Role of parC Mutations at Position 84 on High-Level Delafloxacin Resistance in Methicillin-Resistant Staphylococcus aureus.

High-level delafloxacin-resistant (H-L DLX-R) Staphylococcus aureus isolates (minimum inhibitory concentration ≥1 mg/L) associated with mutations affecting position 84 of ParC have emerged. We aimed to elucidate the role of these mutations as a mechanism of H-L DLX resistance in methicillin-resistant S. aureus (MRSA) isolates recovered from blood cultures. Susceptibility to DLX was determined in 75 MRSA isolates by E-test, and an rt-PCR was developed to detect mutations affecting position 84 of ParC to screen a further 185 MRSA isolates. The genomes of 48 isolates, including all DLX-R isolates or with alterations at position 84, and also a subset of DLX-susceptible isolates were analyzed. Among the 75 isolates studied, 77.34% were DLX-susceptible and only 4 H-L DLX-R isolates were found. Seven (3.8%) isolates with alterations at position 84 of ParC were detected by rt-PCR. Genomic analysis showed that 89.9% (8/9) of isolates with the substitution E84K/G in ParC, together with other mutations in gyrA and parC, were H-L DLX-R. However, the E84K substitution in ParC alone or with other alterations was found in two isolates without H-L DLX-R. Alterations at position 84 of ParC are rare but play a key role in H-L DLX resistance in MRSA but only when other alterations in GyrA are present.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates.

BACKGROUND: The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. RESULTS: Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6')-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. CONCLUSIONS: Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Multi-Facet analysis of analytical and numerical models to resolve sustainable artificial recharge rates in unconfined aquifers.

Managed aquifer recharge (MAR) has emerged as a potential solution to resolve water insecurity, globally. However, integrated studies quantifying the surplus source water, suitable recharge sites and safe recharge capacity is limited. In this study, a novel methodology is presented to quantify transient injection rates in unconfined aquifers and generate MAR suitability maps based on estimated surplus water and permissible aquifer recharge capacity (PARC). Subbasin scale monthly surplus surface runoff was estimated at 75% dependability using a SWAT model. A linear regression model based on numerical solution was used to capture the aquifer response to injection and to calculate PARC values at subbasin level. The available surplus runoff and PARC values was then used to determine the suitable site and recharge rate during MAR operation. The developed methodology was applied in the semi-arid region of Lower Betwa River Basin (LBRB), India. The estimated surplus runoff was generally confined to the monsoon months of June to September and exhibited spatial heterogeneity with an average runoff rate of 5000 m3/d in 85% of the LBRB. Analysis of the PARC results revealed that thick alluvial aquifers had large permissible storage capacity and about 50% of the LBRB was capable of storing over 3500 m3/d of water. This study revealed that sufficient surplus runoff was generated in the LBRB, but it lacked the adequate safe aquifer storage capacity to conserve it. A total 65 subbasins was identified as the best suited sites for MAR which had enough surplus water and storage capacity to suffice 20% of the total water demand in the LBRB. The developed methodology was computationally efficient, could augment the field problem of determining scheduled recharge rates and could be used as a decision-making tool in artificial recharge projects.

Nondestructive testing of runny salted egg yolk based on improved ConvNeXt-T.

Salted egg yolks from salted duck eggs are widely utilized in the domestic and international food industry as both raw materials and ingredients. When salted egg yolks are not fully cured and matured, they exist in a fluid state, with a mixture of solid and liquid internally. Due to this composition, they are susceptible to deterioration during storage and usage, necessitating their detection and classification. In this study, a dataset specifically for salted egg yolks was established, and the ConvNeXt-T model, employed as the benchmark model, underwent two notable improvements. First, a lightweight location-aware circular convolution (ParC) was introduced, utilizing a ParC-block to replace a portion of the original ConvNeXt-T block. This enhancement aimed to overcome the limitations of convolution in extracting global feature information while integrating the global sensing capability of vision transformer and the localization capability of convolution. Additionally, the activation function was modified through substitution. These improvements resulted in the final model. Experimental results indicate that the enhanced model exhibits faster convergence on the custom salted egg yolk dataset compared to the baseline model. Furthermore, a significant reduction of model parameters by a factor of 4 led to a 2.167 percentage point improvement in the accuracy of the test set. The ParC-ConvNeXt-SMU-T model achieved an accuracy of 96.833% with 26.8 million parameters. Notably, the improved model demonstrates exceptional effectiveness in recognizing salted egg yolks. PRACTICAL APPLICATION: This study can be widely applied in the process of salted egg yolk production and quality inspection, which can improve the actual sorting efficiency of salted egg yolks and reduce the labor cost at the same time. It can also be used for nondestructive testing of salted egg yolks by governmental enterprises and other regulatory authorities.

Chemokine ligand 18 predicts all-cause mortality in patients hospitalized with chest pain of suspected coronary origin.

Introduction: Chemokines mediate recruitment and activation of leucocytes. Chemokine ligand 18 (CCL18) is mainly expressed by monocytes/macrophages and dendritic cells. It is highly expressed in chronic inflammatory diseases, and locally in atherosclerotic plaques, particularly at sites of reduced stability, and systemically in acute coronary syndrome patients. Reports on its prognostic utility in the latter condition, including myocardial infarction (MI), are scarce. Aim: To assess the utility of CCL18 as a prognostic marker of recurrent cardiovascular events in patients hospitalized with chest pain of suspected coronary origin. Methods: The population consisted of 871 consecutive chest-pain patients, of whom 386 were diagnosed with acute myocardial infarction (AMI) based on Troponin-T (TnT) levels >50 ng/L. Stepwise Cox regression models, applying normalized continuous loge/SD values, were fitted for the biomarkers with cardiac mortality within 2 years and total mortality within 2 and 7 years as the dependent variables. Results: Plasma samples from 849 patients were available. By 2 years follow-up, 138 (15.8%) patients had died, of which 86 were cardiac deaths. Univariate analysis showed a positive, significant association between CCL18 and total death [HR 1.55 (95% 1.30-1.83), p < 0.001], and for cardiac death [HR 1.32 (95% 1.06-1.64), p = 0.013]. Associations after adjustment were non-significant. By 7 years follow-up, 332 (38.1%) patients had died. CLL18 was independently associated with all-cause mortality [HR 1.14 (95% CI, 1.01-1.29), p = 0.030], but not with MI (n = 203) or stroke (n = 55). Conclusion: CCL18 independently predicts long-term all-cause mortality but had no independent prognostic bearing on short-term cardiac death and CVD events.

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Adaptive evolution of chloroplast division mechanisms during plant terrestrialization.

Despite extensive research, the origin and evolution of the chloroplast division machinery remain unclear. Here, we employ recently sequenced genomes and transcriptomes of Archaeplastida clades to identify the core components of chloroplast division and reconstruct their evolutionary histories, respectively. Our findings show that complete division ring structures emerged in Charophytes. We find that Glaucophytes experienced strong selection pressure, generating diverse variants adapted to the changing terrestrial environments. By integrating the functions of chloroplast division genes (CDGs) annotated in a workflow developed using large-scale multi-omics data, we further show that dispersed duplications acquire more species-specific functions under stronger selection pressures. Notably, PARC6, a dispersed duplicate CDG, regulates leaf color and plant growth in Solanum lycopersicum, demonstrating neofunctionalization. Our findings provide an integrated perspective on the functional evolution of chloroplast division machinery and highlight the potential of dispersed duplicate genes as the primary source of adaptive evolution of chloroplast division.

Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.

Comparison of gradient diffusion and molecular methods using Allplex™ NG&DR assay (Seegene®) for macrolide and fluoroquinolone screening resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1 mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06 mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods.

Detection of macrolide and fluoroquinolone resistance-associated 23S rRNA and parC mutations in Mycoplasma genitalium by nested real-time PCR.

Background: Traditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge. Objective: Sequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance. Results: 105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001). Conclusions: The results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.

Changes in eotaxin-3 and pulmonary and activation-regulated chemokine levels in patients after dupilumab treatment: a systematic review and meta-analysis.

Introduction: Dupilumab is approved for a variety of type 2 inflammatory diseases. Changes in chemokine levels during treatment require further analysis. Aim: We evaluated changes in eotaxin-3 and PARC levels after dupilumab treatment through a meta-analysis, aiming to provide more comprehensive results. Material and methods: Databases were searched to select eligible publications. The study quality was assessed after inclusion. The standardized mean difference (SMD) was used for evaluation. Results: Four studies were included. Eotaxin-3 levels were not seen significantly decreased at weeks 1 and 12, with SMD = -0.39 (95% CI: -1.78, 0.99) and -2.60 (95% CI: -5.77, 0.57), respectively (p > 0.05). Eotaxin-3 levels decreased significantly at weeks 2, 4, 8, 16, 24, 36, and 52, with SMD = -0.94 (95% CI: -1.61, -0.27); -1.17 (95% CI: -1.49, -0.84); -1.20 (95% CI: -1.52, -0.88); -1.31 (95% CI: -1.83, -0.79); -4.57 (95% CI: -6.90, -2.33); -5.28 (95% CI: -5.52, -5.04); and -4.03 (95% CI: -4.22, -3.85) (p < 0.05), respectively. PARC levels decreased significantly at weeks 4, 8, 12, and 16, with SMD = -1.08 (95% CI: -1.59, -0.58); -1.17 (95% CI: -1.68, -0.66); -1.11 (95% CI: -1.61, -0.60); and -1.15 (95% CI: -1.66, -0.64) (p < 0.05), respectively. Conclusions: Eotaxin-3 and PARC levels can be significantly reduced in patients treated with dupilumab.

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology.

The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

BlaTEM-positive Salmonella enterica serovars Agona and Derby are prevalent among food-producing animals in Chongqing, China.

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of ß-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of ß-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6')-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with ß-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Molecular and Functional Characterization of Different BrainSphere Models for Use in Neurotoxicity Testing on Microelectrode Arrays.

The currently accepted methods for neurotoxicity (NT) testing rely on animal studies. However, high costs and low testing throughput hinder their application for large numbers of chemicals. To overcome these limitations, in vitro methods are currently being developed based on human-induced pluripotent stem cells (hiPSC) that allow higher testing throughput at lower costs. We applied six different protocols to generate 3D BrainSphere models for acute NT evaluation. These include three different media for 2D neural induction and two media for subsequent 3D differentiation resulting in self-organized, organotypic neuron/astrocyte microtissues. All induction protocols yielded nearly 100% NESTIN-positive hiPSC-derived neural progenitor cells (hiNPCs), though with different gene expression profiles concerning regional patterning. Moreover, gene expression and immunocytochemistry analyses revealed that the choice of media determines neural differentiation patterns. On the functional level, BrainSpheres exhibited different levels of electrical activity on microelectrode arrays (MEA). Spike sorting allowed BrainSphere functional characterization with the mixed cultures consisting of GABAergic, glutamatergic, dopaminergic, serotonergic, and cholinergic neurons. A test method for acute NT testing, the human multi-neurotransmitter receptor (hMNR) assay, was proposed to apply such MEA-based spike sorting. These models are promising tools not only in toxicology but also for drug development and disease modeling.

Prevalence of Mycoplasma genitalium infection with antimicrobial resistance mutations among gay sex workers in China.

BACKGROUND: Gay sex workers (GSWs) within the population of men who have sex with men in China are known as money boys (MBs). Limited research has been conducted to investigate the infection rate and antimicrobial resistance of Mycoplasma genitalium (M. genitalium) among GSWs in China. This study aimed to evaluate the status of M. genitalium infection among in GSWs. METHODS: This study was performed among 349 GSWs who were followed up for four years by internet-based sampling collection. The participants were asked to complete an online questionnaire using a mobile app, and trained interviewers took urethral, anorectal, and saliva swab specimens. STIs, including HIV and M. genitalium, were detected. Detection of resistance-associated mutations (RAMs) to macrolides and fluoroquinolones was performed via Sanger sequencing of the 23S rRNA, parC and gyrA genes. RESULTS: GSWs were enrolled by identifying 10 initial "seeds" from the Blued and WeChat apps. Face-to-face interviews were conducted with 349 GSWs from June 2017 to July 2021. The prevalence of M. genitalium and HIV positivity was 92/349 (26.4%, 95% confidence interval [CI] 21.7-31.0) and 71/349 (20.3%, 95% CI 16.3-24.4), respectively. The proportion of GSWs with M. genitalium infection alone in urethral swabs was 16, and the proportion with symptoms was 2/16 (12.5%). The proportion of GSWs with M. genitalium infection alone in anorectal swabs was 36, and the proportion with symptoms was 3/36 (8.3%). Multivariate regression analysis showed that using new types of drugs in the past 3 months and inconsistent condom usage with clients in the past 30 days were associated with M. genitalium infection. Macrolide resistance within the 23S rRNA gene was detected in 73/88 (83.0%) of the M. genitalium-positive GSWs. Moreover, 79.8% (71/89) of parC and 21.1% (19/90) of gyrA genes had mutations responsible for fluoroquinolone resistance. Three cases had no mutations in any of the three genes, 11 cases had mutations in all three genes, five cases had gyrA and parC gene mutations with no mutation in the 23S rRNA gene, and 42 cases had 23S rRNA and parC gene mutations with no mutation in the gyrA gene. CONCLUSION: M. genitalium infections in our study displayed a high prevalence and very high levels of macrolide and fluoroquinolone resistance among GSWs in China. Asymptomatic M. genitalium infections are quite common among GSWs. Routine resistance testing of M. genitalium-positive specimens and antimicrobial resistance surveillance are crucial.

Fluoroquinolone-resistance mechanisms and molecular epidemiology of ciprofloxacin-resistant Klebsiella pneumoniae isolates in Iran.

Klebsiella pneumoniae is an important cause of nosocomial infections and displays increasing resistance to fluoroquinolones (FQ). This study surveyed the mechanisms of FQ resistance and molecular typing of K. pneumoniae isolates from intensive care units patients in Tehran, Iran. A total of 48 ciprofloxacin (CIP) resistant K. pneumoniae isolates from urine samples were included in this study. Broth microdilution assays revealed high-level CIP resistance (MIC > 32 µg/mL) in 31.25% of the isolates. Plasmid-mediated quinolone resistance genes were detected in 41 (85.4%) isolates. Among which, qnrS (41.67%) was the most prevalent followed by qnrD (35.42%), qnrB (27.1%), qnrA (25%), qepA (22.9%), aac(6')-Ib-cr (20.83%), and qnrC (6.25%). Target site mutations (gyrA and parC) were assessed using PCR and sequencing on all isolates. A single mutation in gyrA (S83I) was found in 13 (27.1%) isolates and two isolates harbored six simultaneous mutations. Fourteen isolates (29.2%) had mutations in parC and S129A and A141V mutations were the most prevalent. Real time PCR showed an increase in the expression level of acrB and oqxB efflux genes in 68.75 and 29.16% isolates, respectively. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed 14 genotypes and 11 of them were classified by multilocus sequence typing (MLST) into 11 different sequence types belonging to seven clonal complexes and two singletons, most of them have not been reported in Iran yet. We are concerned about the spread of these clones throughout our country. Most FQ resistance mechanisms were detected among our isolates. However, target site mutation had the greatest effect on CIP resistance among our isolates.