Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise.

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

d-serine levels in Alzheimer's disease: implications for novel biomarker development.

Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-ß oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-ß/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis.

Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of transthyretin-related oculoleptomeningeal amyloidosis.

Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease characterized by the accumulation of transthyretin (TTR) amyloid within the central nervous system. The mechanisms underlying the pathogenesis of OA, and in particular how amyloid triggers neuronal damage, are still unknown. Here, we show that amyloid fibrils formed by a mutant form of TTR, A25T, activate microglia, leading to the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and nitric oxide. Further, we found that A25T amyloid fibrils induce the activation of Akt, culminating in the translocation of NFκB to the nucleus of microglia. While A25T fibrils were not directly toxic to neurons, the exposure of neuronal cultures to media conditioned by fibril-activated microglia caused synapse loss that culminated in extensive neuronal death via apoptosis. Finally, intracerebroventricular (i.c.v.) injection of A25T fibrils caused microgliosis, increased brain TNF-α and IL-6 levels and cognitive deficits in mice, which could be prevented by minocycline treatment. These results indicate that A25T fibrils act as pro-inflammatory agents in OA, activating microglia and causing neuronal damage.

Aß oligomers induce glutamate release from hippocampal neurons.

Soluble oligomers of the amyloid-ß peptide (AßOs) accumulate in Alzheimer's disease (AD) brain and have been implicated in mechanisms of pathogenesis. The neurotoxicity of AßOs appears to be, at least in part, due to dysregulation of glutamate signaling. Here, we show that AßOs promote extracellular accumulation of glutamate and d-serine, a co-agonist at glutamate receptors of the N-methyl-d-aspartate subtype (NMDARs), in hippocampal neuronal cultures. The increase in extracellular glutamate levels induced by AßOs was blocked by the sodium channel blocker tetrodotoxin (TTX), by the NMDAR blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) and by removal of Ca(2+) from the extracellular medium, indicating dependence on excitatory neuronal activity. AßOs enhanced both the release of pre-synaptic vesicles labeled by FM1-43 and spontaneous post-synaptic activity measured by whole-cell patch-clamp. Activation of inhibitory GABA(A) receptors by taurine blocked the increase in extracellular glutamate levels, suggesting that selective pharmacological inhibition of neuronal activity can counteract the impact of AbOs on glutamate dyshomeostasis. Results reveal a novel mechanism by which Ab oligomers promote abnormal release of glutamate in hippocampal neurons, which may contribute to dysregulation of excitatory signaling in the brain.

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure.

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biological phenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biologicalphenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

Evaluation of the patterns of Schistosoma mansoni infection and re-infection in Senegal, from faecal egg counts and serum concentrations of circulating anodic antigen.

Infection and re-infection patterns were evaluated in a recent Schistosoma mansoni focus in northern Senegal, by determining concentrations of serum circulating anodic antigen (CAA), as a measure of worm burden, and counting eggs in faeces before, 6 or 12 weeks and 1 year after praziquantel treatment in two subsequent cohorts (cohort A and B). No differences in egg counts and CAA concentrations or their relationship were found between the cohorts, which were examined 2 years apart. Within both cohorts, CAA concentrations showed the same, typical, age-related patterns as egg counts, with a peak in children and a strong decline in adults. These trends were apparent both before and 1 year after treatment. The results indicate that an age-related resistance to infection and to re-infection has been firmly established, at a steady level, in the recent S. mansoni focus investigated, with no indication of a gradual development of immunity or anti-fecundity immunity over a period of 2 years. Both shortly and 1 year after treatment, the decrease in egg counts was stronger than that in CAA concentrations, indicating that that there had been a reduction in worm fecundity after treatment. The possibility that praziquantel may induce anti-fecundity immunity has important implications for the use and interpretation of the results of (egg-count-based) re-infection studies designed to follow the development of naturally acquired immunity.

PABMB Lecture. Protein dynamics, folding and misfolding: from basic physical chemistry to human conformational diseases.

Proteins exhibit a variety of motions ranging from amino acid side-chain rotations to the motions of large domains. Recognition of their conformational flexibility has led to the view that protein molecules undergo fast dynamic interconversion between different conformational substates. This proposal has received support from a wide variety of experimental techniques and from computer simulations of protein dynamics. More recently, studies of the subunit dissociation of oligomeric proteins induced by hydrostatic pressure have shown that the characteristic times for subunit exchange between oligomers and for interconversion between different conformations may be rather slow (hours or days). In such cases, proteins cannot be treated as an ensemble of rapidly interconverting conformational substates, but rather as a persistently heterogeneous population of different long-lived conformers. This is reminiscent of the deterministic behavior exhibited by macroscopic bodies, and may have important implications for our understanding of protein folding and biological functions. Here, we propose that the deterministic behavior of proteins may be closely related to the genesis of conformational diseases, a class of pathological conditions that includes transmissible spongiform encephalopathies, Alzheimer's disease and other amyloidosis.

Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase.

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.

Dual role of glutamatergic neurotransmission on amyloid beta(1-42) aggregation and neurotoxicity in embryonic avian retina.

The effects of glutamate receptor antagonists on the toxicity of the beta-amyloid peptide (Abeta(1-42)) in embryonic chick retina were investigated. When used alone or in combination, the N-methyl-D-asparate antagonist, MK-801, the (+/-)-alphaamino-3-hydroxyl-5-methylisoxazole-4-propionic acid/kainate antagonist, DNQX, and the metabotropic receptor antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid, blocked the neurotoxicity of Abeta(1-42). Aggregation of Abeta(1-42) was significantly increased in the presence of acidic glutamate solutions, but not in the presence of other neurotransmitters. These results point to a dual role of glutamatergic transmission in Alzheimer's disease (AD): (i) Abeta neurotoxicity requires activation of glutamate receptors and its blockade prevents cell death; (ii) high concentrations of glutamate in the synaptic cleft indirectly enhance Abeta aggregation through acidification of the medium, resulting in increased amounts of neurotoxic amyloid fibrils. These results suggest that glutamatergic neurotransmission may represent a novel target for therapeutic approaches in AD.

Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies.

The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.

Crystal structures of bovine beta-lactoglobulin in the orthorhombic space group C222(1). Structural differences between genetic variants A and B and features of the Tanford transition.

The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.

Selective neoglycosylation increases the structural stability of vicilin, the 7S storage globulin from pea seeds.

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine epsilon-amino groups with various carbohydrates. Average modification levels of 13.4 +/- 3.0, 11.1 +/- 3.6, 7.5 +/- 4.2, and 4.7 +/- 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046-4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (deltaG(diss)) and molar volume change (deltaV(diss)) of dissociation of vicilin subunits. For unmodified vicilin, deltaG(diss) = 18.2 kcal/mol and deltaV(diss) = -102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with deltaG(diss) of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in deltaV(diss) were found for the glucose and galactose derivatives, whereas deltaV(diss) of -128 and -135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

Preliminary X-ray diffraction studies of rabbit muscle triose phosphate isomerase (TIM).

Triose phosphate isomerase (TIM) is responsible for the interconversion between GAP and DHAP in the glycolytic pathway. Two crystal forms belonging to space group P2(1)2(1)2(1) were obtained by the hanging-drop method and were designated A and B. Synchrotron X-ray diffraction data were collected for both forms. Form A had unit-cell parameters a = 65.14, b = 72.45, c = 93.24 A and diffracted to 2.25 A at 85 K, whereas form B had unit-cell parameters a = 73.02, b = 79.80, c = 172.85 A and diffracted to 2.85 A at room temperature. Molecular replacement was employed to solve the structures, using human TIM as a search model. Further refinement of both structures is under way and is expected to shed light on the recently reported conformational studies for rabbit TIM.

Specificity of circulating antigen detection for schistosomiasis mansoni in Senegal and Burundi.

The specificity of schistosome circulating antigen detection was determined in negative individuals from two S. mansoni- endemic countries, Senegal and Burundi, and compared with results from Dutch control individuals. A nearly absolute specificity was achieved for circulating anodic antigen (CAA) detection in serum, irrespective of the target population or sample pretreatment method. Circulating cathodic antigen (CCA) detection in serum and urine resulted in a lower specificity than serum CAA detection. Apparent large differences in specificity of CCA detection between countries were mainly due to pretreatment methods. Apparently, the alkaline/heating pretreatment method is not as effective as trichloroacetic acid (TCA)-pretreatment in removing (certain) interfering components, which may vary between populations. In view of the development of the urine CCA assay into a noninvasive screening test, a slightly lower specificity may still be acceptable. For precise epidemiological analyses the highly specific serum CAA assay remains the method of choice.