Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III.

A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.

Long-range fluorescence quenching by gold nanoparticles in a sandwich immunoassay for cardiac troponin T.

We report the first homogeneous sandwich immunoassay with gold nanoparticles (AuNPs) as fluorescence quenchers. The sandwich assay is designed for the detection of the protein cardiac troponin T (cTnT) by its simultaneous interaction with two different antibodies, one attached to AuNPs and the other labeled with fluorescent dyes. We demonstrate the working principle of the assay and using time-resolved fluorescence spectroscopy, we determine the quenching efficiency of the gold nanoparticles. In spite of the relatively large separation distance between dye molecules and AuNPs, ranging from 3 to 22 nm, the AuNPs quench the fluorescence with efficiencies as high as 95%. A limit of detection of 0.02 nM (0.7 ng/mL) was obtained for cTnT, which is the lowest value reported for a homogeneous sandwich assay for cTnT. These results illustrate the use of metallic nanoparticles as fluorescence quenchers in immunoassays where the large biomolecules involved impose distances for which energy transfer between fluorophores would be inefficient.

Optimized variants of the cold shock protein from in vitro selection: structural basis of their high thermostability.

The bacterial cold shock proteins (Csp) are widely used as models for the experimental and computational analysis of protein stability. In a previous study, in vitro evolution was employed to identify strongly stabilizing mutations in Bs-CspB from Bacillus subtilis. The best variant found by this approach contained the mutations M1R, E3K and K65I, which raised the midpoint of thermal unfolding of Bs-CspB from 53.8 degrees C to 83.7 degrees C, and increased the Gibbs free energy of stabilization by 20.9 kJ mol(-1). Another selected variant with the two mutations A46K and S48R was stabilized by 11.1 kJ mol(-1). To elucidate the molecular basis of these stabilizations, we determined the crystal structures of these two Bs-CspB variants. The mutated residues are generally well ordered and provide additional stabilizing interactions, such as charge interactions, additional hydrogen bonds and improved side-chain packing. Several mutations improve the electrostatic interactions, either by the removal of unfavorable charges (E3K) or by compensating their destabilizing interactions (A46K, S48R). The stabilizing mutations are clustered at a contiguous surface area of Bs-CspB, which apparently is critically important for the stability of the beta-barrel structure but not well optimized in the wild-type protein.

Optimization of the gbeta1 domain by computational design and by in vitro evolution: structural and energetic basis of stabilization.

Computational design and in vitro evolution are major strategies for stabilizing proteins. For the four critical positions 16, 18, 25, and 29 of the B domain of the streptococcal protein G (Gbeta1), they identified the same optimal residues at positions 16 and 25, but not at 18 and 29. Here we analyzed the energetic contributions of the residues from these two approaches by single and double mutant analyses and determined crystal structures for a variant from the calculation (I16/L18/E25/K29) and from the selection (I16/I18/E25/F29). The structural analysis explains the observed differences in stabilization. Residues 16, 18, and 29 line an invagination, which results from a packing defect between the helix and the beta-sheet of Gbeta1. In all stabilized variants, residues with larger side-chains occur at these positions and packing is improved. In the selected variant, packing is better optimized than in the computed variant. Such differences in side-chain packing strongly affect stability but are difficult to evaluate by computation.

Elevated serum S100B levels indicate a higher risk of hemorrhagic transformation after thrombolytic therapy in acute stroke.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage constitutes an often fatal sequela of thrombolytic therapy in patients with ischemic stroke. Early blood-brain barrier disruption may play an important role, and the astroglial protein S100B is known to indicate blood-brain barrier dysfunction. We investigated whether elevated pretreatment serum S100B levels predict hemorrhagic transformation (HT) in thrombolyzed patients with stroke. METHODS: We retrospectively included 275 patients with ischemic stroke (mean age of 69+/-13 years; 46% female) who had received thrombolytic therapy within 6 hours of symptom onset. S100B levels were determined from pretreatment blood samples. Follow-up brain scans were obtained 24 hours after admission, and HT was classified as either hemorrhagic infarction (1, 2) or parenchymal hemorrhage (1, 2). RESULTS: HT occurred in 80 patients (29%; 45 hemorrhagic infarction, 35 parenchymal hemorrhage). Median S100B values were significantly higher in patients with HT (0.14 versus 0.11 mug/L; P=0.017). An S100B value in the highest quintile corresponded to an OR for any HT of 2.87 (95% CI: 1.55 to 5.32; P=0.001) in univariate analysis and of 2.80 (1.40 to 5.62; P=0.004) after adjustment for age, sex, symptom severity, timespan from symptom onset to hospital admission, vascular risk factors, and storage time of serum probes. A pretreatment S100B value above 0.23 mug/L had only a moderate sensitivity (0.46) and specificity (0.82) for predicting severe parenchymal bleeding (parenchymal hemorrhage 2). CONCLUSIONS: Elevated S100B serum levels before thrombolytic therapy constitute an independent risk factor for HT in patients with acute stroke. Unfortunately, the diagnostic accuracy of S100B is too low for it to function in this context as a reliable biomarker in clinical practice.

In vitro evolution of a hyperstable Gbeta1 variant.

An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.

The correlation between protein stability and dipole moment: a critical test.

Improving the stability of proteins is a major aim in basic and applied protein science. Querol and coworkers calculated changes in the quasi-electric dipole moment of a protein and used it as a simple criterion to predict stabilizing charge mutations. They employed this method to propose for the bacterial cold shock protein Bc-Csp a number of charge mutations that should have a strong influence on stability. We produced eight variants of Bc-Csp with such mutations and measured their stabilities experimentally. However, we could not find a correlation between the stability and the quasi dipole moment of these variants. Possibly, the quasi dipole moment reflects only a secondary aspect of the changes that are caused by charge mutations in a protein.

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions.

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.

Evolutionary protein stabilization in comparison with computational design.

Two major strategies are currently used for stabilizing proteins: in vitro evolution and computational design. Here, we used gene libraries of the beta1 domain of the streptococcal protein G (Gbeta1) and Proside, an in vitro selection method, to identify stabilized variants of this protein. In the Gbeta1 libraries, the codons for the four boundary positions 16, 18, 25, and 29 were randomized. Many Gbeta1 variants with strongly increased thermal stabilities were found in 11 selections performed with five independent libraries. Previously, Mayo and co-workers used computational design to stabilize Gbeta1 by sequence optimization at the same positions. Their best variant ranked third within the panel of the selected variants. None of the ten computed sequences was found in the Proside selections, because several computed residues for positions 18 and 29 were not optimal for stability.

Neuron-specific enolase and tau protein as neurobiochemical markers of neuronal damage are related to early clinical course and long-term outcome in acute ischemic stroke.

OBJECTIVES: Analyses of neuron-specific enolase (NSE) and tau protein in patients with hyperacute ischemic stroke, their association with infarct volume, severity of the neurological deficit, the neurovascular status and functional outcome. PATIENTS AND METHODS: In 66 consecutive patients, serial venous blood samples were taken at 3, 6, 12, 18, 24, 48, 72, 96, and 120 h after stroke onset. The neurovascular status was assessed by repetitive extra- and transcranial duplex sonography. Neurological deficits were quantified by the NIH stroke scale, and functional outcome was assessed with the modified Rankin scale (mRS). RESULTS: After a first rise within 3 h, NSE decreased followed by a secondary increase until Day 5. Tau protein concentrations showed a continuous increase from admission onward. NSE and tau release were highly correlated with severity of neurological deficits and infarct volume (P = 0.001). NSE, but not tau protein, release was associated to the neurovascular status on admission. NSE and tau protein values were significantly correlated with the functional outcome at 3 months (P < 0.001). CONCLUSION: Release kinetics of NSE and tau protein are associated with patients' clinical deficits and infarct volume, and may be used as an additional predictor of the early course and functional outcome.

Release of brain-type and heart-type fatty acid-binding proteins in serum after acute ischaemic stroke.

This study aimed at an analysis of the release of Braintype and Heart-type Fatty Acid- Binding Proteins (B-FABP and HFABP) in acute ischaemic stroke and their potential value as neurobiochemical markers of brain damage. We investigated 42 consecutive patients admitted within 6 hours after ischaemic stroke. Serial venous blood samples were taken hourly between 1 to 6 hours, and at 12, 18, 24, 48, 72, 96, and 120 hours after stroke onset. In all patients lesion topography was assessed and infarct volume was calculated. The neurological deficit was quantified by the National Institutes of Health stroke scale score, and functional outcome was assessed with the modified Rankin Scale 3 months after stroke. H-FABP and B-FABP concentrations showed peak values already 2 to 3 hours after stroke onset and remained elevated up to last measurements at 120 hours.Unlike BFABP, early H-FABP concentrations were significantly associated with the severity of the neurological deficit and the functional outcome. High H-FABP release was associated with large infarction on CT. Our study shows for the first time quantitative data of serum BFABP and H-FABP being elevated early in acute ischaemic stroke indicating that especially H-FABP might have the potential to be a rapid marker of brain damage and clinical severity. As both FABPs indicate damage to neuronal and glial tissue but are not specific for cerebral infarction, further investigations are needed to better understand the prolonged release of both in ischaemic stroke which is in contrast to the transient increase after myocardial infarction and can not be explained by their renal extraction.

Release of neurobiochemical markers of brain damage is related to the neurovascular status on admission and the site of arterial occlusion in acute ischemic stroke.

OBJECTIVES: The study aimed at an analysis of the kinetics of protein S100B and neuron-specific enolase (NSE) and their relation to the site of arterial occlusion in patients with acute ischemic stroke. METHODS: We investigated 32 consecutive patients admitted within 6 h after stroke onset. Serial venous blood samples were taken hourly between 1 and 6 h, and at 12, 18, 24, 48, 72, 96, and 120 h after stroke onset. The neurovascular status was assessed on admission and monitored by repetitive extracranial and transcranial duplex sonography. In all patients, infarct volume was calculated. The neurological deficit was quantified by the National Institutes of Health stroke scale score, and functional outcome after 3 months was assessed with the modified Rankin Scale. RESULTS: Patients with normal flow velocities in basal cerebral arteries at admission showed significantly less S100B release than those with main stem or multiple branch occlusions (p<0.01). S100B cut-off values of 0.15 microg/l (between 6 and 18 h), 0.21 microg/l (between 24 and 48 h) and 0.5 microg/l (from 72 to 120 h) differentiated best between patients with initially normal and pathological sonographic vessel findings. The release of S100B and NSE was highly correlated with the severity of the corresponding neurological deficit as well as with the final infarct volume. S100B concentrations from 6 h on were associated with the functional outcome. S100B values 48 h after stroke above 0.2 microg/l indicated a poor functional status 3 months after stroke. CONCLUSIONS: Protein S100B may serve as a monitoring parameter in acute ischemic stroke, especially with respect to the neurovascular status. Furthermore, S100B obtains additional information about functional outcome.

S100B serum levels are closely correlated with body mass index: an important caveat in neuropsychiatric research.

Elevated blood levels of S100B in neuropsychiatric disorders have so far been mainly attributed to glial pathologies. However, increases or dysfunction of adipose tissue may be alternatively responsible. Our study assessed S100B serum levels in 60 adult subjects without a prior history of neuropsychiatric disorders. S100B concentrations were closely correlated with the body mass index (BMI, range 18-45 kg/m(2)) as well as levels of leptin and adipocyte-type fatty acid-binding protein (A-FABP/FABP4) that are well-known adipose-related factors. Effect sizes as measured by Cohen's d indicated medium (0.8 > d > 0.5) to strong effects (d > 0.9) of BMI on S100B blood levels. In conclusion, physiological S100B levels in humans appear to closely reflect adipose tissue mass, which should therefore be considered as an important confounding factor in clinical studies examining the role of S100B.

Dimer formation of a stabilized Gbeta1 variant: a structural and energetic analysis.

In previous work, a strongly stabilized variant of the beta1 domain of streptococcal protein G (Gbeta1) was obtained by an in vitro selection method. This variant, termed Gbeta1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gbeta1 gene libraries were performed, and the crystal structure of Gbeta1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gbeta1 by contributions of between 1.6 and 6.0 kJ mol(-1) (at 70 degrees C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gbeta1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-A crystal structure of Gbeta1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gbeta1-M2 molecules via six intermolecular hydrogen bonds between the two beta strands 2 and 2' and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120 degrees rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gbeta1 variants.

A new pathophysiological aspect of S100B in schizophrenia: potential regulation of S100B by its scavenger soluble RAGE.

BACKGROUND: Several studies have reported elevated S100B serum levels in schizophrenia. Our study focused on its scavenger, soluble receptor for advanced glycation end products (sRAGE). Given the benefits of sRAGE in metabolic and inflammatory diseases, we hypothesized a similar effect in schizophrenia. METHODS: S100B and sRAGE concentrations were explored during acute paranoid schizophrenia and during reconvalescence. Serum samples from 26 inpatients were investigated on hospital admission (T0) and 6 weeks posttreatment (T6) by S100B-immunoluminometry and sRAGE-ELISA. Thirty-two matched healthy individuals served as controls. Psychopathology was monitored using the Positive and Negative Syndrome Scale (PANSS). RESULTS: S100B (p = .021) and sRAGE (p = .020) were elevated in schizophrenic patients at T0. S100B levels normalized under antipsychotic treatment (p = .003), whereas sRAGE increased further by T6 (p = .005). Changes of S100B during treatment correlated inversely with DeltasRAGE (r = -.422, p = .032). PANSS was negatively associated with sRAGE at T0 (positive score: r = -.415, p = .035; total score: r = -.395, p = .046). CONCLUSIONS: Our results provide support for a reduction of S100B levels during reconvalescence from acute paranoid schizophrenia that is regulated by its scavenger sRAGE. This mechanism could provide novel treatment strategies.

Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles.

We report on a competitive, homogeneous immunoassay for the detection of the hapten digoxigenin. The assay is based on competitive fluorescence quenching by gold nanoparticles. Digoxigenin is indirectly labeled with the fluorophore Cy3B through bovine serum albumin and used as a marker. Gold nanoparticles functionalized with anti-digoxigenin antibodies serve as fluorescence quenchers. Free digoxigenin molecules in the analyte solution compete with the labeled markers for antibodies on the gold nanoparticles. The fluorescence signal depends linearly on the free digoxigenin concentration within a range of concentration from 0.5 to 3 ng mL(-1). The limit of detection is estimated as 0.2 ng mL(-1) and the limit of quantitation is estimated as 0.6 ng mL(-1). The method can be used to detect digoxin, a drug used to cure cardiac arrhythmia.

A folded and functional protein domain in an amyloid-like fibril.

The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.

Gold nanostoves for microsecond DNA melting analysis.

In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.

Conservative medical treatment and intravenous thrombolysis in acute stroke from carotid T occlusion.

BACKGROUND: We aimed to analyse the course of early recanalization and corresponding functional outcome in patients with an acute occlusion of the carotid T who were treated conservatively or underwent intravenous thrombolysis. METHODS: Forty-two patients with an acute occlusion of the carotid T within 6 h were recruited from consecutive admissions to a neurological department participating in the Duplex Sonography in Acute Stroke study. All patients underwent a standardized admission and follow-up procedure. Colour-coded duplex sonography was performed on admission, 30 min after thrombolysis, and at 6 and 24 h after onset of symptoms. Recanalization of the carotid T was classified as complete, partial and absent. Functional outcome was rated with the modified Rankin scale (mRS) at 3 months as favourable (mRS 0-2) or poor (mRS 3-6). RESULTS: Within 6 h, complete or partial recanalization occurred in 1 of 27 patients treated conservatively and in 6 of 15 thrombolysed patients. Intravenous thrombolysis predicted early recanalization also after adjustment for age, sex, cardioembolic stroke aetiology and time to treatment (adjusted odds ratio, OR, 39.7; 95% confidence interval, CI, 2.0-801.7; p = 0.016). An early recanalization was the only selected predictor of a favourable outcome (OR, 13.6; 95% CI, 1.0-179.0; p = 0.047) at regression analysis, and was achieved in 3 thrombolysed patients but in none with conservative medical treatment. CONCLUSIONS: In patients treated conservatively, functional outcome is poor and early recanalization rarely occurs. The latter can be achieved by intravenous thrombolysis with a rate comparable to that found at an intra-arterial approach without major intracranial bleeding complications. Early recanalization is associated with a better functional outcome.

Protein S-100B and neuron specific enolase as early neurobiochemical markers of the severity of traumatic brain injury.

The present study aimed at the predictive value of early release patterns of protein S-100B and neuron specific enolase (NSE) in patients with traumatic brain injury. We investigated 69 patients who were admitted to the Department of Neurosurgery following traumatic brain injury. Both NSE and S-100B serum concentrations during the first three days after admission were highly and significantly correlated with Glasgow Coma and Coma Remission Scale scores at the respective blood sampling times as well as 2 weeks later. Signs of intracranial pathology as evaluated by CCT or MRI scans showed no association with NSE or S-100B release patterns. Our data support the hypothesis that NSE and protein S-100B are useful and sensitive neurobiochemical markers for the early clinical outcome of traumatic brain injury.