Printed circuit board based segmented quadrupole ion guide

Collisional damping quadrupole ion guides have become popular tools in mass spectrometry. In such devices, ions are cooled by collisions with a buffer gas and focused to trajectories near the axis of the ion guide under the influence of a radio frequency (RF) field. This produces a narrow beam of low-energy ions which can be transported with high efficiency. Typically, quadrupole ion guides are constructed of four parallel rods to which RF voltages are applied. To overcome the dampening of the axial velocity component resulting from collisions with neutral gas particles, an additional static axial field is provided by dividing the rods in several segments and applying an electric potential to each segment. However, this method is mechanically complex, requiring a precise alignment of all segments, and several separate connections for the DC und RF voltages to all segments. In this work, we present a simple and low-cost segmented quadrupole ion guide design that is based on standard printed circuit boards (PCB) including both the segmented electrodes as well as the signal distribution network. Furthermore, we present simulations of the ion movement inside this PCB quadrupole and experimentally evaluate the ion transfer. Our measurements show that the segmented PCB quadrupole with planar electrodes reaches similar ion transmissions in comparison with conventional quadrupole ion guides built from segmented circular rods

Regarding the Influence of Additives and Additional Plasma-Induced Chemical Ionization on Adduct Formation in ESI/IMS/MS

Ion mobility spectrometers (IMS) separate ions based on their ion mobility, which depends mainly on collision cross-section, mass, and charge of the ions. However, the performance is often hampered in electrospray ionization (ESI) by the appearance of multiple ion mobility peaks in the spectrum for the same analyte due to clustering and additional sodium adducts. In this work, we investigate the influence of solvents and buffer additives on the detected ion mobility peaks using ESI. Additionally, we investigate the effects of an additional chemical ionization (CI) induced by plasma ionization on the ions formed by electrospray. For this purpose, we coupled our high-resolution IMS with a resolving power of Rp = 100 to a time-of-flight mass spectrometer. Depending on the analyte and the chosen additives, the ionization process can be influenced during the electrospray process. For the herbicide isoproturon, the addition of 5 mM sodium acetate results in the formation of the sodium adduct [M + Na]+, which is reflected in the ion mobility K0 of 1.22 cm2/(V·s). In contrast, the addition of 5 mM ammonium acetate yields the protonated species [M + H]+ and a correspondingly higher K0 of 1.29 cm2/(V·s). In some cases, as with the herbicide pyrimethanil, the addition of sodium acetate can completely suppress ionizations. By carefully choosing the solvent additive for ESI-IMS or additional CI, the formation of different ion mobility peaks can be observed. This can facilitate the assignment of ions to ion mobility peaks using IMS as a compact, stand-alone instrument, e.g., for on-site analysis

Positive Reactant Ion Formation in High Kinetic Energy Ion Mobility Spectrometry (HiKE-IMS)

In contrast to classical Ion Mobility Spectrometers (IMS) operating at ambient pressure, the High Kinetic Energy Ion Mobility Spectrometer (HiKE-IMS) is operated at reduced pressures of between 10 and 40 mbar and higher reduced electric field strengths of up to 120 Td. Thus, the ion–molecule reactions occurring in the HiKE-IMS can significantly differ from those in classical ambient pressure IMS. In order to predict the ionization pathways of specific analyte molecules, profound knowledge of the reactant ion species generated in HiKE-IMS and their dependence on the ionization conditions is essential. In this work, the formation of positive reactant ions in HiKE-IMS is investigated in detail. On the basis of kinetic and thermodynamic data from the literature, the ion–molecule reactions are kinetically modeled. To verify the model, we present measurements of the reactant ion population and its dependence on the reduced electric field strength, the operating pressure, and the water concentration in the sample gas. All of these parameters significantly affect the reactant ion population formed in HiKE-IMS

Fasudil attenuates aggregation of α-synuclein in models of Parkinson’s disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (alpha-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced alpha-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of alpha-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on alpha-Syn pathology in vivo in a transgenic mouse model overexpressing human alpha-Syn bearing the A53T mutation (alpha-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in alpha-Syn(A53T) mice as determined by Catwalk (TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of alpha-Syn pathology in the midbrain of alpha-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with alpha-Syn and attenuates alpha-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies

In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats

Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG) and the receptor activator for nuclear factor-kappaB ligand (RANKL). Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA) molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery▿

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis

Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy

Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy

Therapeutic Drug Monitoring of Second- and Third-Generation Antipsychotic Drugs—Influence of Smoking Behavior and Inflammation on Pharmacokinetics

Both inflammation and smoking can influence a drug’s pharmacokinetic properties, i.e., its liberation, absorption, distribution, metabolism, and elimination. Depending on, e.g., pharmacogenetics, these changes may alter treatment response or cause serious adverse drug reactions and are thus of clinical relevance. Antipsychotic drugs, used in the treatment of psychosis and schizophrenia, should be closely monitored due to multiple factors (e.g., the narrow therapeutic window of certain psychotropic drugs, the chronicity of most mental illnesses, and the common occurrence of polypharmacotherapy in psychiatry). Therapeutic drug monitoring (TDM) aids with drug titration by enabling the quantification of patients’ drug levels. Recommendations on the use of TDM during treatment with psychotropic drugs are presented in the Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology; however, data on antipsychotic drug levels during inflammation or after changes in smoking behavior—both clinically relevant in psychiatry—that can aid clinical decision making are sparse. The following narrative review provides an overview of relevant literature regarding TDM in psychiatry, particularly in the context of second- and third-generation antipsychotic drugs, inflammation, and smoking behavior. It aims to spread awareness regarding TDM (most pronouncedly of clozapine and olanzapine) as a tool to optimize drug safety and provide patient-tailored treatment