Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

Comparison of Two Measurement Paradigms to Determine Electrically Evoked Cochlear Nerve Responses and Their Correlation to Cochlear Nerve Cross-section in Infants and Young Children With Cochlear Implant.

INTRODUCTION: Electrically evoked compound action potentials (ECAPs) are used for intra-/postoperative monitoring with intracochlear stimulation of cochlear implants. ECAPs are recorded in MED-EL (Innsbruck, Austria) implants using auditory response telemetry (ART), which has been further developed with automatic threshold determination as AutoART. The success of an ECAP measurement also depends on the number of available spiral ganglion cells and the bipolar neurons of the cochlear nerve (CN). It is assumed that a higher population of spiral ganglion cell implies a larger CN cross-sectional area (CSA), which consequently affects ECAP measurements. METHODS: Intraoperative ECAP measurements from 19 implanted ears of children aged 8 to 18 months were retrospectively evaluated. A comparison and correlation of ART/AutoART ECAP thresholds/slopes at electrodes E2 (apical), E6 (medial), E10 (basal), and averaged E1 to E12 with CN CSA on magnetic resonance imaging was performed. RESULTS: A Pearson correlation of the ART/AutoART ECAP thresholds/slopes for E2/E6/E10 and the averaged electrodes E1 to E12 showed a significant correlation. The CN CSA did not correlate significantly with the averaged ART/AutoART ECAP thresholds/slopes across all 12 electrodes. SUMMARY: AutoART provides reliable measurements and is therefore a suitable alternative to ART. No significant influence of CN CSA on ECAP thresholds/slopes was observed. A predictive evaluation of the success of ECAP measurements based on CN CSA for a clinical setting cannot be made according to the present data.

ART and AutoART ECAP measurements and cochlear nerve anatomy as predictors in adult cochlear implant recipients.

PURPOSE: The purpose of this retrospective study is to compare the results of electrically evoked compound action potential (ECAP) measurements using automatic auditory response telemetry (AutoART) with those obtained by ART in adults. The study also aimed to evaluate the predictive value of intraoperative ART and AutoART ECAPs for speech intelligibility (SI) and hearing success (HS), and to determine if cochlear nerve (CN) cross-sectional area (CSA) obtained preoperatively by magnetic resonance imaging (MRI) scans could predict ART and AutoART ECAPs and SI and HS outcome. METHODS: The study analyzed and correlated ART and AutoART ECAP thresholds at electrodes E2, E6, and E10, as well as averaged ECAP thresholds over electrodes E1-E12, using data from 32 implants. Correlations were also examined for ART and AutoART ECAP slopes. In addition, averaged ART and AutoART ECAP thresholds and slopes over all 12 electrodes for each participant were correlated with CN CSA measured from MRI sequences. SI of the monosyllabic Freiburg Speech Test at 65 dB sound pressure level was examined along with averaged ART and AutoART thresholds and slopes over all 12 electrodes. A parallel analysis was performed for HS, derived from the difference between baseline and 6-month SI. Finally, correlations between CN CSA and SI, as well as CN CSA and HS were examined. RESULTS: The results of the study showed a significant positive correlation between ART and AutoART ECAP thresholds and as well as slopes for E2, E6, E10 and averaged thresholds and slopes of E1-E12. However, no significant correlation was observed between ART and AutoART averaged ECAP thresholds and slopes and either SI and HS or CN CSA. Furthermore, no significant correlation was found between CN CSA and SI and HS. CONCLUSION: While AutoART is a reliable and safe program for measuring ECAPs in adults, the study found no preoperative prognostic information on intraoperative ECAP results using parameters extracted from current MRI sequences or pre-/intraoperative information on subsequent hearing outcome using ECAP and CN CSA.

In vitro screening technologies for the discovery and development of novel drugs against Toxoplasma gondii.

INTRODUCTION: Toxoplasmosis constitutes a challenge for public health, animal production and welfare. Since more than 60 years, only a limited panel of drugs has been in use for clinical applications. AREAS COVERED: Herein, the authors describe the methodology and the results of library screening approaches to identify inhibitors of Toxoplasma gondii and related strains. The authors then provide the reader with their expert perspectives for the future. EXPERT OPINION: Various library screening projects, in particular those using reporter strains, have led to the identification of numerous compounds with good efficacy and specificity in vitro. However, only few compounds are effective in suitable animal models such as rodents. Whereas no novel compound has cleared the hurdle to applications in humans, the few compounds with known indication and application profiles in human patients are of interest for further investigations. Taken together, drug repurposing as well as high-throughput screening of novel compound libraries may shorten the way to novel drugs against toxoplasmosis.

Single and combination treatment of Toxoplasma gondii infections with a bumped kinase inhibitor and artemisone in vitro and with artemiside in experimentally infected mice.

In previous studies, the artemisinin derivatives artemisone, its pro-drug artemiside and the bumped-kinase inhibitor BKI-1748 were effective against T. gondii via different modes of action. This suggests that they may act synergistically resulting in improved efficacies in vitro and in vivo. To test this hypothesis, the compounds were applied alone and in combination to T. gondii infected human fibroblast host cells in order to determine their inhibition constants and effects on cellular ultrastructure. In addition, the efficacy of either single- or combined treatments were assessed in an acute TgShSp1-oocyst infection model based on CD1 outbred mice. Whereas the IC50 of the compounds in combination (42 nM) was close to the IC50 of BKI-1748 alone (46 nM) and half of the IC50 of artemisone alone (92 nM), the IC90 of the combination was half of the values found with the single compounds (138 nM vs. ca. 270 nM). Another indication for synergistic effects in vitro were distinct alterations of the cellular ultrastructure of tachyzoites observed in combination, but not with the single compounds. These promising results could not be reproduced in vivo. There was no decrease in number of T. gondii positive brains by either treatment. However, the levels of infection in these brains, i. e. the number of tachyzoites, was significantly decreased upon BKI-1748 treatment alone, and the combination with artemiside did not produce any further decrease. The treatment with artemiside alone had no significant effects. A vertical transmission model could not be established since artemiside strongly interfered with pregnancy and caused abortion. These results show that is difficult to extrapolate from promising in vitro results to the situation in vivo.

Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains.

In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene.

[Speech perception as a function of cochlear coverage-comparison in bimodally hearing cochlear implant patients. German version]. / Sprachverstehen in Abhängigkeit von der cochleären Abdeckung ­ Vergleich bei bimodal versorgten Cochleaimplantatpatienten.

BACKGROUND: Hearing success in bimodally hearing patients with a cochlear implant (CI) and a hearing aid (HA) exhibits different results: while some benefit from bimodal CI and HA, others do not. OBJECTIVE: The aim of this study was to investigate hearing success in terms of speech perception in bimodally fitted patients in relation to the cochlear coverage (CC) of the CI electrodes. MATERIALS AND METHODS: Using the OTOPLAN software (CAScination AG, Bern, Switzerland), CC was retrospectively measured from CT scans of the temporal bone of 39 patients, who were then categorized into two groups: CC ≤ 65% (CC500) and CC > 65% (CC600). Monaural speech intelligibility for monosyllables at a sound pressure level (SPL) of 65 dB in open field was assessed at various timepoints, preoperatively with HA and postoperatively with CI, and compared between the groups. In addition, speech intelligibility was correlated with CC in the entire cohort before surgery and during follow-up (FU). RESULTS: Overall, no significant differences in speech intelligibility were found between CC500 and CC600 patients at any of the FU timepoints. However, both CC500 and CC600 patients showed a steady improvement in speech intelligibility after implantation. While CC600 patients tended to show an earlier improvement in speech intelligibility, CC500 patients tended to show a slower improvement during the first 3 months and a steeper learning curve thereafter. The two patient groups converged during FU, with no significant differences in speech intelligibility. There was no significant relationship between unimodal/unilateral free-field speech intelligibility and CC. However, patients with a CC of 70-75% achieved maximum speech intelligibility. CONCLUSION: Despite a nonsignificant correlation between CC and speech discrimination, patients seem to reach their maximum in unimodal/unilateral speech understanding mainly at 70-75% coverage. However, there is room for further investigation, as CC500 was associated with a shorter cochlear duct length (CDL), and long and very long electrodes were used in both groups.

[Revision surgery after stapedectomy]. / Revisionsoperationen nach Stapesplastik.

Revision stapes surgery is considered to be significantly more demanding than primary stapes surgery, both in terms of the indication and the surgical approach. This article reviews common indications for revision after stapedectomy as well as the surgical approaches and intraoperative findings. A distinction is made between revision surgeries, which are usually carried out because of conductive hearing loss a long time after stapes surgery, and acute or subacute revisions that become necessary in the immediate postoperative course. With the shortening of postoperative observation times under inpatient conditions as a result of increasing economization and the associated shift of the immediate postoperative phase to the outpatient setting, the recognition of postoperative irregularities is also becoming increasingly important for otorhinolaryngologists in private practice, even if they do not perform these highly specialized interventions themselves.

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours.

Toxoplasma gondii infection: novel emerging therapeutic targets.

INTRODUCTION: Toxoplasmosis constitutes a challenge for public health, animal production, and welfare. So far, only a limited panel of drugs has been marketed for clinical applications. In addition to classical screening, the investigation of unique targets of the parasite may lead to the identification of novel drugs. AREAS COVERED: Herein, the authors describe the methodology to identify novel drug targets in Toxoplasma gondii and review the literature with a focus on the last two decades. EXPERT OPINION: Over the last two decades, the investigation of essential proteins of T. gondii as potential drug targets has fostered the hope of identifying novel compounds for the treatment of toxoplasmosis. Despite good efficacies in vitro, only a few classes of these compounds are effective in suitable rodent models, and none has cleared the hurdle to applications in humans. This shows that target-based drug discovery is in no way better than classical screening approaches. In both cases, off-target effects and adverse side effects in the hosts must be considered. Proteomics-driven analyses of parasite- and host-derived proteins that physically bind drug candidates may constitute a suitable tool to characterize drug targets, irrespectively of the drug discovery methods.

Hotspots and future trends of phosphorus recycling from livestock manure: A bibliometric review.

In recent decades, the importance of managing the earth's dwindling phosphorus (P) has grown exponentially, as have efforts to develop a circular economy. Livestock manure represents a P-rich waste product, so recycling P from livestock manure has garnered the attention of scholars worldwide. Based on a global database from 1978 to 2021, this study presents the current status of recycling P from livestock manure and proposes strategies for efficient P utilization. Unlike traditional review articles, this work establishes a visual collaborative network on P recycling from livestock manure of research areas, countries, institutions, and authors through a bibliometric analysis using Citespace and VOSviewer software. The co-citation analysis of literature revealed the development of the main research content in this field, and further clustering analysis illustrated the current key research directions. Keyword co-occurrence analysis identified the hotspots and new frontiers of research in this field. According to the results, the United States was the most influential and actively contributing nation, and China was the country with the tightest international ties. The most popular research area was environmental science, and the Bioresource Technology published the largest number of papers in this area. The research priority was the technologies development of P recycling from livestock manure, of which the most used method was struvite precipitation and biochar adsorption. Subsequently, evaluation is also essential, including the economic benefits and environmental impacts of the recycling process by life cycle assessment and substance flow analysis, as well as the agronomic efficiency of the recycled products. New directions for technological innovation in recycling P from livestock manure and potential risks in the recycling process are explored. The results of this study may provide a framework for understanding the mechanisms of P utilization in livestock manure, and support the overall popularization of P recycling technology from livestock manure.

Low-cost drum granulator for mechanized seedball production.

Seed granulation is a coating technique, which turns a raw material mixture of sand, loam, water, seeds, and fertilizers into seedballs. It enhances the seedling establishment and early growth of crops, like pearl millet, in nutrient-poor soil. Mechanization is highly required, as large-scale production poses challenges to local farmers due to time constraints and labor demand. The prototype of a drum granulator for seeds, also known as a seedball machine, essentially consists of a metal frame and a drum. The seedballs are formed by a rotational motion of the drum. The construction and operation of the machine were designed to be simple. In this study, the combined effect of different factors, such as substrate composition, rotational speed and residence time was taken into account. This study revealed that the amount of loam and the rotational speed of the drum appeared to be the most influencing factors on seedball production and quality. The machine had a production capacity of seedballs ten times higher than manual production. The machine-made seedballs were also of high quality, exceeding 98% germination rate under greenhouse conditions. Besides pearl millet, the machine can be potentially used for other small-sized seeds, such as cotton or sesame.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis.

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Comparison of phosphorus species in livestock manure and digestate by different detection techniques.

Phosphorus (P) species characterize the effectiveness of the P fertilizer. In this study, the P species and distribution in different manures (pig manure, dairy manure and chicken manure) and their digestate were systematically investigated through combined characterization methods of Hedley fractionation (H2OP, NaHCO3-P, NaOH-P, HCl-P, and Residual), X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) techniques. The results from Hedley fractionation showed that >80 % of P in the digestate was inorganic and the HCl-P content in manure increased significantly during anaerobic digestion (AD). XRD manifested that insoluble hydroxyapatite and struvite belonging to HCl-P were presented during AD, which was in agreement with the result of Hedley fractionation. 31P NMR spectral analysis revealed that some orthophosphate monoesters were hydrolyzed during AD, meanwhile the orthophosphate diester organic phosphorus like DNA and phospholipids content has increased. After characterizing P species by combining these methods, it was found that chemical sequential extraction could be an effective way to fully understand the P in livestock manure and digestate, with other methods used as auxiliary tool depending on the purpose of studies. Meanwhile, this study provided a basic knowledge of utilizing digestate as P fertilizer and minimizing the risk of P loss from livestock manure. Overall, applying digestates can minimize the risk of P loss from directly applied livestock manure while satisfying plant demands, and is an environmentally friendly P fertilizer.

Determining the heat of desorption for cassava products based on data measured by an automated gravimetric moisture sorption system.

BACKGROUND: The isosteric heat of desorption is vital in evaluating the energy performance of food dryers. The isosteric heat of desorption was investigated for different cassava (Manihot esculenta Crantz) products prepared as flour or starch, with and without fermentation. An automated moisture sorption gravimetric analyser was used to measure the desorption isotherms over 10-90% relative humidity of the drying air at temperatures ranging from 25 to 65 °C. RESULTS: Analysis of variance showed an imperceptible contribution of the preparation method in the measured desorption data. This finding also agreed with microscopical images, which revealed the lack of compelling structural differences among different products. A set of empirical sorption equations suggested by the ASAE standard was examined over the measured desorption isotherms. The standard error of estimation was found to be in the acceptable range of 2.36-3.71%. Furthermore, the fulfilment of the enthalpy-entropy compensation theory was considered as an additional criterion in the thermodynamic results of different sorption equations, besides their fitting adequacy. The modified Chung-Pfost equation has proved to be the most suitable equation for cassava products, as it is capable of reflecting the temperature dependency of the isosteric heat of desorption. The net isosteric heat of desorption obtained was in the range of 540-1110 kJ kg-1 for 0.10 kg kg-1 dry-basis moisture content and 52-108 kJ kg-1 for 0.25 kg kg-1 dry-basis moisture content. CONCLUSION: These findings are technologically relevant for optimising common drying technologies such as flash and flatbed dryers. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation.

The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 µM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin.

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.

Adult Cochlear Implant Users Versus Typical Hearing Persons: An Automatic Analysis of Acoustic-Prosodic Parameters.

PURPOSE: The aim of this study was to investigate the speech prosody of postlingually deaf cochlear implant (CI) users compared with control speakers without hearing or speech impairment. METHOD: Speech recordings of 74 CI users (37 males and 37 females) and 72 age-balanced control speakers (36 males and 36 females) are considered. All participants are German native speakers and read Der Nordwind und die Sonne (The North Wind and the Sun), a standard text in pathological speech analysis and phonetic transcriptions. Automatic acoustic analysis is performed considering pitch, loudness, and duration features, including speech rate and rhythm. RESULTS: In general, duration and rhythm features differ between CI users and control speakers. CI users read slower and have a lower voiced segment ratio compared with control speakers. A lower voiced ratio goes along with a prolongation of the voiced segments' duration in male and with a prolongation of pauses in female CI users. Rhythm features in CI users have higher variability in the duration of vowels and consonants than in control speakers. The use of bilateral CIs showed no advantages concerning speech prosody features in comparison to unilateral use of CI. CONCLUSIONS: Even after cochlear implantation and rehabilitation, the speech of postlingually deaf adults deviates from the speech of control speakers, which might be due to changed auditory feedback. We suggest considering changes in temporal aspects of speech in future rehabilitation strategies. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.21579171.

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.