Deciphering environment effects in peptide bond solvation dynamics by experiment and theory.

Most proteins work in aqueous solution and the interaction with water strongly affects their structure and function. However, experimentally the motion of a specific single water molecule is difficult to trace by conventional methods, because they average over the heterogeneous solvation structure of bulk water surrounding the protein. Here, we provide a detailed atomistic picture of the water rearrangement dynamics around the -CONH- peptide linkage in the two model systems formanilide and acetanilide, which simply differ by the presence of a methyl group at the peptide linkage. The combination of picosecond pump-probe time-resolved infrared spectroscopy and molecular dynamics simulations demonstrates that the solvation dynamics at the molecular level is strongly influenced by this small structural difference. The effective timescales for solvent migration triggered by ionization are mainly controlled by the efficiency of the kinetic energy redistribution rather than the shape of the potential energy surface. This approach provides a fundamental understanding of protein hydration and may help to design functional molecules in solution with tailored properties.

Tick abundance: a one year study on the impact of flood events along the banks of the river Danube, Austria.

The abundance of questing ticks depends on various factors. In this study, the impact of a major flood event on tick abundance and activity was observed. Ticks were collected on a weekly basis in two approximately 2 km2 large floodplain areas on the inner and the outer bank of the river Danube north of Vienna, Austria. In 2013 before a 200 year flood event, an average of 55 ticks per hour was collected in the area on the outer bank and 21 ticks per hour in the area on the inner bank. After the flood event the tick activity was massively reduced, with 12 ticks per hour on the outer bank and 1.1 ticks per hour on the inner bank. The most distinctive factor between the two areas was the level of sediment after the flooding, with almost no sediment in the outer bank, whereas on the inner bank the average height of sediment was 270 mm. Our data indicate the residual sediment has a greater impact on tick abundance and activity than the flooding itself. Besides the direct effect of ticks being buried under the sediment, there may be important indirect effects of the sediment on the habitat of the ticks and/or the host animals. We assume that this is the reason for the generally significantly lower numbers of questing ticks in this area on the inner bank of the Danube in this region, with periodical flood events.

Solvent migration in microhydrated aromatic aggregates: ionization-induced site switching in the 4-aminobenzonitrile-water cluster.

The dependence of the preferred microhydration sites of 4-aminobenzonitrile (4ABN) on electronic excitation and ionization is determined through IR spectroscopy of its clusters with water (W) in a supersonic expansion and through quantum chemical calculations. IR spectra of neutral 4ABN and two isomers of its hydrogen-bonded (H-bonded) 4ABN-W complexes are obtained in the ground and first excited singlet states (S0, S1) through IR depletion spectroscopy associated with resonance-enhanced multiphoton ionization. Spectral analysis reveals that electronic excitation does not change the H-bonding motif of each isomer, that is, H2O binding either to the CN or the NH site of 4ABN, denoted as 4ABN-W(CN) and 4ABN-W(NH), respectively. The IR spectra of 4ABN(+)-W in the doublet cation ground electronic state (D0) are measured by generating them either in an electron ionization source (EI-IR) or through resonant multiphoton ionization (REMPI-IR). The EI-IR spectrum shows only transitions of the most stable isomer of the cation, which is assigned to 4ABN(+)-W(NH). The REMPI-IR spectrum obtained through isomer-selective resonant photoionization of 4ABN-W(NH) is essentially the same as the EI-IR spectrum. The REMPI-IR spectrum obtained by ionizing 4ABN-W(CN) is also similar to that of the 4ABN(+)-W(NH) isomer, but differs from that calculated for 4ABN(+)-W(CN), indicating that the H2O ligand migrates from the CN to the NH site upon ionization with a yield of 100%. The mechanism of this CN→NH site-switching reaction is discussed in the light of the calculated potential energy surface and the role of intracluster vibrational energy redistribution.

Solvation dynamics of a single water molecule probed by infrared spectra--theory meets experiment.

The dynamics and energetics of water at interfaces or in biological systems plays a fundamental role in all solvation and biological phenomena in aqueous solution. In particular, the migration of water molecules is the first step that controls the overall process in the time domain. Experimentally, the dynamics of individual water molecules is nearly impossible to follow in solution, because signals from molecules in heterogeneous environments overlap. Although molecular dynamics simulations do not have this restriction, there is a lack of experimental data to validate the calculated dynamics. Here, we demonstrate a new strategy, in which the calculated dynamics are verified by measured time-resolved infrared spectra. The coexistence of fast and slow migrations of water molecules around a CONH peptide linkage is revealed for a model system representative of a hydrate peptide.

IR spectroscopy of the 4-aminobenzonitrile-Ar cluster in the S0, S1 neutral and D0 cationic states.

The S1-S0 resonant enhanced multiphoton ionization (REMPI) spectrum as well as the infrared (IR) spectra in the S0 and S1 states of 4-aminobenzonitrile (4ABN) and its van der Waals complex with Ar (4ABN-Ar) were measured by means of IR depletion spectroscopy (REMPI-IR). The IR spectrum of 4ABN-Ar in S0 shows symmetric and antisymmetric NH stretching vibrations (ν(s) and ν(a)) of the amino group at the same positions as those in the 4ABN monomer. This suggests that the Ar ligand locates above the benzene ring by van der Waals interactions (π-bound). The same coincidence of vibrational frequencies was found in S1, and the π-bound geometry was kept by the electronic excitation. The REMPI-IR spectrum of 4ABN(+)-Ar was also measured, and three major vibrational transitions were found. From the comparison to the IR dissociation spectrum with an electron impact source (EI-IR), they were assigned to ν(s), ν(a) and an NH-bending overtone of the π-bound structure. It is concluded that photoionization of 4ABN(+)-Ar does not promote site-switching of Ar from the π-site to the H-site.

Emergence of sandflies (Phlebotominae) in Austria, a Central European country.

The possible existence of autochthonous sandfly populations in Central Europe north of the Alps has long been excluded. However, in the past years, sandflies have been documented in Germany, Belgium, and recently, also in Austria, close to the Slovenian border. Moreover, autochthonous human Leishmania and Phlebovirus infections have been reported in Central Europe, particularly in Germany. From 2010 to 2012, sandfly trapping (740 trap nights) was performed at 53 different capture sites in Austria using battery-operated CDC miniature light traps. Sites were chosen on the basis of their climate profile in the federal states Styria, Burgenland, and Lower Austria. Sandfly specimens found were transferred to 70% ethanol for conservation. Identification was based on morphological characters of the male genitalia and the female spermathecae, respectively. Altogether, 24 specimens, 22 females and 2 males, all identified as Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, were found at six different sampling sites in all three federal states investigated. The highest number of catches was made on a farm in Lower Austria. Altogether, the period of sandfly activity in Austria was shown to be much longer than presumed, the earliest capture was made on July 3rd and the latest on August 28th. Sandflies have been autochthonous in Austria in small foci probably for long, but in the course of global warming, further spreading may be expected. Although P. mascittii is only an assumed vector of Leishmania spp.-data on its experimental transmission capacity are still lacking-the wide distribution of sandflies in Austria, a country thought to be free of sandflies, further supports a potential emergence of sandflies in Central Europe. This is of medical relevance, not only with respect to the transmission of Leishmania spp. for which a reservoir is given in dogs, but also with respect to the phleboviruses.

Ionization-induced solvent migration in acetanilide-methanol clusters inferred from isomer-selective infrared spectroscopy.

We present the resonance-enhanced multiphoton ionization, infrared-ultraviolet hole burning (IR-UV HB), and IR dip spectra of the trans-acetanilide-methanol (AA-MeOH) cluster in the S(0), S(1), and cationic ground state (D(0)) in a supersonic jet. The IR-UV HB spectra demonstrate the co-existence of two isomers in S(0,1), in which MeOH binds either to the NH or the CO site of the peptide linkage in AA, denoted as AA(NH)-MeOH and AA(CO)-MeOH. When AA(CO)-MeOH is selectively ionized, its IR spectrum in D(0) is the same as that measured for AA(+) (NH)-MeOH. Thus, photoionization of AA(CO)-MeOH induces migration of MeOH from the CO to the NH site with 100% yield.

Correlations between rCBF and symptoms in two independent cohorts of drug-free patients with schizophrenia.

We report on the correlations between whole brain rCBF and the positive and negative symptoms of schizophrenia in two cohorts of patients who were scanned while free of antipsychotic medication. We hypothesized that positive symptoms would correlate with rCBF in limbic and paralimbic regions, and that negative symptoms would correlate with rCBF in frontal and parietal regions. Both cohorts of patients with schizophrenia (Cohort 1: n=32; Cohort 2: n=23) were scanned using PET with H(2)(15)O while free of antipsychotic medication for an average of 21 and 15 days, respectively. Both groups were scanned during a resting state. Using SPM99, we conducted pixel by pixel linear regression analyses between BPRS scores and whole brain rCBF. As hypothesized, positive symptoms correlated with rCBF in the anterior cingulate cortex (ACC) in a positive direction and with the hippocampus/parahippocampus in a negative direction in both patient groups. When the positive symptoms were further divided into disorganization and hallucination/delusion scores, similar positive correlations with ACC and negative correlations with hippocampus rCBF were found. In both cohorts, the disorganization scores correlated positively with rCBF in Broca's area. As expected, negative symptoms correlated inversely with rCBF in frontal and parietal regions. This study provides evidence that limbic dysfunction may underlie the production of positive symptoms. It suggests that abnormal function of Broca's area may add a specific language-related dimension to positive symptoms. This study also provides further support for an independent neurobiological substrate of negative symptoms distinct from positive symptoms. The involvement of both frontal and parietal regions is implicated in the pathophysiology of negative symptoms.

First report of Rickettsia raoultii in field collected Dermacentor reticulatus ticks from Austria.

In a set of pooled field collected Dermacentor reticulatus ticks, Rickettsia raoultii, the causative agent of Tick-borne lymphadenopathy/Dermacentor-borne necrosis erythema and lymphadenopathy, was found for the first time in Austria. The coordinates of the positive locations for tick and pathogen abundance are given and shown in a map.

The effects of a subanesthetic dose of ketamine on verbal memory in normal volunteers.

RATIONALE: N-methyl-D: -aspartate (NMDA) glutamate receptor antagonists have been reported to induce schizophrenia-like symptoms in humans, including memory impairments. Although the NMDA receptor has been shown to impair memory acquisition by disrupting long-term potentiation (LTP), limited research has been done on studying the effects of NMDA antagonists on the post-LTP cascade of events implicated in consolidation as measured by administering the drug after the initial learning experience. OBJECTIVE: The purpose of this experiment was to examine the effect of ketamine on mental status and to identify NMDA antagonist-induced memory deficits by comparing the recall performance of items presented both immediately before and during ketamine infusion. METHODS: Thirteen normal controls received a 60-min infusion of ketamine in a randomized double-blind, cross-over design. Mental status was evaluated with the Brief Psychiatric Rating Scale and the Clinician-Administered Dissociative States Scale. The first 12-item word list was presented immediately before infusion, and two lists were subsequently presented during the infusion. Verbal memory performance was assessed by measuring the delayed cued recall of each list 30 min after its presentation. RESULTS: At the beginning, subjects experienced perceptual and reality distortion symptoms, followed later by mild subjective effects. Ketamine significantly reduced the delayed recall of words presented immediately before, but not during, drug infusion. Ketamine-induced decrements in verbal recall correlated significantly with plasma ketamine levels. CONCLUSION: This study characterizes the behavioral effects associated with ketamine and suggests that ketamine decreases verbal memory performance by interfering with early consolidation processes.

Subtle effects of ketamine on memory when administered following stimulus presentation.

RATIONALE: N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., PCP, ketamine) have been shown to impair learning/memory. Well documented in animal models, only limited research in humans has been reported. Findings to date are similar to results of animal studies; however, antagonists are typically administered before the learning experience. This may be problematic as memory failure could be secondary to inattention induced by the psychotomimetic effects of these drugs and/or alterations in sensory processing which can degrade the quality of the stimulus, thereby affecting the accuracy of recall. OBJECTIVE: The objective of the study is to compare the effects of ketamine vs placebo on recall for words when administered after stimulus presentation. METHODS: In this double-blind crossover study, 24 normal controls were given bolus injections of ketamine (0.3 mg/kg) or placebo. Immediately prior to infusion, subjects were administered a verbal memory test. Delayed recall was measured 45 min postinfusion. Mental status changes were assessed using the Brief Psychiatric Rating Scale. RESULTS: Subjects experienced a significant increase in psychiatric symptoms that peaked at 20 min. Results indicate no differences between the drug and placebo conditions for the memory task. However, reminiscence (i.e., recall of previously unrecalled items with repeated testing) was significantly reduced following ketamine administration compared to placebo. CONCLUSIONS: Findings suggest that aspects of memory consolidation are affected by drugs that interfere with NMDA receptor function.

Functional effects of single dose first- and second-generation antipsychotic administration in subjects with schizophrenia.

Using PET with (15)O water, we characterized the time course of functional brain changes following the acute administration of a first- and a second-generation antipsychotic. Volunteers with schizophrenia were scanned while drug-free (baseline) and after single dose administration of haloperidol (n=6) or olanzapine (n=6) during a time course adapted to their plasma kinetics. To obtain brain location information, we contrasted each post-drug scan to baseline-acquired scans. We plotted the regional cerebral blood flow (rCBF) extracted in these locations and calculated the kinetic characteristics of the curves. Further, we compared and contrasted the rCBF changes induced by the drugs over the first 4 h post-drug administration. Dorsal and ventral striatum, thalamus and anterior cingulate cortex were activated with haloperidol, while frontal, temporal and cerebellum regions evidenced reduced flow. With olanzapine, ventral striatum, anterior cingulate and temporal cortices evidenced increases, and thalamus and lingual cortex decreases, in rCBF. Both drugs activated the caudate nucleus. Haloperidol induced greater activation of the dorsal striatum than did olanzapine. These data reveal important differences in patterns of brain activation between the drugs. Differences in the involvement in basal ganglia parallel known differences between the drugs in the emergence of acute EPS upon emergency administration.

Functional effects of antipsychotic drugs: comparing clozapine with haloperidol.

BACKGROUND: Using positron emission tomography (PET) with (15)O water, we compared regional cerebral blood flow (rCBF) patterns induced by clozapine or haloperidol in individuals with schizophrenia. Based on the known clinical characteristics of each drug, we hypothesized that brain regions where the drugs show similar rCBF patterns are among those mediating their antipsychotic actions; whereas, regions where the drugs produce different rCBF patterns are among those mediating their different drug actions, namely, haloperidol's motor side effects or clozapine's unique therapeutic action. METHODS: Persons with schizophrenia were scanned using PET with (15)O water, first after withdrawal of all psychotropic medication (n = 6), then again after treatment with therapeutic doses of haloperidol (n = 5) or clozapine (n = 5). RESULTS: Both drugs increased rCBF in the ventral striatum and decreased rCBF in hippocampus and ventrolateral frontal cortex. The rCBF increase associated with haloperidol was greater than that with clozapine in the dorsal and ventral striatum; the rCBF increase with clozapine was greater than that with haloperidol in cortical regions, including anterior cingulate and dorsolateral frontal cortex. CONCLUSIONS: These data suggest that the rCBF increase in ventral striatum and/or the decrease in hippocampus and/or ventrolateral frontal cortex mediate a common component of antipsychotic action of these drugs. The increased rCBF in dorsal striatum by haloperidol could well be associated with its prominent motor side effects, whereas the increased rCBF in the anterior cingulate or dorsolateral frontal cortex may mediate the superior antipsychotic action of clozapine. The proposals based on these preliminary observations require further study.

Effects of ketamine on leading saccades during smooth-pursuit eye movements may implicate cerebellar dysfunction in schizophrenia.

OBJECTIVE: Ketamine has proved a useful probe in the study of schizophrenia. Recent studies have shown that ketamine causes abnormalities in eye tracking similar to those seen in patients with schizophrenia. The authors examined the effects of ketamine on leading saccadic eye movements, a specific component of the smooth-pursuit response shown to be abnormal in schizophrenia patients and their relatives. METHOD: Twelve normal healthy volunteers received a 0.1 mg/kg bolus injection of ketamine or placebo in double-blind fashion during a smooth-pursuit eye-movement task. The number of leading saccades and the ratios of leading saccades to smooth-pursuit response time and to total saccadic eye-movement time were measured. RESULTS: Ketamine significantly increased the number of leading saccades and increased the leading saccade ratios for more slowly moving targets. Similar nonsignificant effects were noted at higher target speeds. Ketamine-induced abnormalities were similar to those observed in relatives of schizophrenia patients under drug-free conditions. CONCLUSIONS: These results suggest that neurotransmission mediated by N-methyl-D-aspartate (NMDA) is involved in eye-tracking abnormalities. The generation of disruptive leading saccades during smooth pursuit is thought to be mediated by frontal-thalamic-cerebellar circuitry. Evidence that the locus of this and other ketamine-induced smooth-pursuit eye-movement deficits involves NMDA receptor functioning in the cerebellum is suggested.

Clozapine but not haloperidol Re-establishes normal task-activated rCBF patterns in schizophrenia within the anterior cingulate cortex.

Our previous work has identified that unmedicated volunteers with schizophrenia have regional cerebral blood flow (rCBF) activation patterns inappropriately related to the cognitive demand of a task in anterior cingulate cortex (ACC). Using positron emission tomography (PET) with (15)O water, we compared task-induced rCBF patterns induced by haloperidol or clozapine in individuals with schizophrenia. We hypothesized that clozapine, given its superior clinical action, would tend to normalize the abnormal task-activated response in ACC more than haloperidol. Schizophrenia volunteers (SVs) (n=6) and normal volunteers (NVs) (n=12) were trained to perform a tone discrimination task with 70-80% accuracy. They were then scanned during three task conditions: (1). Rest, (2). sensory motor control (SMC) task, and (3). decision task (DEC). SVs were initially scanned after withdrawal of all psychotropic medication and again after treatment with therapeutic doses of haloperidol (n=5) and/or clozapine (n=5). rCBF values, sampled in the grown maxima of the task-activated ACC cluster, were analyzed between groups and task conditions. Task performance was similar across the unmedicated, haloperidol- and clozapine-medicated SV groups. There was a reduction in accuracy in the haloperidol SV group compared to the NVs. Group and task conditions affected rCBF in the ACC. Clozapine, but not haloperidol, reversed the abnormal ACC rCBF pattern in unmedicated SV to normal. The clozapine-treated SV group showed a rCBF pattern similar to the NV group in that ACC activation was not observed during the control task but occurred during the decision condition. The pattern seen in the haloperidol-treated SV group was similar to the unmedicated SV group in that ACC activation was seen during the control task and no further activation was seen during the DEC. We report that clozapine, but not haloperidol, normalizes anterior cingulate rCBF patterns in schizophrenia during a cognitive task. Based on these preliminary data, we propose that this pattern may account for the superior therapeutic effect of clozapine and represents a surrogate marker of this action.

Antipsychotic Drugs Alter Functional Connectivity between the Medial Frontal Cortex, Hippocampus, and Nucleus Accumbens as Measured by H215O PET.

To evaluate changes in functional connectivity as a result of treatment with antipsychotic drugs (APDs) in subjects with schizophrenia (SZ), we identified a limited number of regions that have been implicated in the mechanism of action of APDs and that are part of a neuronal network known to be modulated by dopamine (DA). These regions consisted of the nucleus accumbens (NAcc), the hippocampus (Hip), and the medial frontal cortex (MFC). SZ participants were blindly randomized into a haloperidol treatment group (n = 12) and an olanzapine treatment group (n = 17). Using PET with 15O, we evaluated changes in functional connectivity between these regions during rest and task performance at three treatment time points: (1) at baseline, after withdrawal of all psychotropic medication (2 weeks), (2) after 1 week on medication, and (3) after 6 weeks on medication. Results from the two treatment groups were combined during analysis to investigate the common effects of APDs on functional connectivity. We found that the functional connectivity between MFC and NAcc significantly increased at week one, and then significantly decreased from week one to week 6. The functional connectivity between MFC and Hip significantly decreased at week one and week 6 relative to baseline. Critically, the strength of the functional connectivity between the MFC and Hip after 1 week of treatment was predictive of treatment response. This pattern of changes may represent an important biomarker for indexing treatment response. The regulation by APDs of the balance between prefrontal and limbic inputs to the striatum may be crucial to restoring adaptive behavior.

Gamma and delta neural oscillations and association with clinical symptoms under subanesthetic ketamine.

Several electrical neural oscillatory abnormalities have been associated with schizophrenia, although the underlying mechanisms of these oscillatory problems are unclear. Animal studies suggest that one of the key mechanisms of neural oscillations is through glutamatergic regulation; therefore, neural oscillations may provide a valuable animal-clinical interface on studying glutamatergic dysfunction in schizophrenia. To identify glutamatergic control of neural oscillation relevant to human subjects, we studied the effects of ketamine, an N-methyl-D-aspartate antagonist that can mimic some clinical aspects of schizophrenia, on auditory-evoked neural oscillations using a paired-click paradigm. This was a double-blind, placebo-controlled, crossover study of ketamine vs saline infusion on 10 healthy subjects. Clinically, infusion of ketamine in subanesthetic dose significantly increased thought disorder, withdrawal-retardation, and dissociative symptoms. Ketamine significantly augmented high-frequency oscillations (gamma band at 40-85 Hz, p=0.006) and reduced low-frequency oscillations (delta band at 1-5 Hz, p<0.001) compared with placebo. Importantly, the combined effect of increased gamma and reduced delta frequency oscillations was significantly associated with more withdrawal-retardation symptoms experienced during ketamine administration (p=0.02). Ketamine also reduced gating of the theta-alpha (5-12 Hz) range oscillation, an effect that mimics previously described deficits in schizophrenia patients and their first-degree relatives. In conclusion, acute ketamine appeared to mimic some aspects of neural oscillatory deficits in schizophrenia, and showed an opposite effect on scalp-recorded gamma vs low-frequency oscillations. These electrical oscillatory indexes of subanesthetic ketamine can be potentially used to cross-examine glutamatergic pharmacological effects in translational animal and human studies.

Modulation of limbic circuitry predicts treatment response to antipsychotic medication: a functional imaging study in schizophrenia.

The regional neuronal changes taking place in the early and late stages of antipsychotic treatment are still not well characterized in humans. In addition, it is not known whether these regional changes are predictive of or are correlated with treatment response. Using PET with 15O, we evaluated the time course of regional cerebral blood flow (rCBF) patterns generated by a first (haloperidol) and a second (olanzapine) generation antipsychotic drug in patients with schizophrenia during a 6-week treatment trial. Patients were initially scanned after withdrawal of all psychotropic medication (2 weeks), and then blindly randomized to treatment with haloperidol (n=12) or olanzapine (n=17) for a period of 6 weeks. Patients were scanned again after 1 and 6 weeks of treatment. All assessments, including scanning sessions, were obtained in a double-blind manner. As hypothesized, we observed rCBF changes that were common to both the drugs, implicating cortico-subcortical and limbic neuronal networks in antipsychotic action. In addition, in these regions, some patterns seen at weeks 1 and 6 were distinctive, indexing neuronal changes related to an early (ventral striatum, hippocampus) and consolidated (anterior cingulate/medial frontal cortex) stage of drug response. Finally, both after 1 and 6 weeks of treatment, we observed differential patterns of rCBF activation between good and poor responders. After 1 week of treatment, greater rCBF increase in the ventral striatum and greater decrease in the hippocampus were associated with good response.