405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence‐guided stereotactic biopsy of brain tumors

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5‐aminolevulinic‐acid‐induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50‐fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well‐defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose‐gel phantoms. Even in direct contact, blood layers of 20–40 µm between probe and tissue must be expected, obscuring 405‐nm‐excited PpIX fluorescence almost completely, but reducing the 633‐nm‐excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX‐assisted detection of high‐grade gliomas in stereotactic biopsy.

PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation.     

Dynamical Model of Drug Accumulation in Bacteria: Sensitivity Analysis and Experimentally Testable Predictions

We present a dynamical model of drug accumulation in bacteria. The model captures key features in experimental time courses on ofloxacin accumulation: initial uptake; two-phase response; and long-term acclimation. In combination with experimental data, the model provides estimates of import and export rates in each phase, the time of entry into the second phase, and the decrease of internal drug during acclimation. Global sensitivity analysis, local sensitivity analysis, and Bayesian sensitivity analysis of the model provide information about the robustness of these estimates, and about the relative importance of different parameters in determining the features of the accumulation time courses in three different bacterial species: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The results lead to experimentally testable predictions of the effects of membrane permeability, drug efflux and trapping (e.g., by DNA binding) on drug accumulation. A key prediction is that a sudden increase in ofloxacin accumulation in both E. coli and S. aureus is accompanied by a decrease in membrane permeability.     

Disruption of the redox balance with either oxidative or anti-oxidative overloading as a promising target for cancer therapy

Oxidative stress acts as a double-edged sword by being both a promoter and a suppressor of cancer. Moderate oxidative stress is beneficial for cancer cell proliferative and invasiveness features, while overexposure of the cells to oxidative insults could induce cancer cell apoptosis and reduce hypoxia along with modulating the immune system for regression of tumor. Cancer cells and cancer stem cells have highly efficient redox systems that make them resistant to oxidative insults. The redox disruptive approach is an area of current research and key for oxidative targeted cancer therapies. This disruption is applicable by using either oxidative or anti-oxidative overloading strategies, specifically on cancer cells without influencing normal cells or tissues around tumor. The activity of tumor suppressor cells within tumor microenvironment is needed to be maintained in patients receiving such approaches.     

Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity

An ecotropic virus was chemically modified in order to determine whether its target cell specificity could be altered. We hypothesized that chemical coupling of galactose residues to a virus might permit specific infection of hepatocytes mediated by asialoglycoprotein receptors unique to these cells. To test this hypothesis, we took advantage of the fact that: 1) artificial asialoglycoproteins can be created by chemical coupling of lactose to proteins; and 2) viruses that are ecotropic have a narrow species specificity. An ecotropic, rodent-specific, replication-defective murine leukemia virus containing the gene for beta-galactosidase was chemically modified with lactose to contain 5.9 mumol of lactose per mg of viral RNA. Modified and unmodified viruses were incubated for 5 days with HepG2, a human hepatoma line that possesses asialoglycoprotein receptors, and SK Hep1, a human cell line that does not. As expected from the ecotropism, unmodified virus did not produce beta-galactosidase activity in either cell type. Modified virus did not produce beta-galactosidase activity in SK Hep1 cells. However, modified virus did produce beta-galactosidase activity, 71.2 units/mg of cell protein, in the human receptor (+) HepG2 cells. Interestingly, modification of the virus also resulted in decreased enzyme activity in previously susceptible host rodent cells. Competition with modified virus by an excess of an asialoglycoprotein completely prevented development of enzymatic activity in HepG2 cells. Histochemical treatment of cells with 5-bromo-4-chloro-3-indoyl beta-D-galactoside to detect in situ beta-galactosidase activity demonstrated that only HepG2 cells treated with modified virus were positive and that 36% of these cells were stained after 5 days. These data indicate that chemical modification of a virus can result in a redirection of the infectivity of the virus toward hepatocyte-derived cells mediated by the presence of asialoglycoprotein receptors.     

KIR diversity in Māori and Polynesians: populations in which HLA-B is not a significant KIR ligand

HLA class I molecules and killer cell immunoglobulin-like receptors (KIR) form a diverse system of ligands and receptors that individualize human immune systems in ways that improve the survival of individuals and populations. Human settlement of Oceania by island-hopping East and Southeast Asian migrants started ~3,500 years ago. Subsequently, New Zealand was reached ~750 years ago by ancestral Māori. To examine how this history impacted KIR and HLA diversity, and their functional interaction, we defined at high resolution the allelic and haplotype diversity of the 13 expressed KIR genes in 49 Māori and 34 Polynesians. Eighty KIR variants, including four ‘new’ alleles, were defined, as were 35 centromeric and 22 telomeric KIR region haplotypes, which combine to give >50 full-length KIR haplotypes. Two new and divergent variant KIR form part of a telomeric KIR haplotype, which appears derived from Papua New Guinea and was probably obtained by the Asian migrants en route to Polynesia. Māori and Polynesian KIR are very similar, but differ significantly from African, European, Japanese, and Amerindian KIR. Māori and Polynesians have high KIR haplotype diversity with corresponding allotype diversity being maintained throughout the KIR locus. Within the population, each individual has a unique combination of HLA class I and KIR. Characterizing Māori and Polynesians is a paucity of HLA-B allotypes recognized by KIR. Compensating for this deficiency are high frequencies (>50 %) of HLA-A allotypes recognized by KIR. These HLA-A allotypes are ones that modern humans likely acquired from archaic humans at a much earlier time.     

Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in C. elegans

Parkinson''s disease (PD), the second most prevalent neurodegenerative disease after Alzheimer''s disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.     

Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate: FASTER UNFOLDING AND CHANGE OF PATHWAY*

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and β-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.Deposition of proteins in the form of extracellular amyloid fibrils is a consistent mechanism underlying a group of diverse human diseases, including neurodegenerative disorders and non-neuropathic conditions (1). From a pathogenetic standpoint, these disorders differ by type of aggregated protein and by type of organs involved in amyloid deposition. Among the most prominent neurodegenerative conditions are Alzheimer and Creuzfeldt Jakob diseases, which affect the central nervous system via extracellular deposits. Examples of non-neuropathic systemic amyloidosis are light chain amyloidosis and type II diabetes, where deposits are found in joints, skeletal tissue, and several organs (e.g. heart and kidney). Each of these disorders can be traced back to the aberrant conversion of one specific protein or peptide from its soluble, native state into amyloid structures (1). Numerous biochemical and genetic studies have established a widely accepted causative link between pathological symptoms and amyloid structure formation and deposition (2).Amyloid fibrils are often localized in close proximity to basement membranes, a specialized component of the extracellular matrix that is mainly built of collagens and glycosaminoglycans (GAGs),³ often attached to a protein core to form the proteoglycans (3–5). GAGs are long unbranched polysaccharides that often occur, with the exception of hyaluronic acid existing in a free form, as O- or N-linked side chains of proteoglycans, where they regulate the activity of several proteins. Since they have been found physically associated with all types of amyloid deposits in vivo so far analyzed, they have been attributed fundamental relevance in amyloidogenesis (3, 4). Of the different types of natural GAGs, heparan sulfate (HS) is among the most important cofactors in amyloid deposits. First, it has been established as a universal component of amyloid, since it has been found to be associated with amyloid deposits of different proteins, including the serum amyloid A protein (6), the immunoglobin light chain (7), transthyretin (8), cystatin C (9), the amyloid β peptide (10), the islet amyloid polypeptide (11), and the prion protein (PrP) (12). More importantly, it has been attributed an active role in amyloidogenesis. Its ability to promote fibrillogenesis has been reported for both the 42- and 40-residue forms of the amyloid β peptide (13, 14), mature islet amyloid polypeptide and proislet amyloid polypeptide 1–48 (15), α-synuclein (16), the 173–243 fragment of D187N gelsolin (17), β2-microgloblulin (18), and the tau protein (19). HS has also been found to shift the secondary structure of a subtype of serum amyloid A protein from a random coil to a β-sheet, presumably aggregated, structure (20, 21) and to convert the prion protein from the PrP^C to the PrP^SC form (22).Despite the large body of data supporting the importance of HS in amyloidogenesis, little is known about the precise mechanism by which HS promotes amyloid formation and the effect that this GAG has on the various phases of the aggregation process and on the overall aggregation pathway. In the current work, human muscle acylphosphatase (mAcP) is utilized to study the impact of HS on amyloid aggregation, with particular attention to the various kinetic phases observed in the presence of this GAG. mAcP represents an enzyme unrelated to any human disease but a particularly suitable model for amyloid aggregation studies for a number of reasons. First, it is small in size (98 residues) and lacks disulfide bridges, trans-peptidyl-prolyl bonds, non-proteinaceous cofactors, and other complexities (23, 24). Second, it can form in the presence of 25% (v/v) trifluoroethanol (TFE) amyloid-like fibrils with extensive β-sheet structure and Congo Red birefringence (25). Third, its aggregation process has been studied using a variety of experimental approaches (25–33) and has been shown to be dramatically influenced by heparin, the highly sulfated form of HS (34).In the presence of 25% (v/v) TFE, mAcP has been shown to unfold rapidly into a denatured state enriched with α-helical structure (25). This partially unfolded state assembles to form, on a time scale of 1–2 h, amyloid-like protofibrils that develop very slowly to form, after a period of several days, long amyloid protofilaments that then associate further to form higher order structures (35). Even the early, protofibrillar aggregates that form within 1–2 h have the ability to bind Congo Red and thioflavin T (ThT) and have an extensive β-sheet structure, as detected with far-UV CD and Fourier transform infrared spectroscopy (25). This indicates that these protofibrillar structures have the essential structural characteristics of amyloid. The unfolding of the native state into a partially unfolded state is required to initiate aggregation, as shown by the need to use denaturing conditions to start aggregation (27, 35), by the finding that mutations destabilizing the native state promote aggregation (26), and by the observation that ligands binding to and stabilizing the native state have the opposite effect (29). Importantly, the TFE-denatured state of mAcP, which is the most commonly used to trigger aggregation of this protein and will also be used here, is not the only aggregation-competent state of mAcP, since other denatured states of mAcP have been shown to be capable of amyloid fibril formation (27).The present study aims at investigating the mechanism through which HS influences mAcP aggregation into amyloid-like aggregates. We will investigate both the unfolding and aggregation phases of mAcP in the presence of HS and will monitor them using a variety of biophysical methods. We will show that HS accelerates unfolding in addition to promoting aggregation of the resulting TFE-denatured state, thus playing a double-faced role in the context of its proaggregating effect. We will also show that HS is responsible for the appearance of parallel phases in the aggregation process of this protein and that its effect is not limited to a simple acceleration of the overall process. Following these findings, we will emphasize that a full understanding of the newly generated kinetics is essential for a correct interpretation of the effects of HS on amyloid formation.     

Characterizing pathogenic processes in Batten disease: use of small eukaryotic model systems

The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders. Nevertheless, small model organisms, including those lacking a nervous system, have proven invaluable in the study of mechanisms that underlie the disease and in studying the functions of the conserved proteins associated to each disease. From the single-celled yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to the worm, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster, biochemical and, in particular, genetic studies on these organisms have provided insight into the NCLs.     

Extra domains in secondary transport carriers and channel proteins

"Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.