A strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma

Many of the emerging technologies for the global evaluation of gene expression, at both the RNA and protein level, are being applied to the problem of finding biomarkers for human disease progression. These analyses can be made difficult, however, by variation between samples that arises from both technical and nondisease related physiological or genetic causes. In an effort to identify serum polypeptides whose presence or absence correlates with the clinical status of patients at high risk for hepatocellular carcinoma (HCC), we have developed a strategy that helps to focus the analysis on meaningful changes in protein levels above the background of variation. For the current study we divided the patient population into four clinically defined diagnostic groups that represent a generally increasing risk for HCC. Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC and our groups included patients with no indication of liver disease (healthy), those with inactive chronic HBV, those with active chronic HBV, and patients with a diagnosis of HCC and history of chronic HBV infection. Serum polypeptides from these patients were first analyzed in two-dimensional gels by combining the serum from patients in each of the four groups to generate composite gel profiles. Analysis of these composite gels allowed us to identify two relatively abundant features that were reduced in the HCC group as compared to the healthy group. Tryptic fragment mass fingerprinting identified the features as a carboxy terminal fragment of complement C3 and an isoform of apolipoprotein A1. These two features were examined by two-dimensional gel electrophoresis of serum from each individual in the four groups in order to verify that the inter-group differences seen in composite gels reported changes in abundance for most members of the group, rather than extreme changes for a small fraction of the group. These preliminary studies suggest that a proteomic methodology can be used for the identification of serum biomarkers for HCC and other liver disease.     

Perspectives for subunit vaccines for the control of ticks

For the first time, successful vaccination against a tick has been carried out using a single defined antigen. Further, it has been shown that it is feasible to produce active antigenic material by recombinant DNA technology. This represents a significant advance towards the development of an alternative means of tick control. Nevertheless, as with any new product and new technology, much developmental work still has to be done before one can be confident that a practical means of tick control will result. From the published information, it does not seem that research on vaccines against other tick species is as advanced as that on Boophilus microplus. The work on B. microplus may, however, provide a short cut to the development of further tick vaccines.     

Template for developing guidelines for the evaluation of the clinical efficacy of psychophysiological interventions

An essential function of both the Association for Applied Psychophysiology and Biofeedback (AAPB) and the Society for Neuronal Regulation (SNR) is the systematic evaluation of psychophysiological interventions that have been developed for the treatment of medical and psychiatric disorders. In order to address scientific concerns regarding the efficacy of specific clinical applications of biofeedback, these two societies formed and Efficacy Task Force. The process to be used in the assessment of treatment efficacy, specificity and clinical utility is presented in the form of a template that will serve as the foundation for a series of scientific reviews and practice guidlines to be published by both societies.     

What's good for the host is good for the bug

Tuberculosis, caused by Mycobacterium tuberculosis, kills approximately two million people each year. The infection is characterized by an inflammatory response culminating in the formation of a granuloma, a collection of immune cells that controls the infection. However, the granuloma can be the source of immunopathology that encourages transmission. Recent data support the idea that mycobacterial products can positively and negatively regulate the inflammatory response. Our contention is that induction of the immune response and subsequent granuloma formation is beneficial to the host for control of infection, and is also beneficial to the bacillus, as a place to hide and as a means for transmitting the infection to naive hosts.     

The case for the hybrid capital approach for the measurement of the welfare and sustainability

This paper provides an overview of the plethora of approaches that are available to measure welfare and sustainable development. Many methods exist but there is no consensus on the ‘correct’ approach. Furthermore, we also show that the wide variety of sustainable development indictor (SDI) sets which have been adopted also show significant differences. We argue that this is mostly because many of these studies do not use a theoretical approach. We argue that the ‘capital approach’, which is used in the sustainability debate, is the most promising method to enhance international harmonization. Support is mounting in the scientific, policy and statistical communities for this approach in which economic capital, human capital, natural capital and social capital are distinguished. Many applications of this method express these capital stocks in monetary units (the ‘monetary capital approach’). This paper argues that the ‘hybrid capital approach’, in which the capital stocks can also be measured in non-monetary units, is probably more likely to achieve consensus over a large number of countries and institutes. Also a number of challenges remain for the capital approach. We argue that ideally the indicators should be based on satellite accounts of the national accounting framework. Also the capital approach could be further expanded to current welfare, progress of societies, inequality, and the international dimension of sustainability. We conclude that if the hybrid capital approach is adopted it may become easier to make consistent, theoretically sound and policy relevant comparisons between countries.     

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.     

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.     

A model for the functioning of the striatum

A model is presented for the operation of the striatum. The model posits that the basal ganglia are responsible for driving smooth transitions of state for an organism. We propose that this is accomplished through the computation of a potential function within the striatum on which a gradient descent is performed toward the goal state. The model suggests that various somatotopic regions of the striatum correspond to state spaces, each of which pertains to a different aspect of the organism. This paper discusses this model only in the context of motor control, i.e., egomotion and limb movement. The model appears to account for a variety of experimental results, and for some unusual properties of the striatum.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.     

Response surfaces for overdispersion in the study of the conditions for fish eggs hatching

Response surface methodology, originally developed for determining optimal conditions in industrial experiments, was early adapted to experiments in marine ecology. However, these involved studying the shape of the complete response surface, not only detecting the optimum, and often had counts or durations as the response variable. Thus, nonlinear, nonnormal response models were required. For counts, binomial and beta-binomial models have been used, the latter because of substantial overdispersion. In closely controlled experiments, overdispersion among units held under the same conditions might indicate that some mishap has occurred in conducting the study. One possible check is to model the dispersion as a second response surface. This procedure is used to show that overdispersion in fish egg hatching experiments has a biological explanation in that it occurs only under suboptimal hatching conditions.     

Ligand-induced modulation of the hepatic receptor for asialoglycoproteins. Evidence for the role of cell surface hyposialylation

In a previous paper, we reported a ligand-induced modulation of the asialoglycoprotein receptors on HepG2 cells whereby these receptors were rendered functionally inert while remaining immunologically intact on the plasma membrane. At that time, it was speculated that the loss in receptor-binding ability was a direct result of a concomitant decrease in cell surface sialic acid residues. Experiments designed to test that hypothesis are presented. The results revealed: 1) an identical response in binding activity and sialic acid content in cells subjected to minimal exposure to neuraminidase; 2) a parallel and synchronous recovery of both parameters following modulation; 3) an invariant binding of high affinity ligands, and 4) the ability of galactose oxidase to restore, at least partially, the cell's ability to bind asialoglycoprotein. These results indicate that ligand-induced surface hyposialylation directly diminishes expression of the asialoglycoprotein receptor.     

The role of mutants in the search for the photoreceptor for phototropism in higher plants

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.     

A reformulation of the social brain theory for schizophrenia: the case for out-group intolerance

This article proposes a reformulation of the social brain theory of schizophrenia. Contrary to those who consider schizophrenia to be an inherently human condition, we suggest that it is a relatively recent phenomenon, and that the vulnerability to it remained hidden among our hunter-gatherer ancestors. Hence, we contend that schizophrenia is the result of a mismatch between the post-Neolithic human social environment and the design of the social brain. We review the evidence from human evolutionary history of the importance of the distinction between ingroup and out-group membership that lies at the heart of intergroup conflict, violence, and xenophobia. We then review the evidence for the disparities in schizophrenia incidence around the world and for the higher risk of this condition among immigrants and city dwellers. Our hypothesis explains a range of epidemiological findings on schizophrenia related to the risk of migration and urbanization, the improved prognosis in underdeveloped countries, and variations in the prevalence of the disorder. However, although this hypothesis may identify the ultimate causation of schizophrenia, it does not specify the proximate mechanisms that lead to it. We conclude with a number of testable and refutable predictions for future research.     

Characterisation of the gene for Drosophila amphiphysin

A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes. Messenger RNA of this amphiphysin is expressed widely during embryogenesis and has elevated expression in a number of sites including the foregut, hindgut and epidermis, but not in the central nervous system. Taken together, these data are consistent with a role for Drosophila amphiphysin in endocytosis, but the details of this role may differ from that of vertebrate amphiphysins.     

Origin of the genes for the isoforms of creatine kinase

Creatine kinase (CK) is a member of a family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases which play a central role in cellular energy homeostasis. There are three CK isoform gene groups, each coding for proteins targeted to different intracellular compartments--cytoplasmic (CytCK), mitochondrial (MtCK) and flagellar (FlgCK). The former two CKs are either dimeric or octameric while FlgCKs are contiguous trimers consisting of three fused, complete CK domains. Conventional wisdom supports the view that CKs evolved from a cytoplasmic, monomeric ancestral protein closely related to a phosphagen kinase homologue, arginine kinase (AK). Recently, it has been shown that a demosponge (Phylum Porifera) expresses a true MtCK and two dimeric, protoflagellar CKs (protoflgCK) with great similarity to FlgCKs. To further probe the early evolution of CK, we have obtained additional sequences for Mt- and protoflgCKs from two more demosponges and from three hexactinellid (glass) sponges as well as an MtCK sequence from a basal metazoan cnidarian. Phylogenetic analyses using Maximum Likelihood (ML) of these new CK sequences with other CKs and phosphagen kinases yielded a consensus tree containing an assemblage of MtCKs and a supercluster consisting of protoflg-, Flg- and CytCKs. The MtCKs appear basal in the tree topology consistent with prior results. Within the protoflg-, Flg- and CytCK supercluster, the protoflgCKs appear to be allied to the domains of the FlgCKs, although the support is not robust. PCR amplification of genomic DNA and sequencing of the genes for Mt- and protoflgCK from the demosponge Suberites fuscus showed that the sponge MtCK shares four-five common intron:exon boundaries with invertebrate, protochordate and vertebrate MtCKs supporting a common ancestry and the extreme conservation of intron:exon organization in MtCK genes. The protoflgCK gene organization was highly divergent in relation to other CK genes but shares a common intron:exon boundary with domain 2 of the gene for the FlgCK from the tunicate Ciona intestinalis, providing support for the linkage of the protoflgCKs with the FlgCKs. Our results show that the two, major CK gene lineages are present in arguably the oldest, extant metazoan group, the hexactinellid sponges, indicating that these two genes are ancient and confirming prior work that the MtCK gene is likely basal and ancestral.     

Strategies for the identification of loci responsible for the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is a chronic, debilitating disease, which manifests itself by de-myelination of the central nervous system (CNS). MS is predominantly found in Caucasians of European decent and is more prominent in females than males. MS is one of the most prevalent causes of disability of young adults in the world. The exact cause of MS is not known, however genetic susceptibility to MS is linked to the major histocompability complex (MHC). Self reactive CD4+ T cells, specific for CNS antigens, such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP), are detectable in MS patients along with pathogenic autoantibodies specific to these CNS antigens produced by B cells. These observations suggest that MS is an autoimmune disease. Epidemiology of MS along with the analysis of sibling pairs and twins suggest that the multiple genetic factors and their interaction with environment contribute to disease susceptibility. Recent developments and advancements in genetic analysis may aid in accurate determination of genetic risk factors for the development of MS. We review these developments, advances in technology and discuss recent results in this article. These authors contributed equally to this paper     

A method for the calculation of induced band power: implications for the significance of brain oscillations

A method for the calculation of significant changes in induced band power (IBP) is presented. In contrast to traditional measures of event-related band power (ERBP) which are composed of evoked and not evoked EEG components, the proposed measure for IBP is deprived from phase locked (or evoked) EEG activity. It is assumed that changes in IBP reflect the modulation of brain oscillations that are largely independent from ERPs. The results of a visual oddball task show that significant changes in IBP can be observed in response to the presentation of a warning signal (preceding a target or nontarget) and the imperative stimulus (i.e. a target or nontarget) in the α, θ and δ band. Only a few significant changes in IBP were obtained for the warning signal in the θ band although highly significant changes in ERBP were found. Our findings document that changes in IBP may be considered a phenomenon that is largely independent from the occurrence of ERPs. They underline the significance of oscillatory processes and suggest that induced rhythms are modulated by stimuli and/or events in a not phase locked way.     

Selective forces for the origin of the eukaryotic nucleus

The origin of the eukaryotic cell nucleus and the selective forces that drove its evolution remain unknown and are a matter of controversy. Autogenous models state that both the nucleus and endoplasmic reticulum (ER) derived from the invagination of the plasma membrane, but most of them do not advance clear selective forces for this process. Alternative models proposing an endosymbiotic origin of the nucleus fail to provide a pathway fully compatible with our knowledge of cell biology. We propose here an evolutionary scenario that reconciles both an ancestral endosymbiotic origin of the eukaryotic nucleus (endosymbiosis of a methanogenic archaeon within a fermentative myxobacterium) with an autogenous generation of the contemporary nuclear membrane and ER from the bacterial membrane. We specifically state two selective forces that operated sequentially during its evolution: (1) metabolic compartmentation to avoid deleterious co-existence of anabolic (autotrophic synthesis by the methanogen) and catabolic (fermentation by the myxobacterium) pathways in the cell, and (2) avoidance of aberrant protein synthesis due to intron spreading in the ancient archaeal genome following mitochondrial acquisition and loss of methanogenesis.     

Xeroprotectants for the stabilization of biomaterials

With the advancement of science and technology, it is crucial to have effective preservation methods for the stable long-term storage of biological material (biomaterials). As an alternative to cryopreservation, various techniques have been developed, which are based on the survival mechanism of anhydrobiotic organisms. In this sense, it has been found that the synthesis of xeroprotectants can effectively stabilize biomaterials in a dry state. The most widely studied xeroprotectant is trehalose, which has excellent properties for the stabilization of certain proteins, bacteria, and biological membranes. There have also been attempts to apply trehalose to the stabilization of eukaryotic cells but without conclusive results. Consequently, a xeroprotectant or method that is useful for the stable drying of a particular biomaterial might not necessarily be suitable for another one. This article provides an overview of recent advances in the use of new techniques to stabilize biomaterials and compare xeroprotectants with other more standard methods.     

NAD-sensitive hydrogel for the release of macromolecules

A hydrogel membrane containing immobilized ligands and receptors was synthesized and investigated for the controlled diffusion of test proteins (cytochrome C and hemoglobin). Both Cibacron blue (ligand) and lysozyme (receptor) were covalently linked to dextran molecules that were subsequently crosslinked to form a gel. The resulting stable hydrogels contained both covalent and affinity crosslinks such that their intrinsic porosities were sensitive to competitive displacers of the affinity interaction between lysozyme and Cibacron blue. Transport experiments in a twin chamber diffusion cell showed that as NAD was added to the donor side, the dissociation of the binding sites between the Cibacron blue and the lysozyme led to an increase in protein diffusion through the hydrogel. The results showed that addition of NAD caused a saturable concentration-dependent increase in the transport of both cytochrome C and hemoglobin. This effect was shown to be both specific and reversible.