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Abstract Objective We aim to validate and evaluate a new rapid and simplified method, called Blood-rsCDM, for the detection and characterization of carbapenemase using 3-aminophenylboronic acid (APBA) and ethylenediaminetetraacetic acid (EDTA) β-lactamase inhibitors from positive blood cultures. Method We utilized a panel of 172 Enterobacterales strains, including blaKPC (77), blaNDM (48), blaIMP (9), blaVIM (2), blaOXA-181 (2), blaKPC and blaNDM (6), as well as 28 carbapenem-susceptible Enterobacterales isolates, to assess the performance of Blood-rsCDM and the EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined using specific inhibitors at 4 h and 6 h by Blood-rsCDM. Results Blood-rsCDM exhibited a sensitivity of 97.9% at both time points, with a specificity of 100%, regardless of the culture duration. The sensitivity of eCIM was 94.4%, with a specificity of 100%. Blood-rsCDM accurately characterized KPC-producing isolates as 77/77, metallo-β-lactamases (MBLs) as 58/59, and KPC and NDM carbapenemases as 6/6 at 4 h. There was no difference in results between the 4 h and 6 h time points. However, Blood-rsCDM could not differentiate OXA-181-producing strains. For eCIM, the characterization numbers for KPC-, OXA-181-, and MBLs-producing strains were 77/77, 2/2, and 57/59, respectively, but it failed to detect the coproduction of KPC and NDM isolates. Conclusion Blood-rsCDM accurately discriminates carbapenemase within 4 h and is capable of directly differentiating multi-enzyme (KPC and NDM) presence from positive blood culture broths. Therefore, Blood-rsCDM represents a rapid, simple, easy-to-read, and accurate tool that can be utilized in resource-limited settings.

The clinical role of Acinetobacter baumannii has been highlighted in numerous infectious syndromes with a high mortality rate, due to the high prevalence of multidrug-resistant (MDR) isolates. The treatment and eradication of this pathogen is hindered by biofilm-formation, providing protection from noxious environmental factors and antimicrobials. The aim of this study was to assess the antibiotic susceptibility, antiseptic susceptibility and biofilm-forming capacity using phenotypic methods in environmental A. baumannii isolates. One hundred and fourteen (n = 114) isolates were collected, originating from various environmental sources and geographical regions. Antimicrobial susceptibility testing was carried out using the disk diffusion method, while antiseptic susceptibility was performed using the agar dilution method. Determination of biofilm-forming capacity was carried out using a microtiter-plate based method. Resistance in environmental A. baumannii isolates were highest for ciprofloxacin (64.03%, n = 73), levofloxacin (62.18%, n = 71) and trimethoprim-sulfamethoxazole (61.40%, n = 70), while lowest for colistin (1.75%, n = 2). Efflux pump overexpression was seen in 48.25% of isolates (n = 55), 49.12% (n = 56) were classified as MDR. 6.14% (n = 7), 9.65% (n = 11), 24.65% (n = 28) and 59.65% (n = 68) of isolates were non-biofilm producers, weak, medium, and strong biofilm producers, respectively. No significant differences were observed between non-MDR vs. MDR isolates regarding their distribution of biofilm-producers (P = 0.655). The MIC ranges for the tested antiseptics were as follows: benzalkonium chloride 16–128 μg mL−1, chlorhexidine digluconate 4–128 μg mL−1, formaldehyde 64–256 μg mL−1 and triclosan 2–16 μg mL−1, respectively. The conscientious use of antiseptics, together with periodic surveillance, is essential to curb the spread of these bacteria, and to maintain current infection prevention capabilities. [ABSTRACT FROM AUTHOR]

Genetically engineered mouse models have the potential to unravel fundamental biological processes and provide mechanistic insights into the pathogenesis of human diseases. We have previously observed that germline genetic variation at the TULP4 locus influences clinical characteristics in patients with myeloproliferative neoplasms. To elucidate the role of TULP4 in pathological and physiological processes in vivo, we generated a Tulp4 knockout mouse model. Systemic Tulp4 deficiency exerted a strong impact on embryonic development in both Tulp4 homozygous null (Tulp4−/−) and heterozygous (Tulp4+/−) knockout mice, the former exhibiting perinatal lethality. High‐resolution episcopic microscopy (HREM) of day 14.5 embryos allowed for the identification of multiple developmental defects in Tulp4−/− mice, including severe heart defects. Moreover, in Tulp4+/− embryos HREM revealed abnormalities of several organ systems, which per se do not affect prenatal or postnatal survival. In adult Tulp4+/− mice, extensive examinations of hematopoietic and cardiovascular features, involving histopathological surveys of multiple tissues as well as blood counts and immunophenotyping, did not provide evidence for anomalies as observed in corresponding embryos. Finally, evaluating a potential obesity‐related phenotype as reported for other TULP family members revealed a trend for increased body weight of Tulp4+/− mice. Research Highlights: To study the role of the TULP4 gene in vivo, we generated a Tulp4 knockout mouse model.Correlative analyses involving HREM revealed a strong impact of Tulp4 deficiency on murine embryonic development. [ABSTRACT FROM AUTHOR]

The Protein- O -mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis , the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O -mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis , severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O -mannosyltransferase activity in bacteria , in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily. [ABSTRACT FROM AUTHOR]

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

Tau prions feature in the brains of patients suffering from Alzheimer's disease and other tauopathies. For the development of therapeutics that target the replication of tau prions, a high-content, fluorescence-based cell assay was developed. Using this high-content phenotypic screen for nascent tau prion formation, a 4-piperazine isoquinoline compound (1) was identified as a hit with an EC50 value of 390 nM and 0.04 K p,uu. Analogs were synthesized using a hypothesis-based approach to improve potency and in vivo brain penetration resulting in compound 25 (EC50 = 15 nM; K p,uu = 0.63). We investigated the mechanism of action of this series and found that a small set of active compounds were also CDK8 inhibitors.

Djin-Ye Oh, Jeanette Milde, Youngsun Ham, Julia Patricia Ramos Calderón, Marianne Wedde, Ralf Dürrwald, Susanne C Duwe Robert Koch Institute, Department 1: Infectious Diseases, Unit 17: Influenza and Other Respiratory Viruses, National Influenza Center, Berlin, GermanyCorrespondence: Susanne C Duwe, Robert Koch Institute, Department 1: Infectious Diseases, Unit 17: Influenza Viruses and Other Respiratory Viruses | National Influenza Center, Seestr. 10, Berlin, 13353, Germany, Tel +49 30 18754 2283, Fax +49 30 18754 2699, Email DuweS@rki.dePurpose: The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized.Methods: Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established.Results: While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥ 5-fold (influenza B) increase of NAI IC50, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC50 increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant.Conclusion: Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.Keywords: influenza viruses, antiviral resistance, phenotypic assay, genotypic assay, surveillance, neuraminidase, cap-dependent endonuclease, baloxavir marboxil, molecular resistance marker

Activated glia are known to exhibit either neuroprotective or neurodegenerative effects, depending on their phenotype, while participating in chronic pain regulation. Until recently, it has been believed that satellite glial cells and astrocytes are electrically slight and process stimuli only through intracellular calcium flux that triggers downstream signaling mechanisms. Though glia do not exhibit action potentials, they do express both voltage- and ligand-gated ion channels that facilitate measurable calcium transients, a measure of their own phenotypic excitability, and support and modulate sensory neuron excitability through ion buffering and secretion of excitatory or inhibitory neuropeptides (i.e., paracrine signaling). We recently developed a model of acute and chronic nociception using co-cultures of iPSC sensory neurons (SN) and spinal astrocytes on microelectrode arrays (MEAs). Until recently, only neuronal extracellular activity has been recorded using MEAs with a high signal-to-noise ratio and in a non-invasive manner. Unfortunately, this method has limited compatibility with simultaneous calcium transient imaging techniques, which is the most common method for monitoring the phenotypic activity of astrocytes. Moreover, both dye-based and genetically encoded calcium indicator imaging rely on calcium chelation, affecting the culture's long-term physiology. Therefore, it would be ideal to allow continuous and simultaneous direct phenotypic monitoring of both SNs and astrocytes in a high-to-moderate throughput non-invasive manner and would significantly advance the field of electrophysiology. Here, we characterize astrocytic oscillating calcium transients (OCa2+Ts) in mono- and co-cultures of iPSC astrocytes as well as iPSC SN-astrocyte co-cultures on 48 well plate MEAs. We demonstrate that astrocytes exhibit OCa2+Ts in an electrical stimulus amplitude- and duration-dependent manner. We show that OCa2+Ts can be pharmacologically inhibited with the gap junction antagonist, carbenoxolone (100 µM). Most importantly, we demonstrate that both neurons and glia can be phenotypically characterized in real time, repeatedly, over the duration of the culture. In total, our findings suggest that calcium transients in glial populations may serve as a stand-alone or supplemental screening technique for identifying potential analgesics or compounds targeting other glia-mediated pathologies. [ABSTRACT FROM AUTHOR]

Activated glia are known to exhibit either neuroprotective or neurodegenerative effects, depending on their phenotype, while participating in chronic pain regulation. Until recently, it has been believed that satellite glial cells and astrocytes are electrically slight and process stimuli only through intracellular calcium flux that triggers downstream signaling mechanisms. Though glia do not exhibit action potentials, they do express both voltage- and ligand-gated ion channels that facilitate measurable calcium transients, a measure of their own phenotypic excitability, and support and modulate sensory neuron excitability through ion buffering and secretion of excitatory or inhibitory neuropeptides (i.e., paracrine signaling). We recently developed a model of acute and chronic nociception using co-cultures of iPSC sensory neurons (SN) and spinal astrocytes on microelectrode arrays (MEAs). Until recently, only neuronal extracellular activity has been recorded using MEAs with a high signal-to-noise ratio and in a non-invasive manner. Unfortunately, this method has limited compatibility with simultaneous calcium transient imaging techniques, which is the most common method for monitoring the phenotypic activity of astrocytes. Moreover, both dye-based and genetically encoded calcium indicator imaging rely on calcium chelation, affecting the culture’s long-term physiology. Therefore, it would be ideal to allow continuous and simultaneous direct phenotypic monitoring of both SNs and astrocytes in a high-to-moderate throughput non-invasive manner and would significantly advance the field of electrophysiology. Here, we characterize astrocytic oscillating calcium transients (OCa2+Ts) in mono- and co-cultures of iPSC astrocytes as well as iPSC SN-astrocyte co-cultures on 48 well plate MEAs. We demonstrate that astrocytes exhibit OCa2+Ts in an electrical stimulus amplitude- and duration-dependent manner. We show that OCa2+Ts can be pharmacologically inhibited with the gap junction antagonist, carbenoxolone (100 μM). Most importantly, we demonstrate that both neurons and glia can be phenotypically characterized in real time, repeatedly, over the duration of the culture. In total, our findings suggest that calcium transients in glial populations may serve as a stand-alone or supplemental screening technique for identifying potential analgesics or compounds targeting other glia-mediated pathologies.

Background: Carbapenem resistance among gram negative bacilli (GNB) is a major threat to human health. Rapid and accurate detection of carbapenemase production is very important for clinical management, epidemiological purposes and infection prevention and control issues. Direct Carba NP test (CNPt) is a modified CNPt that allows rapid detection of carbapenemases at a lower cost and improved sensitivity. This work aimed to evaluate direct-CNPt for detection of cabapenemases production as a screening and confirmatory test in GNB versus multiplex PCR as the gold standard test. Methods: This study was conducted on 50 clinical isolates of GNB derived from different clinical samples according to their resistance to meropenem (zone of inhibition ≤ 23 mm). Direct CNPt was performed on all 50 clinical isolates using bacterial colonies directly. A change in colour was observed after (̰ 2 h). A multiplex PCR was performed on all isolates to detect bla KPC, bla IMP, bla VIM and bla NDM, and bla OXA-48 genes. Results: The overall sensitivity and specificity of direct CNPt as compared to multiplex PCR were 95.2% and 100% respectively. The sensitivity to OXA-48 and IMP genes were relatively lower (91.6 % and 85.7 % respectively) than other genes which had 100% sensitivity for each. Conclusion: Direct CNPt is a reliable and rapid method that allows detection of different carbapenemases at a reduced cost. It could be used in combination with other phenotypic or genotypic assays in settings where OXA-48 and IMP are of high prevalence.

Background: Worldwide, multi-drug resistant Klebsiella pneumoniae (K. pneumonia) and their virulence’s were contributed more in the multi-drug resistant effect. According to the World Health organization report, it is an emerging thread in developing countries and also comes under first ever critical list. In this context, the current study was concentrated on detection of extended spectrum beta lactamase (ESBL) producing strain and their antimicrobial susceptibility study of K. pneumoniae. Materials and methods: Firstly, the multi-drug resistant effect of the K. pneumoniae was identified from specific CLSI guidelines recommended antibiotics by disc diffusion method. Consecutively, the primary ESBL identification test was performed using ceftazidime and cefotaxime, followed by double disc combination and phenotypic confirmation tests using ceftazidime/clavulanic acid and cefotaxime/clavulanic acid. Finally, the minimum inhibition concentration of some important sensitive antibiotics were performed against selected K. pneumoniae was confirmed by micro broth dilution method. Results and conclusions: The current result was most favorable to selected K. pneumoniae with more multi drug resistant characteristic nature. All the performed antibiotics were almost more sensitive to selected K. pneumoniae. The effective antibiotics of piperacillin was also exhibited more resistant effect against tested bacteria and it cleaved the bacterial enzyme clearly. The present result of primary ESBL identification test result was exhibited with ≤22 mm and ≤27 mm against ceftazidime and cefotaxime were observed respectively. Followed result of double disc combination and phenotypic confirmation experiments results were clearly stated that the selected K. pneumoniae was ESBL producer. The ceftazidime, cefotaxime and ceftazidime/clavulanic acid and cefotaxime/clavulanic acid were exhibited with merged zones and ≥5 mm zones around the combination disc when compared with disc alone were observed. All the ESBL detection test results were clearly indicated that the selected K. pneumoniae strain was ESBL producer.

Background: Recent years, the treatment of multi-drug resistant bacteria and their effect are very difficult due to the virulence factors modification. Based on the world wide thread, we have tried to identify the ESBLs producing bacteria from urinary tract infection patients. In result, the highly antibiotic resistant effect of Pseudomonas aeruginosa and Klebsiella pneumoniae were identified. Methods: Initially, Hexa disc diffusion method was performed to detect the multi-drug resistant bacteria using respective antibiotics of HX066, HX033 and HX077, HX012 discs. Consecutively, the ESBL producing ability of confirmed multi drug resistant bacteria was performed to detect their ESBL producing ability using specific extended spectrum beta lactamase (ESBLs) detection discs of Hexa G-minus 24. Furthermore, the ESBL producing ability of the bacteria was confirmed by ESBLs detection Ezy MIC™ E-test stripe method. Results and conclusions: In result, 10, 5 and 4 mm and 10, 14 and 8 mm zone of inhibition against imipenem (IPM), Ticarcillin/Clavulanic acid (TCC), Cefoperazone (CPZ) and Ampicillin (AMP), Norfloxacin (NX), Nalidixic acid (NA) antibiotics for P. aeruginosa and 16, 22 and 10, 18 mm zone of inhibition against ceftazidime (CAZ), methicillin (MET), ampicillin amoxyclav (AMC), co-trimoxazole (COT) of the HX077 HX012 for K. pneumoniae were observed. Based on the Clinical & Laboratory Standards Institute (CLSI) guidelines, both the bacteria were more resistant to tested antibiotics and it could be developed more resistant against all the tested antibiotics. In addition, the phenotypic detection of ESBL production effect was also performed against both the selected uropathogens, and the results were shown ≥22 mm, ≥27 zone of inhibition against all the tested antibiotics. Further, the genetic identification of multi plux PCR result was shown TEM, SHV and CTX-m genes were present in both the selected uropathogens. Finally, our results were correlated each other and concluded that the selected uropathogens were multi drug resistant effect and also ESBLs producer.

Mitophagy is the selective degradation of mitochondria by autophagy. It promotes the turnover of mitochondria and prevents the accumulation of dysfunctional mitochondria, which can lead to cellular degeneration. Mitophagy is known to be altered in several pathological conditions, especially in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We recently demonstrated an increase in autophagy flux in lymphoblasts from ALS patients bearing a mutation in SOD1. Thus, the identification of mitophagy inhibitors may be a therapeutic option to recover mitochondrial homeostasis. Here, using a phenotypic mitophagy assay, we identified a new mitophagy inhibitor, the small molecule named IGS2.7 from the MBC library. Interestingly, the treatment of different cellular and in vivo models of ALS with mutations on SOD1 and TARDBP with this inhibitor restores autophagy to control levels. These results point mitophagy inhibitors, especially IGS2.7, to a new therapeutic approach for familial ALS patients. [ABSTRACT FROM AUTHOR]

The formation of a protective biofilm by Pseudomonas aeruginosa (PA) is one of the hallmarks of their survival both in vivo and in harsh environmental conditions, thus, biofilm-eradication has relevance from therapeutic perspectives and for infection control. The aim of our study was to investigate the possible relationship between antibiotic resistance, biofilm-forming capacity and virulence factors in n = 166 PA isolates of environmental origin. Antimicrobial susceptibility testing and the phenotypic detection of resistance determinants were carried out using standard protocols. The biofilm-forming capacity of PA was tested using a standardized crystal violet microtiter plate-based method. Motility (swimming, swarming, and twitching) and siderophore production of the isolates were also assessed. Resistance rates were highest for ciprofloxacin (46.98%), levofloxacin (45.18%), ceftazidime (31.92%) and cefepime (30.12%); 19.28% of isolates met the criteria to be classified as multidrug-resistant (MDR). Efflux pump overexpression, AmpC overexpression, and modified Hodge-test positivity were noted in 28.31%, 18.07% and 3.61%, respectively. 22.89% of isolates were weak/non-biofilm producers, while 27.71% and 49.40% were moderate and strong biofilm producers, respectively. Based on MDR status of the isolates, no significant differences in biofilm-production were shown among environmental PA (non-MDR OD570 [mean ± SD]: 0.416 ± 0.167 vs. MDR OD570: 0.399 ± 0.192; p > 0.05). No significant association was observed between either motility types in the context of drug resistance or biofilm-forming capacity (p > 0.05). 83.13% of isolates tested were positive for siderophore production. The importance of PA as a pathogen in chronic and healthcare-associated infections has been described extensively, while there is increasing awareness of PA as an environmental agent in agriculture and aquaculture. Additional studies in this field would be an important undertaking to understand the interrelated nature of biofilm production and antimicrobial resistance, as these insights may become relevant bases for developing novel therapeutics and eradication strategies against PA. [ABSTRACT FROM AUTHOR]

Combretastatin A-4 (CA-4, 1) is an antimicrotubule agent used as a prototype for the design of several synthetic analogues with anti-tubulin activity, such as LASSBio-1586 (2). A series of branched and unbranched homologs of the lead-compound 2, and vinyl, ethinyl and benzyl analogues, were designed and synthesized. A comparison between the cytotoxic effect of these homologs and 2 on different human tumor cell lines was performed from a cell viability study using MTT with 48 h and 72 h incubations. In general, the compounds were less potent than CA-4, showing CC50 values ranging from 0.030 μM to 7.53 μM (MTT at 72 h) and 0.096 μM to 8.768 μM (MTT at 48 h). The antimitotic effect of the target compounds was demonstrated by cell cycle analysis through flow cytometry, and the cellular mechanism of cytotoxicity was determined by immunofluorescence. While the benzyl homolog 10 (LASSBio-2070) was shown to be a microtubule stabilizer, the lead-compound 2 (LASSBio-1586) and the methylated homolog 3 (LASSBio-1735) had microtubule destabilizing behavior. Molecular docking studies were performed on tubulin protein to investigate their binding mode on colchicine and taxane domain. Surprisingly, the benzyl homolog 10 was able to modulate EGFR phosphorylate activity in a phenotypic model. These data suggest LASSBio-2070 (10) as a putative dual inhibitor of tubulin and EGFR. Its binding mode with EGFR was determined by molecular docking and may be useful in lead-optimization initiatives. [ABSTRACT FROM AUTHOR]

ABSTRACT The gut microbiome exerts a powerful influence on human health and disease. Elucidating the underlying mechanisms of the microbiota’s influence is hindered by the immense complexity of the gut microbial community and the glycans they forage. Despite a wealth of genomic and metagenomic sequencing information, there remains a lack of informative phenotypic measurements. Pudlo NA, Urs K, Crawford R, Pirani A, et al. (mSystems 7: e00947-21, 2022, https://doi.org/10.1128/msystems.00947-21) decode this complexity by introducing a scalable assay to measure specific carbohydrate utilization in the dominant microbiota phylum Bacteroidetes. The results reveal a mosaic structure of glycan utilization, both genetic and functional, underpinning niche construction in the human gastrointestinal tract. This Commentary highlights the significance of their findings in connection to the field’s growing appreciation for competition, cooperation, and horizontal gene transfer in shaping the highly complex microbial community.

Seyyed Askhan Senobar Tahaei,1 Anette Stájer,2 Ibrahim Barrak,2 Eszter Ostorházi,3 Dóra Szabó,3 Márió Gajdács1,3 1Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged, Szeged, 6720, Hungary; 2Department of Periodontology, Faculty of Dentistry, University of Szeged, Szeged, 6720, Hungary; 3Institute of Medical Microbiology, Faculty of Medicine, Semmelweis University, Budapest, 1089, HungaryCorrespondence: Márió Gajdács Tel +36 62-341-330Email gajdacs.mario@szte.huIntroduction: Staphylococcus aureus (S. aureus) is an important causative pathogen in human infections. The production of biofilms by bacteria is an important factor, leading to treatment failures. There has been significant interest in assessing the possible relationship between the multidrug-resistant (MDR) status and the biofilm-producer phenotype in bacteria. The aim of our present study was to assess the biofilm-production rates in clinical methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA] isolates from Hungarian hospitals and the correlation between resistance characteristics and their biofilm-forming capacity.Methods: A total of three hundred (n=300) S. aureus isolates (corresponding to MSSA and MRSA isolates in equal measure) were included in this study. Identification of the isolates was carried out using the VITEK 2 ID/AST system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests, confirmation of MRSA status was carried out using PBP2a agglutination assay. Biofilm-production was assessed using the crystal violet (CV) tube-adherence method and the Congo red agar (CRA) plate method.Results: There were significant differences among MSSA and MRSA isolates regarding susceptibility-levels to commonly used antibiotics (in case of erythromycin, clindamycin and ciprofloxacin: p< 0.001, gentamicin: p=0.023, sulfamethoxazole/trimethoprim: p=0.027, rifampin: p=0.037). In the CV tube adherence-assay, 37% (n=56) of MSSA and 39% (n=58) of MRSA isolates were positive for biofilm-production, while during the use of CRA plates, 41% (n=61) of MSSA and 44% (n=66) of MRSA were positive; no associations were found between methicillin-resistance and biofilm-production. On the other hand, erythromycin, clindamycin and rifampin resistance was associated with biofilm-positivity (p=0.004, p< 0.001 and p< 0.001, respectively). Biofilm-positive isolates were most common from catheter-associated infections.Discussion: Our study emphasizes the need for additional experiments to assess the role biofilms have in the pathogenesis of implant-associated and chronic S. aureus infections.Keywords: Staphylococcus aureus, MSSA, MRSA, biofilm, antibiotic resistance, crystal violet, Congo red agar, phenotypic assay

Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell's physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape. [ABSTRACT FROM AUTHOR]

Background: Carbapenem resistance among gram negative bacilli (GNB) is a major threat to human health. Rapid and accurate detection of carbapenemase production is very important for clinical management, epidemiological purposes and infection prevention and control issues. Direct Carba NP test (CNPt) is a modified CNPt that allows rapid detection of carbapenemases at a lower cost and improved sensitivity. This work aimed to evaluate direct-CNPt for detection of cabapenemases production as a screening and confirmatory test in GNB versus multiplex PCR as the gold standard test. Methods: This study was conducted on 50 clinical isolates of GNB derived from different clinical samples according to their resistance to meropenem (zone of inhibition = 23 mm). Direct CNPt was performed on all 50 clinical isolates using bacterial colonies directly. A change in colour was observed after (? 2 h). A multiplex PCR was performed on all isolates to detect bla KPC, bla IMP, bla VIM and bla NDM, and bla OXA-48 genes. Results: The overall sensitivity and specificity of direct CNPt as compared to multiplex PCR were 95.2% and 100% respectively. The sensitivity to OXA-48 and IMP genes were relatively lower (91.6 % and 85.7 % respectively) than other genes which had 100% sensitivity for each. Conclusion: Direct CNPt is a reliable and rapid method that allows detection of different carbapenemases at a reduced cost. It could be used in combination with other phenotypic or genotypic assays in settings where OXA-48 and IMP are of high prevalence. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to develop a new rapid and simplified carbapenemase detection method (rsCDM) for detection and characterization of carbapenemase with 3-aminophenylboronic acid (APBA), ethylenediaminetetraacetic acid (EDTA), and cloxacillin (CLO) β-lactamase inhibitors. Methods: A panel of 182 carbapenem-resistant Enterobacterales (CRE) strains with blaKPC (88), blaNDM (60), blaIMP (10), blaVIM (3), blaOXA-181 (5), blaKPC , and blaNDM (7), porin changes in combination with an extended-spectrum β-lactamase (ESBL) (3) or AmpC hyper-production (6) and 43 carbapenem-susceptible Enterobacterales isolates were used to evaluate the performance of rsCDM and EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined with specific inhibitors at 4, 6, and 18 h by rsCDM, and the difference between imipenem (IMI) and meropenem (MEM) disks was simultaneously compared. Results: The sensitivity of rsCDM using IMI was 97.1% at the three time points, with a specificity of 100%, independent of the culture duration. Similar to IPM, MEM disk also showed high sensitivity (97.1%) and specificity (100%) at 6 h. And the sensitivity of eCIM was 95.4% and the specificity was 100%. Based on a decision algorithm, the characterization number of IMI and MEM in KPC-producing isolates was 88 vs. 87, metallo-β-lactamases (MBLs) was 73 vs. 72, KPC and NDM carbapenemase was 7 vs. 7 at 4 h, respectively. After 6 h, the category number changed insignificantly except for isolates with combined AmpC overproduction and porin changes, showing an increase in IMI (6) and MEM (2), and there was no difference in the results between 6 and 18 h for the two tablets. OXA-181-producing strains can't be distinguished by rsCDM. For eCIM, the characterization number in KPC-, OXA- 181-, and MBLs-producing strains was 88, 5, and 72, but it failed to detect multi-enzyme-producing isolates (KPC and NDM). Conclusion: rsCDM accurately discriminated carbapenemase within 4 h and could differentiate multi-enzyme (KPC and NDM) and AmpC in conjunction with porin changes strains. Hence, rsCDM represents a rapid, simple, easy readout, and accurate tool that can be used without any specialized equipment. [ABSTRACT FROM AUTHOR]

The production of biofilms is a critical factor in facilitating the survival of Staphylococcus spp. in vivo and in protecting against various environmental noxa. The possible relationship between the antibiotic-resistant phenotype and biofilm-forming capacity has raised considerable interest. The purpose of the study was to assess the interdependence between biofilm-forming capacity and the antibiotic-resistant phenotype in 299 Staphylococcus spp. (S. aureus n = 143, non-aureus staphylococci [NAS] n = 156) of environmental origin. Antimicrobial susceptibility testing and detection of methicillin resistance (MR) was performed. The capacity of isolates to produce biofilms was assessed using Congo red agar (CRA) plates and a crystal violet microtiter-plate-based (CV-MTP) method. MR was identified in 46.9% of S. aureus and 53.8% of NAS isolates (p > 0.05), with resistance to most commonly used drugs being significantly higher in MR isolates compared to methicillin-susceptible isolates. Resistance rates were highest for clindamycin (57.9%), erythromycin (52.2%) and trimethoprim-sulfamethoxazole (51.1%), while susceptibility was retained for most last-resort drugs. Based on the CRA plates, biofilm was produced by 30.8% of S. aureus and 44.9% of NAS (p = 0.014), while based on the CV-MTP method, 51.7% of S. aureus and 62.8% of NAS were identified as strong biofilm producers, respectively (mean OD570 values: S. aureus: 0.779±0.471 vs. NAS: 1.053±0.551; p < 0.001). No significant differences in biofilm formation were observed based on MR (susceptible: 0.824 ± 0.325 vs. resistant: 0.896 ± 0.367; p = 0.101). However, pronounced differences in biofilm formation were identified based on rifampicin susceptibility (S: 0.784 ± 0.281 vs. R: 1.239 ± 0.286; p = 0.011). The mechanistic understanding of the mechanisms Staphylococcus spp. use to withstand harsh environmental and in vivo conditions is crucial to appropriately address the therapy and eradication of these pathogens. [ABSTRACT FROM AUTHOR]

ObjectiveThis study aimed to develop a new rapid and simplified carbapenemase detection method (rsCDM) for detection and characterization of carbapenemase with 3-aminophenylboronic acid (APBA), ethylenediaminetetraacetic acid (EDTA), and cloxacillin (CLO) β-lactamase inhibitors.MethodsA panel of 182 carbapenem-resistant Enterobacterales (CRE) strains with blaKPC (88), blaNDM (60), blaIMP (10), blaVIM (3), blaOXA-181 (5), blaKPC, and blaNDM (7), porin changes in combination with an extended-spectrum β-lactamase (ESBL) (3) or AmpC hyper-production (6) and 43 carbapenem-susceptible Enterobacterales isolates were used to evaluate the performance of rsCDM and EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined with specific inhibitors at 4, 6, and 18 h by rsCDM, and the difference between imipenem (IMI) and meropenem (MEM) disks was simultaneously compared.ResultsThe sensitivity of rsCDM using IMI was 97.1% at the three time points, with a specificity of 100%, independent of the culture duration. Similar to IPM, MEM disk also showed high sensitivity (97.1%) and specificity (100%) at 6 h. And the sensitivity of eCIM was 95.4% and the specificity was 100%. Based on a decision algorithm, the characterization number of IMI and MEM in KPC-producing isolates was 88 vs. 87, metallo-β-lactamases (MBLs) was 73 vs. 72, KPC and NDM carbapenemase was 7 vs. 7 at 4 h, respectively. After 6 h, the category number changed insignificantly except for isolates with combined AmpC overproduction and porin changes, showing an increase in IMI (6) and MEM (2), and there was no difference in the results between 6 and 18 h for the two tablets. OXA-181-producing strains can’t be distinguished by rsCDM. For eCIM, the characterization number in KPC-, OXA- 181-, and MBLs-producing strains was 88, 5, and 72, but it failed to detect multi-enzyme-producing isolates (KPC and NDM).ConclusionrsCDM accurately discriminated carbapenemase within 4 h and could differentiate multi-enzyme (KPC and NDM) and AmpC in conjunction with porin changes strains. Hence, rsCDM represents a rapid, simple, easy readout, and accurate tool that can be used without any specialized equipment.

This paper describes a microscale fibroplasia and contraction model that is based on fibrin-embedded lung fibroblasts and provides a convenient visual readout of fibrosis. Cell-laden fibrin microgel drops are formed by aqueous two-phase microprinting. The cells deposit extracellular matrix (ECM) molecules such as collagen while fibrin is gradually degraded. Ultimately, the cells contract the collagen-rich matrix to form a compact cell-ECM spheroid. The size of the spheroid provides the visual readout of the extent of fibroplasia. Stimulation of this wound-healing model with the profibrotic cytokine TGF-β1 leads to an excessive scar formation response that manifests as increased collagen production and larger cell-ECM spheroids. Addition of drugs also shifted the scarring profile: the FDA-approved fibrosis drugs (nintedanib and pirfenidone) and a PAI-1 inhibitor (TM5275) significantly reduced cell-ECM spheroid size. Not only is the assay useful for evaluation of antifibrotic drug effects, it is relatively sensitive; one of the few in vitro fibroplasia assays that can detect pirfenidone effects at submillimolar concentrations. Although this paper focuses on lung fibrosis, the approach opens opportunities for studying a broad range of fibrotic diseases and for evaluating antifibrotic therapeutics. [ABSTRACT FROM AUTHOR]

Worldwide, multi-drug resistant Klebsiella pneumoniae (K. pneumonia) and their virulence's were contributed more in the multi-drug resistant effect. According to the World Health organization report, it is an emerging thread in developing countries and also comes under first ever critical list. In this context, the current study was concentrated on detection of extended spectrum beta lactamase (ESBL) producing strain and their antimicrobial susceptibility study of K. pneumoniae. Firstly, the multi-drug resistant effect of the K. pneumoniae was identified from specific CLSI guidelines recommended antibiotics by disc diffusion method. Consecutively, the primary ESBL identification test was performed using ceftazidime and cefotaxime, followed by double disc combination and phenotypic confirmation tests using ceftazidime/clavulanic acid and cefotaxime/clavulanic acid. Finally, the minimum inhibition concentration of some important sensitive antibiotics were performed against selected K. pneumoniae was confirmed by micro broth dilution method. The current result was most favorable to selected K. pneumoniae with more multi drug resistant characteristic nature. All the performed antibiotics were almost more sensitive to selected K. pneumoniae. The effective antibiotics of piperacillin was also exhibited more resistant effect against tested bacteria and it cleaved the bacterial enzyme clearly. The present result of primary ESBL identification test result was exhibited with ≤22 mm and ≤27 mm against ceftazidime and cefotaxime were observed respectively. Followed result of double disc combination and phenotypic confirmation experiments results were clearly stated that the selected K. pneumoniae was ESBL producer. The ceftazidime, cefotaxime and ceftazidime/clavulanic acid and cefotaxime/clavulanic acid were exhibited with merged zones and ≥5 mm zones around the combination disc when compared with disc alone were observed. All the ESBL detection test results were clearly indicated that the selected K. pneumoniae strain was ESBL producer. [ABSTRACT FROM AUTHOR]

Recent years, the treatment of multi-drug resistant bacteria and their effect are very difficult due to the virulence factors modification. Based on the world wide thread, we have tried to identify the ESBLs producing bacteria from urinary tract infection patients. In result, the highly antibiotic resistant effect of Pseudomonas aeruginosa and Klebsiella pneumoniae were identified. Initially, Hexa disc diffusion method was performed to detect the multi-drug resistant bacteria using respective antibiotics of HX066, HX033 and HX077, HX012 discs. Consecutively, the ESBL producing ability of confirmed multi drug resistant bacteria was performed to detect their ESBL producing ability using specific extended spectrum beta lactamase (ESBLs) detection discs of Hexa G-minus 24. Furthermore, the ESBL producing ability of the bacteria was confirmed by ESBLs detection Ezy MIC™ E-test stripe method. In result, 10, 5 and 4 mm and 10, 14 and 8 mm zone of inhibition against imipenem (IPM), Ticarcillin/Clavulanic acid (TCC), Cefoperazone (CPZ) and Ampicillin (AMP), Norfloxacin (NX), Nalidixic acid (NA) antibiotics for P. aeruginosa and 16, 22 and 10, 18 mm zone of inhibition against ceftazidime (CAZ), methicillin (MET), ampicillin amoxyclav (AMC), co-trimoxazole (COT) of the HX077 HX012 for K. pneumoniae were observed. Based on the Clinical & Laboratory Standards Institute (CLSI) guidelines, both the bacteria were more resistant to tested antibiotics and it could be developed more resistant against all the tested antibiotics. In addition, the phenotypic detection of ESBL production effect was also performed against both the selected uropathogens, and the results were shown ≥22 mm, ≥27 zone of inhibition against all the tested antibiotics. Further, the genetic identification of multi plux PCR result was shown TEM, SHV and CTX-m genes were present in both the selected uropathogens. Finally, our results were correlated each other and concluded that the selected uropathogens were multi drug resistant effect and also ESBLs producer. [ABSTRACT FROM AUTHOR]

Introduction: The unparalleled progress in science of the last decades has brought a better understanding of the molecular mechanisms of diseases. This promoted drug discovery processes based on a target approach. However, despite the high promises associated, a critical decrease in the number of first-in-class drugs has been observed., Areas Covered: This review analyses the challenges, advances, and opportunities associated with the main strategies of the drug discovery process, i.e. based on a rational target approach and on an empirical phenotypic approach. This review also evaluates how the gap between these two crossroads can be bridged toward a more efficient drug discovery process., Expert Opinion: The critical lack of knowledge of the complex biological networks is leading to targets not relevant for the clinical context or to drugs that present undesired adverse effects. The phenotypic systems designed by considering available molecular mechanisms can mitigate these knowledge gaps. Associated with the expansion of the chemical space and other technologies, these designs can lead to more efficient drug discoveries. Technological and scientific knowledge should also be applied to identify, as early as possible, both drug targets and mechanisms of action, leading to a more efficient drug discovery pipeline.

Combretastatin A-4 (CA-4, 1) is an antimicrotubule agent used as a prototype for the design of several synthetic analogues with anti-tubulin activity, such as LASSBio-1586 (2). A series of branched and unbranched homologs of the lead-compound 2, and vinyl, ethinyl and benzyl analogues, were designed and synthesized. A comparison between the cytotoxic effect of these homologs and 2 on different human tumor cell lines was performed from a cell viability study using MTT with 48 h and 72 h incubations. In general, the compounds were less potent than CA-4, showing CC50 values ranging from 0.030 μM to 7.53 μM (MTT at 72 h) and 0.096 μM to 8.768 μM (MTT at 48 h). The antimitotic effect of the target compounds was demonstrated by cell cycle analysis through flow cytometry, and the cellular mechanism of cytotoxicity was determined by immunofluorescence. While the benzyl homolog 10 (LASSBio-2070) was shown to be a microtubule stabilizer, the lead-compound 2 (LASSBio-1586) and the methylated homolog 3 (LASSBio-1735) had microtubule destabilizing behavior. Molecular docking studies were performed on tubulin protein to investigate their binding mode on colchicine and taxane domain. Surprisingly, the benzyl homolog 10 was able to modulate EGFR phosphorylate activity in a phenotypic model. These data suggest LASSBio-2070 (10) as a putative dual inhibitor of tubulin and EGFR. Its binding mode with EGFR was determined by molecular docking and may be useful in lead-optimization initiatives.

Phenotypic drug discovery (PDD) uses biological systems directly for new drug screening. While PDD has proved effective in the discovery of drugs with novel mechanisms, for broader adoption, key challenges need resolution: progression of poorly qualified leads and overloaded pipelines due to lack of effective tools to process and prioritize hits; and advancement of leads with undesirable mechanisms that fail at more expensive stages of discovery. Here I discuss how human-based phenotypic platforms are being applied throughout the discovery process for hit triage and prioritization, for elimination of hits with unsuitable mechanisms, and for supporting clinical strategies through pathway-based decision frameworks. Harnessing the data generated in these platforms can also fuel a deeper understanding of drug efficacy and toxicity mechanisms. As these approaches increase in use, they will gain in power for driving better decisions, generating better leads faster and in turn promoting greater adoption of PDD. [Display omitted] Berg describes how the application of phenotypic assays throughout the stages of drug discovery can improve and streamline the selection process. As data-driven phenotypic approaches become more integrated into drug discovery, they will grow in power to drive better decisions. [ABSTRACT FROM AUTHOR]

Introduction: Staphylococcus aureus (S. aureus) is an important causative pathogen in human infections. The production of biofilms by bacteria is an important factor, leading to treatment failures. There has been significant interest in assessing the possible relationship between the multidrug-resistant (MDR) status and the biofilm-producer phenotype in bacteria. The aim of our present study was to assess the biofilm-production rates in clinical methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA] isolates from Hungarian hospitals and the correlation between resistance characteristics and their biofilm-forming capacity. Methods: A total of three hundred (n=300) S. aureus isolates (corresponding to MSSA and MRSA isolates in equal measure) were included in this study. Identification of the isolates was carried out using the VITEK 2 ID/AST system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests, confirmation of MRSA status was carried out using PBP2a agglutination assay. Biofilm-production was assessed using the crystal violet (CV) tube-adherence method and the Congo red agar (CRA) plate method. Results: There were significant differences among MSSA and MRSA isolates regarding susceptibility-levels to commonly used antibiotics (in case of erythromycin, clindamycin and ciprofloxacin: p< 0.001, gentamicin: p=0.023, sulfamethoxazole/trimethoprim: p=0.027, rifampin: p=0.037). In the CV tube adherence-assay, 37% (n=56) of MSSA and 39% (n=58) of MRSA isolates were positive for biofilm-production, while during the use of CRA plates, 41% (n=61) of MSSA and 44% (n=66) of MRSA were positive; no associations were found between methicillin-resistance and biofilm-production. On the other hand, erythromycin, clindamycin and rifampin resistance was associated with biofilm-positivity (p=0.004, p< 0.001 and p< 0.001, respectively). Biofilm-positive isolates were most common from catheter-associated infections. Discussion: Our study emphasizes the need for additional experiments to assess the role biofilms have in the pathogenesis of implant-associated and chronic S. aureus infections. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (P. aeruginosa) is ubiquitous in nature, and may be a causative agent in severe, life-threatening infections. In >60% of cases, β-lactam antibiotics are used in the therapy of P. aeruginosa infections, therefore the emergence of carbapenem-resistant P. aeruginosa (CRPA) is a significant clinical concern. In this study, phenotypic methods were used to characterize fifty-four (n = 54) P. aeruginosa isolates, which were included based on their suspected non-susceptibility to meropenem. Minimum inhibitory concentrations (MICs) of meropenem, ceftazidime, cefepime, ciprofloxacin, gentamicin, were determined using E-tests, while colistin MICs were determined using broth microdilution. The isolates were subjected to the modified Hodge test (MHT), the modified carbapenem-inactivation method (mCIM) and the imipenem/ EDTA combined disk test (CDT). AmpC and efflux pump overexpression was studied using agar plates containing cloxacillin and phenylalanine-arginine β-naphthylamide (PAβN), respectively. Assessment of biofilm-formation was carried out using the crystal violet tube-adherence method. 38.9% of the strains showed meropenem MICs in the resistant range (>8 mg/L). Efflux-pump overexpression and AmpC-hyperproduction was seen in 44.4% and 35.2% of isolates, respectively. 88.8% of the isolates were characterized as strong biofilm-producers. On the other hand, the presence of carbapenemases was suspected in a minority (16.7%) of tested isolates. As safe and effective therapeutic options in carbapenem-resistant Gram-negative infections are severely limited, characterization of these isolates using phenotypic and molecular-based methods is important to provide insights into the epidemiological features of these pathogens. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen, which may be a causative agent in a wide-range of human pathologies. Car-bapenems are usually considered the last safe and effective choice of drugs for the treatment of Gram-negative infections. The emergence of carbapenem-resistant A. baumannii (CRAB) is a critical public health issue as they leave clinicians with limited therapeutic options. In this study, phenotypic methods were used to characterize sixty-two (n = 62) A. baumannii isolates, which were included based on their suspected non-susceptibility to meropenem. Minimum inhibitory concentrations (MICs) of meropenem, levofloxacin, gentamicin, sulfamethoxazole/trimethoprim, tigecycline were determined using E-tests, while colistin MICs were determined using broth microdilution. The isolates were subjected to the modified Hodge test (MHT), the modified carbapenem-inactivation method (mCIM) and the imipenem/EDTA combined disk test (CDT). Efflux pump overexpression was studied using agar plates containing phenylalanine-arginine β-naphthylamide (PAβN). Assessment of biofilm-formation was carried out using the crystal violet tube-adherence method. 64.5% of the strains showed meropenem MICs in the resistant range (>8 mg/L), resistance rates were similarly high to the other tested antibiotics. The MHT and mCIM assay were positive in 79.0% and 67.7% of cases, respectively; the presence of an MBL was suggested for 29.0% of isolates. Efflux-pump overexpression was seen in 12.9% of isolates. 54.8% of the isolates were characterized as strong biofilm-producers. Microbiology laboratories have an important role in differentiating the distinct mechanisms by which these pathogens develop the CRAB phenotype, as plasmid-borne carbapenemases are significant from the standpoint of public health microbiology. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis is a deadly lung disease, wherein normal lung tissue is progressively replaced with fibrotic scar tissue. An aspect of this process can be recreated in vitro by embedding fibroblasts into a collagen matrix and providing a fibrotic stimulus. This work expands upon a previously described method to print microscale cell-laden collagen gels and combines it with live cell imaging and automated image analysis to enable high-throughput analysis of the kinetics of cell-mediated contraction of this collagen matrix. The image analysis method utilizes a plugin for FIJI, built around Waikato Environment for Knowledge Analysis (WEKA) Segmentation. After cross-validation of this automated image analysis with manual shape tracing, the assay was applied to primary human lung fibroblasts including cells isolated from idiopathic pulmonary fibrosis patients. In the absence of any exogenous stimuli, the analysis showed significantly faster and more extensive contraction of the diseased cells compared to the healthy ones. Upon stimulation with transforming growth factor beta 1 (TGF-β1), fibroblasts from the healthy donor showed significantly more contraction throughout the observation period while differences in the response of diseased cells was subtle and could only be detected during a smaller window of time. Finally, dose-response curves for the inhibition of collagen gel contraction were determined for 3 small molecules including the only 2 FDA-approved drugs for idiopathic pulmonary fibrosis.

Abstract Background Despite the development of new therapies for leishmaniasis, among the 200 countries or territories reporting to the WHO, 87 were identified as endemic for Tegumentary Leishmaniasis and 75 as endemic for Visceral Leishmaniasis. The identification of antileishmanial drug candidates is essential to fill the drug discovery pipeline for leishmaniasis. In the hit molecule LQB-118 selected, the first generation of pterocarpanquinones was effective and safe against experimental visceral and cutaneous leishmaniasis via oral delivery. In this paper, we report the synthesis and antileishmanial activity of the second generation of pterocarpanoquinones. Methods The second generation of pterocarpanquinones 2a-f was prepared through a palladium-catalyzed oxyarylation of dihydronaphtalen and chromens with iodolawsone, easily prepared by iodination of lawsone. The spectrum of antileishmanial activity was evaluated in promastigotes and intracellular amastigotes of L. amazonensis, L. braziliensis, and L. infantum. Toxicity was assessed in peritoneal macrophages and selective index calculated by CC50/IC50. Oxidative stress was measured by intracellular ROS levels and mitochondrial membrane potential in treated cells. Results In this work, we answered two pertinent questions about the structure of the first-generation pterocarpanquinones: the configuration and positions of rings B (pyran) and C (furan) and the presence of oxygen in the B ring. When rings B and C are exchanged, we noted an improvement of the activity against promastigotes and amastigotes of L. amazonensis and promastigotes of L. infantum. As to the oxygen in ring B of the new generation, we observed that the oxygenated compound 2b is approximately twice as active against L. braziliensis promastigotes than its deoxy derivative 2a. Another modification that improved the activity was the addition of the methylenedioxy group. A variation in the susceptibility among species was evident in the clinically relevant form of the parasite, the intracellular amastigote. L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. The pterocarpanoquinones (2b and 2c) that possess the oxygen atom in ring B showed induction of increased ROS production. Conclusions The data presented indicate that the pterocarpanoquinones are promising compounds for the development of new leishmanicidal agents.

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC50 = 23.45 ± 4.54 μM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 ± 5.77 μM). This result suggests that lipophilic substituents on either—meta and/or—para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE50 = 49.22 ± 7.71 μM) presented the highest selectivity index. [ABSTRACT FROM AUTHOR]

Parasitic worms cause very significant diseases in animals and humans worldwide, and their control is critical to enhance health, well-being and productivity. Due to widespread drug resistance in many parasitic worms of animals globally, there is a major, continuing demand for the discovery and development of anthelmintic drugs for use to control these worms. Here, we established a practical, cost-effective and semi-automated high throughput screening (HTS) assay, which relies on the measurement of motility of larvae of the barber’s pole worm (Haemonchus contortus) using infrared light-interference. Using this assay, we screened 80,500 small molecules and achieved a hit rate of 0.05%. We identified three small molecules that reproducibly inhibited larval motility and/or development (IC50 values of ~4 to 41 µM). Future work will critically assess the potential of selected hits as candidates for subsequent optimisation or repurposing against parasitic nematodes. This HTS assay has a major advantage over most previous assays in that it achieves a ≥ 10-times higher throughput (i.e., 10,000 compounds per week), and is thus suited to the screening of libraries of tens of thousands to hundreds of thousands of compounds for subsequent hit-to-lead optimisation or effective repurposing and development. The current assay should be adaptable to many socioeconomically important parasitic nematodes, including those that cause neglected tropical diseases (NTDs). This aspect is of relevance, given the goals of the World Health Organization (WHO) Roadmap for NTDs 2021–2030, to develop more effective drugs and drug combinations to improve patient outcomes and circumvent the ineffectiveness of some current anthelmintic drugs and possible drug resistance.

A direct-seeded rice cultivation system has been widely adopted in Asian countries. Optimum germination and vigorous seedling growth under submergence are key traits for the practice of direct seeding. We studied the post-germination seedling vigour in Vietnamese lowland rice accessions based on three bio-parameters, shoot elongation growth under five-day submergence in water-filled test-tubes, seedling recovery rate five days after transferring submerged seedlings to pots with soil and seedling survival rate 21 days after sowing seeds in nursery beds and immediate incubation under submergence. A large diversity was found in seedling vigour thus estimated among the accessions. Significantly high correlations were observed among all three bio-parameters, verifying the contribution of seedling vigour to the manifestation of submergence tolerance at this critical stage of rice development. To examine the roles of anoxia/hypoxia-responsive genes, the expression of 17 candidate genes was studied by reverse transcription polymerase chain reaction (RT-PCR) and compared between selected vigorous and non-vigorous groups of accessions. Transcripts of all but two genes showed marked accumulation in submerged seedlings. No differences, however, were found between the two contrasting groups. The observed common and coordinate expression of anoxia/hypoxia-induced genes suggests that they might assume roles in attaining baseline tolerance against submergence stress. It was also suggested that some unknown genetic factors are operating in determining cultivar/genotype-specific levels of submergence tolerance as assessed by post-germination seedling vigour.

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2 ) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gorostiola González, Sijben, Dall’ Acqua, Liu, IJzerman, Heitman and van Westen.)

Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocyte's normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (Factin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell's filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3γ and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte- protective agents for future therapeutic development. [ABSTRACT FROM AUTHOR]

[Display omitted] Asarum sieboldii var. seoulense is a plant species under the family Aristolochiaceae and has been used for centuries as an ingredient in a well-known Traditional Chinese medicine (TCM), "Xixin", to treat symptoms of the neurodegenerative condition Parkinson's Disease (PD). Although there have been studies on the neuroprotective effect of this TCM, the phenotypic profiles of its chemical constituents against PD-implicated cellular organelles have not been reported. This research investigated the chemistry of A. sieboldii var. seoulense extract to identify the active small molecules that exhibited perturbation to the cellular compartments related to PD, potentially supporting its traditional application in treating this condition. 1H NMR-guided chemical investigation of this plant yielded twenty secondary metabolites which belong to isobutylamides, lignans and phenolics. The compounds were evaluated against an olfactory cell line derived from a PD patient using phenotypic assay. Several isolates, 2 , 3 , 7 , 11 , 13 – 16 and 18 – 20, were found to induce moderate perturbation to the staining of mitochondria, autophagosome and α-tubulin of the cells. Considering that PD pathogenesis is closely related to these cellular compartments, the results provided a rationale for the traditional application of Xixin in the treatment of PD. [ABSTRACT FROM AUTHOR]

Objectives This study was designed to evaluate the sodium mercaptoacetic acid double disk synergy test (SMA-DDST), the Etest metallo-β-lactamase (MBL) MP/MPI (Etest MP/MPI), and the Mastdiscs ID Carbapenemase Detection Disc Set (MAST-CDS) for the detection of MBL-producing Enterobacteriaceae isolates in Japan. Methods Fifty-one clinical isolates and four reference strains were tested. These isolates included 40, 4, and 11 IMP-, New Delhi MBL (NDM)-, and non-MBL-producers, respectively. SMA-DDST was performed with meropenem (MEPM)-containing disks. Results Sensitivities were 38/44 (86%), 40/44 (91%), and 15/44 (34%), and the cost ratio was 1:9.4:3.8 for MEPM-SMA-DDST:Etest MP/MPI:MAST-CDS, respectively. The specificity was 11/11 (100%) for all assays. MEPM-SMA-DDST detected IMP-producing isolates with high sensitivity (38/40; 95%), but the assay was inadequate for NDM-producing isolates (0/4; 0%). The Etest MP/MPI detected both IMP- (36/40; 90%) and NDM-producing isolates (4/4; 100%), but was the most expensive. MAST-CDS detected IMP-producing isolates with low sensitivity (11/40; 28%), but the assay worked well for NDM-producing isolates (4/4; 100%). Conclusions Our results indicated that MEPM-SMA-DDST was the most cost-effective assay for the detection of IMP-producing isolates. Therefore, we conclude that MEPM-SMA-DDST is the optimal available assay for clinical first-line screening in IMP-endemic areas such as Japan. However, this assay could not detect NDM-producing isolates, whereas the Etest MP/MPI and MAST-CDS could. When MEPM-SMA-DDST is negative, the Etest MP/MPI and MAST-CDS could be used to obtain supportive data and prevent detection failure for NDM-producing isolates. [ABSTRACT FROM AUTHOR]

Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G/G cell cycle arrest of ORL-150 cells through inducing p27. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent. [ABSTRACT FROM AUTHOR]