Population Bottlenecks and Pathogen Extinction: “Make This Everyone's Mission to Mars,Including Yours”

Kapusinszky et al. (J Virol 89:8152–8161, 2015, http://dx.doi.org/10.1128/JVI.00671-15) report that host population bottlenecks may result in pathogen extinction, which provides a compelling argument for an alternative approach to vaccination for the control of virus spread. By comparing the prevalence levels of three viral pathogens in two populations of African green monkeys (AGMs) (Chlorocebus sabaeus) from Africa and two Caribbean Islands, they convincingly show that a major host bottleneck resulted in the eradication of select pathogens from a given host.     

Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?

The aim of this review is to give a broad picture of what is actually known about the synaptotagmin family. Synaptotagmin I is an abundant synaptic vesicle and secretory granule protein in neurons and endocrine cells which plays a key role in Ca(2+)-induced exocytosis. It belongs to the large family of C2 domain-proteins as it contains two internal repeats that have homology to the C2 domain of protein kinase C. Eleven synaptotagmin genes have been described in rat and mouse. Except for synaptotagmin I, and by analogy synaptotagmin II, the functions of these proteins are unknown. In this review we focus on data obtained on the various isoforms without exhaustively discussing the role of synaptotagmin I in neurotransmission. Numerous in vitro interactions of synaptotagmin I with key components of the exocytosis-endocytosis machinery have been reported. Additional data concerning the other synaptotagmins are now becoming available and are reviewed here. Only interactions which have been described for several synaptotagmins, are mentioned. It is unlikely that a single isoform displays all of these potential interactions in vivo and probably the subcellular distribution of the protein will favor some of them and preclude others. Therefore, to discuss the putative role of the various synaptotagmins we have examined in detail published data concerning their localization.     

A Nobel Prize for membrane traffic: Vesicles find their journey’s end

Cell biologists everywhere rejoiced when this year’s Nobel Prize in Physiology or Medicine was awarded to James Rothman, Randy Schekman, and Thomas Südhof for their contributions to uncovering the mechanisms governing vesicular transport. In this article, we highlight their achievements and also pay tribute to the pioneering scientists before them who set the stage for their remarkable discoveries.In 1974, nearly 40 years ago, the Nobel Prize in Physiology or Medicine was awarded to George E. Palade, Albert Claude, and Christian de Duve for work that effectively established a new field, cell biology. Collectively, the efforts of these three pioneers not only defined the essential features of cells but also how to study them. Correlating morphological observations by electron microscopy with biochemical analysis enabled not only the identification of nearly every major organelle in the eukaryotic cell (although endosomes were missed at that time) but also what their respective functions were. Palade’s efforts demonstrated the now-canonical pathway of protein secretion: synthesis in the endoplasmic reticulum (ER), oligosaccharide processing in the Golgi complex, concentration in secretory granules, and release at the plasma membrane. Palade understood implicitly that the ER, Golgi, secretory granules, and plasma membrane had to be interconnected by a series of vesicular carriers that carried cargo from one station to the next—dissociative transport. He also appreciated that the process had to be regulated if compartment specificity was to be maintained. The need for specificity defined the next major conceptual challenges: how do proteins intended for secretion traverse the compartments of the secretory pathway, how are transport vesicles formed, how do vesicles recognize their appropriate destinations, how does fusion occur after the appropriate destination is reached, and, finally, how are the components from the originating compartment returned or recycled to their sites of origin after fusion with the destination compartment? Palade may have framed these problems, but it was left to the next generation of cell biologists to solve them.This year’s Nobel Prize in Physiology or Medicine awarded to James Rothman, Randy Schekman, and Thomas Südof recognizes a truly remarkable body of work that provides superb conceptual clarity and mechanistic insight into virtually all of the issues defined by Palade and colleagues. To a large extent, the award also provides a satisfying degree of recognition to the large community of scientists who established the field of “molecular” cell biology. But it was the intellectual leadership, passion, and courage provided by this year’s awardees (Figs. 1 and ​and2)2) that played a major role in driving the spectacular advances of the past three decades. Particularly in the case of Rothman and Schekman, the scientific dynamic they helped to generate gave the field focus and excitement, from which came great things. The elegance of their experiments together with the exceptionally clear and simple logic that they presented in their papers moved the field ahead quickly and drew many new converts into membrane trafficking.Open in a separate windowFigure 1.Randy Schekman and James Rothman (center) with many of their former trainees at the American Society for Biochemistry and Molecular Biology meeting on “Biochemistry of Membrane Traffic: Secretory and Endocytic Pathways,” October 2010.PHOTOGRAPH COURTESY OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGYOpen in a separate windowFigure 2.Thomas Südhof (top row, center) and his laboratory circa 1993.PHOTOGRAPH COURTESY OF THOMAS SÜDHOFThe first foray into a mechanistic, molecular approach to the cell biological problems defined by Palade was really due to the work of Günter Blobel and his colleagues Peter Walter and Bernhard Dobberstein working at The Rockefeller University. These investigators devised a complex but elegant approach enabling the cell-free reconstitution of the first step of secretion, namely the insertion of newly synthesized proteins into and across the ER membrane. Combined with conventional cold-room biochemistry, Blobel and others were able to provide a detailed understanding of the biochemistry of protein translocation. Blobel was duly awarded the Nobel Prize in Physiology or Medicine for his work in 1999. Influenced by Blobel and also Arthur Kornberg, then chair of the Biochemistry Department at Stanford, Jim Rothman (who was a young faculty member at Stanford in the early 1980s) initiated his courageous effort aimed to reconstitute subsequent steps, namely the transport of secretory and membrane protein cargo to and through the Golgi complex. As is often the case with innovative work that pushes the limits of knowledge, Rothman’s interpretations were on occasion controversial, but there was absolutely no controversy regarding the importance of the various components he and his team identified. These components included soluble factors needed for vesicle formation in the Golgi as well as for vesicle fusion, most notably the COPI coat protein complex, NSF (NEM-sensitive factor) and SNAP (soluble NSF attachment protein). With Richard Scheller, Rothman recognized that the synaptic vesicle–associated proteins cloned and purified by Scheller represented both the docking sites for NSF and SNAP and a key component of the mechanism whereby vesicles recognized and even fused with each other. Indeed, the SNAREs (as these proteins are now called) clearly comprise the core fusion machinery that underlies virtually all membrane fusion events in the cell. SNAREs form a family of proteins that are organelle specific, helping to ensure the specificity of membrane traffic as well as the biochemical and functional identity of individual membrane compartments.If Rothman’s work began as a quintessential biochemical approach, Randy Schekman’s started at the other end of the spectrum: genetics. Again with great courage, Schekman decided to use the yeast Saccharomyces cerevisiae as a genetically tractable eukaryote to dissect the steps and various components associated with the secretory pathway. At the time, few thought that yeast cells were capable of higher-order processes such as secretion or that their activities had anything to do with mechanisms in animal cells. Yet Schekman and his then graduate student Peter Novick designed a deceptively simple screen to identify secretory (or “sec”) mutants. Their approach was to look for cells that could not secrete by reasoning that continued synthesis of secretory cargo would render the mutant cells more dense. The approach worked, and literally dozens of mutants were discovered, a large number of which could be shown to generate intriguing phenotypes and to control key steps in the secretory, or sometimes even the endocytic, pathway. Although the original sec screens done by Schekman and colleagues did not immediately turn up the SNARE proteins, they did reveal the presence of small Ras-related monomeric GTPases of the Rab family that helped enforce the specificity of vesicle interactions. They also uncovered cytoplasmic coat proteins (COPII) and complex cytosolic “tethers” that serve to gather vesicles at their targets before the final fusion step. When an increasing number of sec mutants began to overlap with components identified by Rothman’s independent biochemical purifications of components required for fusion or vesicle budding, it was clear that both groups (and indeed the field) were on the right track and the transport machinery was universal. Through whatever controversies bubbled up over the years, this basic fact remained unchallenged. Schekman too moved toward the same type of functional biochemical analysis championed by Rothman, and the circle was completed.Focused on one of the key problems in neurobiology, Thomas Südhof’s efforts may appear less general but are no less important. The synapse represented a special case in the area of membrane traffic since the realization that neuronal transmission reflected the release of quanta of neurotransmitters due to the action potential–triggered fusion of synaptic vesicles with the presynaptic plasma membrane. The work of Cesare Montecucco and colleagues on bacterial toxins provided an important insight, namely that synaptic vesicle release can be blocked by certain bacterial toxins (e.g., botulinum toxin) that act as specific SNARE proteases. Scheller and Rothman had shown that the SNAREs comprised the basic unit of the fusion machinery, but this insight alone did not explain how secretion in the synapse was coupled so tightly to electrical activity. Thomas Südhof’s remarkable body of work, although not growing out of the molecular cell biology community as much as the neuroscience community, provided the conceptual answer: the synaptotagmins. These proteins were found to associate with SNAREs and serve as Ca²⁺-sensing triggers that temporally linked synaptic vesicle transmission to individual neuronal impulses. In addition, Südhof and colleagues discovered Munc18 in the mouse, which corresponds to the yeast Sec1 protein, and demonstrated that it interacts with the SNARE complex, revealing that Munc18 as well as other members of the Sec1/Munc18-like protein family function as part of the vesicle fusion machinery.Collectively, these are remarkable achievements that provide conceptual and mechanistic understanding of basic cellular processes at the most fundamental level. It is certainly the case that others, for example Scheller and Novick mentioned here, might just as easily have been included in this award. Regrettably only three are permitted, and there can be no doubt but that the three selected are entirely deserving given not only the nature of their findings but also the scientific leadership they contributed in a myriad of intangible ways to the incredible progress we have witnessed in the post-Palade era of cell biology.We congratulate our colleagues and friends Jim, Randy, and Thomas for this well-deserved honor. Mazal tov!     

Health Professionals “Make Their Choice”: Pharmaceutical Industry Leaders’ Understandings of Conflict of Interest

Conflicts of interest, stemming from relationships between health professionals and the pharmaceutical industry, remain a highly divisive and inflammatory issue in healthcare. Given that most jurisdictions rely on industry to self-regulate with respect to its interactions with health professionals, it is surprising that little research has explored industry leaders’ understandings of conflicts of interest. Drawing from in-depth interviews with ten pharmaceutical industry leaders based in Australia, we explore the normalized and structural management of conflicts of interest within pharmaceutical companies. We contrast this with participants’ unanimous belief that the antidote to conflicts of interest with health professionals were “informed consumers.” It is, thus, unlikely that a self-regulatory approach will be successful in ensuring ethical interactions with health professionals. However, the pharmaceutical industry’s routine and accepted practices for disclosing and managing employees’ conflicts of interest could, paradoxically, serve as an excellent model for healthcare.     

Membrane traffic: do cones mark sites of fission?

Membrane fission occurs in eukaryotic cells whenever a vesicle is produced or a larger subcellular compartment is divided into smaller discrete units. Recent evidence suggests this fission event is promoted by enzymes that generate phosphatidic acid and thereby cause a distortion of the lipid bilayer.     

Apparent “mild depolarization of the inner mitochondrial membrane” as a result of a possible generation of the outer membrane potential

Recently reported kinase-linked mild depolarization of mitochondria, which prevents the generation of the reactive oxygen species (ROS) and disappears in various organs of the old mice, has been assumed to represent a crucial component of the mitochondrial anti-aging program. To measure mitochondrial inner membrane potential (IMP), the authors used fluorescent probe safranin O⁺. It is widely accepted that the accumulation of such cationic probes in the mitochondrial matrix depends exclusively on IMP, thus completely ignoring the possibility of the outer membrane potential (OMP) generation. However, computational analysis performed in the presented work suggests that the kinase-linked generation of the positive OMP might take place under the described conditions, because the measured potential includes the algebraic sum of both IMP and OMP. Alternatively to the suggested mild depolarization of mitochondria, the reported experimental data might reflect mainly a change of the positive OMP generated by the VDAC-kinase complexes. We also demonstrate that the reported in the literature mitochondrial hyperpolarization induced by erastin (known to prevent VDAC-tubulin interactions) and the depolarization caused by the mitochondrial VDAC knockdowns in the cancer cells might actually represent a decrease or increase, respectively, of the magnitude of the kinase-linked positive OMP. This is consistent with our hypothesis that VDAC voltage gating by the kinase-linked metabolically-dependent OMP plays a very important physiological role in regulating the cell energy metabolism under normal and pathological conditions, in the maintenance of the cell death resistance and even in the genetic aging program.     

Pathogen recognition and development of particulate vaccines: does size matter?

The use of particulate carriers holds great promise for the development of effective and affordable recombinant vaccines. Rational development requires a detailed understanding of particle up-take and processing mechanisms to target cellular pathways capable of stimulating the required immune responses safely. These mechanisms are in turn based on how the host has evolved to recognize and process pathogens. Pathogens, as well as particulate vaccines, come in a wide range of sizes and biochemical compositions. Some of these also provide 'danger signals' so that antigen 'senting cells (APC), usually dendritic cells (DC), acquire specific stimulatory activity. Herein, we provide an overview of the types of particles currently under investigation for the formulation of vaccines, discuss cellular uptake mechanisms (endocytosis, macropinocytosis, phagocytosis, clathrin-dependent and/or caveloae-mediated) for pathogens and particles of different sizes, as well as antigen possessing and presentation by APC in general, and DC in particular. Since particle size and composition can influence the immune response, inducing humoral and/or cellular immunity, activating CD8 T cells and/or CD4 T cells of T helper 1 and/or T helper 2 type, particle characteristics have a major impact on vaccine efficacy. Recently developed methods for the formulation of particulate vaccines are presented in this issue of Methods, showcasing a range of "cutting edge" particulate vaccines that employ particles ranging from nano to micro-sized. This special issue of Methods further addresses practical issues of production, affordability, reproducibility and stability of formulation, and also includes a discussion of the economic and regulatory challenges encountered in developing vaccines for veterinary use and for common Third World infectious diseases.     

PCR detection of cryptosporidium: the way forward?

In this article, Una Morgan and Andrew Thompson briefly review the latest information on polymerase chain reaction (PCR)detection of Cryptosporidium parvum in both clinical and environmental samples. Current detection methods for Cryptosporidium are cumbersome, time-consuming and lack sensitivity. A variety of PCR tests have been described recently in the literature and this article discusses the advantages and disadvantages of each new technique and their potential for future diagnosis of Cryptosporidium.     

Insulin–eukaryotic model membrane interaction: Mechanistic insight of insulin fibrillation and membrane disruption

Injection of exogenous insulin in the subcutaneous mass has been a proven therapy for type II diabetes. However, chronic administration of insulin often develops local amyloidosis at the injection site, pathologically known as “Insulin Ball”. This reduces the insulin bioavailability and exacerbates the disease pathology. Thus, the molecular interaction between insulin and the recipient's membrane surface plays a co-operative role in accelerating the amyloidosis. This interaction, however, is different from the molecular interaction of insulin with the native membranous environment of the pancreatic β-cells. The differential membrane mediated interaction that directly affects the aggregation kinetics of insulin remains elusive yet intriguing to understand the mechanism of pathological development. In this study we have characterized the interactions of insulin at different states with model eukaryotic membranes using high and low-resolution spectroscopic techniques in combination with microscopic investigation. Our results show that insulin amyloid intermediates are capable of interacting with model membranes with variable functional affinity towards the different compositions. Fluorescence correlation spectroscopy confirms the aggregation states of insulin in presence of the eukaryotic model membranes while solid-state NMR spectroscopy in conjugation with differential scanning calorimetry elucidates the molecular interaction of insulin intermediates with the lipid head groups along with the acyl chains. Additionally, dye leakage assays support the eukaryotic model membrane disruption by insulin intermediates, similar to hIAPP and Aβ40, as previously reported. Thus, the present study establishes the distinct mode of interactions of insulin amyloid with pancreatic β-cell and general mammalian cell mimicking membranes.     

Putative Phage Hyperparasite in the Rickettsial Pathogen of Abalone, “Candidatus Xenohaliotis californiensis”

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host–parasite interactions and associated microbial community structure. The marine rickettsial organism, “Candidatus Xenohaliotis californiensis” (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9–99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are “Candidatus Xenohaliotis californiensis,” and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.     

Branching dendrites with resonant membrane: a “sum-over-trips” approach

Dendrites form the major components of neurons. They are complex branching structures that receive and process thousands of synaptic inputs from other neurons. It is well known that dendritic morphology plays an important role in the function of dendrites. Another important contribution to the response characteristics of a single neuron comes from the intrinsic resonant properties of dendritic membrane. In this paper we combine the effects of dendritic branching and resonant membrane dynamics by generalising the “sum-over-trips” approach (Abbott et al. in Biol Cybernetics 66, 49–60 1991). To illustrate how this formalism can shed light on the role of architecture and resonances in determining neuronal output we consider dual recording and reconstruction data from a rat CA1 hippocampal pyramidal cell. Specifically we explore the way in which an I _h current contributes to a voltage overshoot at the soma.     

Staphylococcus aureus enterotoxins A− and B: binding to the enterocyte brush border and uptake by perturbation of the apical endocytic membrane traffic

Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich “terminal web” region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria.     

Parasite regulation by host hormones: an old mechanism of host exploitation?

Recent experimental evidence suggests that parasites can not only evade immune responses actively but also exploit the hormonal microenvironment within the host to favor their establishment, growth and reproduction. The benefit for parasites of hormonal exploitation is so great that they have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates that can bind to the hormonal metabolites synthesized by the host. This strategy is exemplified by two parasites that respond to adrenal steroids and sexual steroids, respectively: Schistosoma mansoni and Taenia crassiceps. Understanding how the host endocrine system can, under certain circumstances, favor the establishment of a parasite, and characterizing the parasite hormone receptors that are involved might aid the design of hormonal analogs and drugs that affect the parasite exclusively.