genetically and physiologically different members with a global geographic distribution (3, 9, 22, 27, 39). For example, Kong et al. (22) identified two morphologically different “Ca. Accumulibacter” cells of small cocci and large coccobacilli labeled with PAOmix (PAO462, PAO651, and PAO846) in laboratory-scale EBPR reactors. Additional results showing phenotypic and morphological diversities of “Ca. Accumulibacter” cells also existed with respect to the different roles of denitrifying PAO (DPAO) in the EBPR process (3, 9, 23). Carvalho et al. (3) detected two different morphotypes of “Ca. Accumulibacter” with different nitrate reduction capabilities. The presence of other “Ca. Accumulibacter” strains with 15% genome sequence divergence from the dominant strains in metagenomic analyses is likely to explain these morphological and phenotypic differences (12). McMahon et al. (33) suggested the use of the polyphosphate kinase (ppk) gene, which is involved in the production of polyphosphate, for a finer elucidation of “Ca. Accumulibacter” diversity. He et al. (15) grouped “Ca. Accumulibacter” strains into five distinct clades, designated clades I, IIA, IIB, IIC, and IID, using ppk gene sequence information. Flowers and colleagues (9) previously reported that “Ca. Accumulibacter” cells of clade IA had nitrate reduction activity with phosphorus uptake but that “Ca. Accumulibacter” cells of clade IIA did not.FISH-fluorescence activated cell sorting (FACS) techniques have been used for the separation of specific microbial cells from complex microbial consortia and their metabolic gene analysis (14, 46). For example, Miyauchi et al. (35) sorted PAOmix probe-labeled “Ca. Accumulibacter” cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity. In the current study, we found that four different “Ca. Accumulibacter” clades (Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4) were present in the EBPR sludge of laboratory-scale reactors supplied with acetate as the sole carbon source. We analyzed their morphological characteristics and ppk gene sequence information using a suite of FISH and FACS approaches and linked fine-scale phylogenetic diversities of “Ca. Accumulibacter” strains with their morphological characteristics and metabolic genes. This study will be useful for further genetic and physiological studies of different “Ca. Accumulibacter” clades.     

Electrophysiological approach to examine a putative cold- and menthol-sensitive channel in the liverwort Conocephalum conicum

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization.^1,2 The nature of the primary step of its action (alteration in membrane composition,³ transient influx of Ca²⁺ etc.,²) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling⁴ but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca²⁺ and generation of AP series.⁵ TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.Key words: action potential, cold, liverwort, menthol, thermoreceptors, voltage transient