Lower EC50 Values Research Articles

Discovery of polymethoxylated flavonoids in Artemisia argyi as main active components in inhibiting rice blast fungus

BackgroundRice blast is a devastating disease caused by Magnaporthe grisea, and it is not well controlled globally. As a Traditional Chinese Medicine, Artemisia argyi has been proven to have inhibitory effects on a variety of phytopathogenic fungi. Here, we used bioactivity-guided isolation method and transcriptomics to clarify the antifungal active compounds in A. argyi and their antifungal mechanisms.ResultsIn vitro studies showed that the absolute ethanol extract (AEE) of A. argyi had a strong inhibitory effect on the growth of M. grisea, with a low EC50 value of 1.156 mg/mL, and could cause the destruction of hyphae cell membrane and the leakage of cell contents. To identify the active constituents, we fractionated the AEE using macroporous adsorption resin and silica gel column chromatography. The active fraction (Fr.F-1) was fractionated and had even higher antifungal activity than AEE. The results showed that both AEE and Fr.F-1 could impact primary metabolic pathways and reactive oxygen species homeostasis of M. grisea, decrease the content of reducing sugars and downregulate the expression of genes related to starch and sucrose metabolism. Further fractionation of Fr.F-1 led to the identification of two polymethoxylated flavonoids (eupatilin and 3,5,3ʹ-trihydroxy-7,8,4ʹ-trimethoxyflavone), both of which had antifungal activity and they worked synergistically in Fr.F-1. Finally, we also investigated the effect of AEE, Fr.F-1 and eupatilin on rice blast control. The results showed that AEE, Fr.F-1 and eupatilin application strongly enhanced rice resistance to M. grisea.ConclusionIn brief, these findings indicate that Fr.F-1 subfraction from A. argyi and its main components polymethoxylated flavonoids confer inhibiting activities to M. grisea. This discovery can effectively alleviate the degradation pressure of A. argyi by-products, promote the healthy development of its industry, and provide new a strategy for the development of new botanical fungicides to control rice blast.Graphical

The cumulative effect of compound heterozygous variants in TRPV3 caused Olmsted syndrome

BackgroundOlmsted syndrome (OS) is a rare genodermatosis predominantly inherited in an autosomal dominant manner, typically arising from gain-of-function (GOF) variants in the transient receptor potential channel vanilloid 3 (TRPV3). ObjectiveThis study aims to investigate potential mechanisms underlying OS in two cases presenting with an autosomal recessive inheritance pattern. MethodsNext-generation sequencing panel was employed to identify TRPV3 variants. TRPV3 plasmids carrying specific point variations were generated and transiently transfected into HEK293T cells. Electrophysiological patch-clamp techniques were utilized to record voltage-activated and ligand-activated currents. Celltiter-Glo luminescent assay was employed to analyze the cell viabilities. ResultsCompound heterozygous variants, c.1563G>C (p.W521C) and c.1376C>T (p.S459L), as well as c.1773G>C (p.L591F) and c.2186G>A (p.R729Q), were identified in the two OS patients respectively. Electrophysiological analysis of ligand-induced activation of TRPV3 variants demonstrated the closest correlation with clinical manifestations. All four variants displayed GOF channel activity characterized by increased sensitivity. Notably, W521C and L591F exhibited both heightened sensitivity and lower EC50 values for the TRPV3 agonist. Co-transfection with wild-type TRPV3 plasmids significantly rescued these effects. Cells co-transfected with the corresponding compound heterozygous variants exhibited intermediate electrophysiological characteristics. ConclusionsIn this study, we present two cases of OS by autosomal-recessive inheritance of TPRV3 variants. This study presents a notable observation of compound heterozygous GOF variants in TRPV3, highlighting their cumulative impact on clinical manifestations. Additionally, we advocate for the use of ligand-dependent ion channel activity assays to assess the pathogenicity of TRPV3 variants in OS.

Development of Luminescent and Photosensitive Cellulose Microfibers From Palm Sprout Fibers: Antibacterial Efficacy and Dye Adsorption Potential.

A key contemporary challenge is enhancing the value of agro-industrial byproducts. Cellulose, the most abundant renewable resource, offers significant industrial potential due to its versatile properties. Produced in its pure form by various bacteria, cellulose is increasingly utilized in microscale and nanoscale fibers for composite reinforcement. In this study, cellulose microfibers were derived from palm sprout fibers through a series of treatments like pretreatment, high-pressure hydrolysis in an autoclave, and bleaching. These fibers were then characterized using techniques such as UV spectroscopy, FTIR, SEM, and XRD. The study evaluated the antibacterial properties of the treated cellulose microfibers against both gram-positive Staphylococcus aureus (07%) and gram-negative Escherichia coli (10%), finding higher effectiveness against E.coli. Additionally, the microfibers exhibited a lower EC50 value of 0.101 mg/mL, suggesting that although the microfiber extract is effective, the standard antioxidant is somewhat more potent. Adsorption studies revealed that the cellulose microfibers followed the Langmuir isotherm model, with a high determination coefficient of 0.998, and reached maximum dye adsorption for RB 160 within 120 min. In this adsorption study, 50 mg of dye was removed. These results indicate that the treated cellulose microfibers are a promising biosorbent for improving dye removal processes.

Population pharmacokinetic and pharmacodynamic model of evogliptin: Severe uremia increases the bioavailability ofevogliptin.

Uremia, a condition characterized by the retention of uremic toxins due to impaired renal function, may affect drug metabolism mediated by CYP3A4 enzymes. Evogliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor diabetic drug that is primarily metabolized by CYP3A4. This study aimed to construct a population pharmacokinetic (PK) and pharmacodynamic (PD) model for evogliptin in patients with varying degrees of renal disease, including end-stage renal disease on hemodialysis. A total of 688 evogliptin concentration and 598 DPP-4 activity data were available from 46 subjects. PK and PD data analyses were performed using a nonlinear mixed-effects model. The PK of evogliptin was optimally described by a two-compartment model with first-order absorption. The significant covariates in the final model included blood amylase and triglyceride on F1 (relative bioavailability). The simulation findings, together with previously reported PK data, provided evidence of a significant inhibition of the first-pass effect of evogliptin in patients with renal impairment. A direct link sigmoidal Emax model was developed to describe the relationship between evogliptin concentration and DPP-4 inhibition. The PD model predicted significant inhibition of DPP-4 at maximum effect (Emax: 88.9%) and a low EC50 value (1.08 μg/L), indicating the high potency and efficacy of evogliptin. The developed PK/PD model accurately predicted exposure and the resulting DPP-4 activity of evogliptin in renal impairment. The findings of this study suggest that renal impairment and associated biochemical changes may impact the bioavailability of CYP3A4-metabolized drugs.

Propiconazole resistance phenotypes in Geotrichum candidum from South Carolina peaches are linked to point mutations in GcCYP51B gene.

Geotrichum candidum Link (1809) is a yeast-like fungus that causes sour rot of peach (Prunus persica). Outbreaks of the disease have occurred since 2021 in peach fruit kept in cold storage despite post-harvest treatments with propiconazole at a commercial farm in South Carolina (SC). A total of 58 isolates, 40 from symptomatic fruit from cold storage in Saluda County (SC packing house isolates), 11 from three SC orchards in Saluda County, Spartanburg County, and Pickens County (SC non-packing house isolates), and 7 California (CA) isolates (at least 3 from packing houses) were evaluated for propiconazole sensitivity. Mycelial growth assays revealed that 6 of 7 CA isolates had the lowest EC50 values and were considered sensitive (S) to propiconazole with an average EC50 value of 0.02 µg/ml and minimum inhibitory concentration (MIC) values >1 to < 3 µg/ml. Isolate 02J018 from CA and all SC non packing house isolates were considered reduced-sensitive (RS) to propiconazole with an average EC50 value of 0.33 µg/ml and MIC values >10 to <30 µg/ml. SC packing house isolates were considered resistant (R) to propiconazole and had an average EC50 value of 3.55 µg/ml and MIC values >300 µg/ml. Two CYP51 genes, GcCYP51A and GcCYP51B, encoding two demethylase inhibitor (DMI) target enzyme 14α-demethylases were identified, sequenced, and characterized. Two GcCYP51A and three GcCYP51B variants were found. While both GcCYP51A variants were linked to S isolates, the GcCYP51B2 variant possessing the mutation Y143F was found in RS, and the GcCYP51B3 variant possessing Y143F, E126K, and G460S mutations was identified in R isolates. The Y143F and G460S mutations had been associated with DMI fungicide resistance in other plant pathogens. No increased constitutive expression of GcCYP51A or GcCYP51B was observed in RS or R isolates. Detached fruit assays revealed that label rates of propiconazole controlled sour rot caused by S and RS but not R isolates. Our results suggest that sour rot outbreaks in a SC packing house were linked to target gene-induced propiconazole resistance in G. candidum.

Identification Of Pathogen Causing Wilt Disease Of Chinese Yam (Dioscorea Polystachya Turczaninow) In Hunan

This study aims to identify the pathogen causing wilt disease of Chinese yam (Dioscorea polystachya Turczaninow.) in Hunan Province. Diseased stems were collected from the fields in Loudi City in the Hunan Province. Tissue culture method was used for the isolation of the pathogen causing wilt disease of Chinese yam, and was identified as Fusarium asiaticum based on the morphological characteristics and molecular technology. Out of five fungicides, Difenoconazole· Azoxystrobin 40% SC, Hexaconazole 5% SC, and Difenoconazole 10% WDG, demonstrated inhibitory effects on the pathogen. Among them, hexonazole 5% SC had the lowest EC50 value and was the most sensitive to the pathogen of Chinese yam wilt, so it should be selected first for the control of Chinese yam wilt. Bangladesh J. Bot. 53(3): 857-863, 2024 (September) Special

Cytotoxic impacts of seven alternative bisphenols on human in vitro cellular models

Bisphenols (BPs), common in plastics, coatings, and resins, are under scrutiny for potential endocrine disruption. Despite banning bisphenol A (BPA), its perceived safer alternatives may still pose health risks, urging thorough studies on their toxicity mechanisms. This study aimed to investigate the cellular toxicity of the top seven most commonly used BPs, bisphenol S (BPS), bisphenol F (BPF), bisphenol AF (BPAF), bisphenol P (BPP), bisphenol AP (BPAP), bisphenol B (BPB), bisphenol E (BPE) in eight different relevant human in vitro cell models: liver (HepaRG), intestinal (Caco-2), breast (T47D), brain (HMC-3), lungs (MRC-5), kidney (HEK293), endothelial (HMEC-1), and skin (HEK-001) cell lines. BPE manifested the highest cytotoxicity in Caco-2 cells, presenting an EC50 value of roughly 0.2 μM (95% confidence interval). In contrast, HEK293 and HepaRG cells demonstrated significant resilience to BPS (EC50 > 1000 μM). BPAF, BPP, and BPAP had consistently low EC50 values across cell lines (6–27.9 μM, 0.6–134.7 μM, and 3.6–178.8 μM), indicating elevated toxicity. After 24 h, all bisphenols adhered to nominal concentrations except BPAF, BPP, and BPS. BPP's concentration notably decreased (30.82 ± 5.53% of nominal value). The results revealed diverse effects of bisphenol analogs on different cell types. These findings emphasized the considerable cytotoxic potential of specific bisphenol analogs across various human cell models, underlining the necessity for a re-evaluation of their safety and regulatory standards.

Antioxidant, antibacterial, and anti-biofilm activities of selected indigenous plant species against nosocomial bacterial pathogens.

Biofilms are responsible for over 60% of nosocomial infections. The focus of this study was to investigate the antioxidant, antibacterial, antibiofilm, and anti-motility activities of Gardenia volkensii, Carissa bispinosa, Peltophorum africanum, and Senna petersiana. Antioxidant activity was evaluated using free radical (DPPH) scavenging and ferric reducing power assays. Antibacterial and antibiofilm activities were evaluated using the broth micro-dilution and the crystal violet assays, respectively. Anti-motility was evaluated using anti-swarming activities, and the brine shrimp lethality assay was used for cytotoxicity. Gardenia volkensii and C. bispinosa acetone extracts had low EC50 values of 9.59 and 9.99μg ml-1on the free-radical scavenging activity, respectively. All the plant extracts demonstrated broad-spectrum antibacterial activity against Klebsiella pneumoniae, Pseudomonasa aeruginosa, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus [minimum inhibitory concentration (MIC)<0.63mg ml-1]. The initial cell adherence stage of P. aeruginosa and E. coli was the most susceptible stage where sub-MICs resulted in inhibitions>50%. Peltophorum africanum had the least cytotoxic effects. All extracts had anti-motility activity against P. aeruginosa and E. coli. This study showed that not only do the plants have strong antibacterial activity but had noteworthy inhibition (>50%) of initial cell adherence and may be suitable candidates for the treatment of nosocomial pathogens.

Effects of Several Fungicides on the Spore Growth Period of Alternaria dauci, a Carrot Black Leaf Blight Fungus, Using a Rezasulin-based Spore Survival Assay

The effects of five fungicides on the spore growth phase of Alternaria dauci, which causes carrot leaf blight, were tested using the spore viability assay (SVA) and agar dilution method (ADM). The average EC50 values for chlorothalonil against seven isolates of A. dauci examined by SVA and ADM were 14.21 μg/ml and more than 100 μg/ml. Dithianon and folpet also had lower EC50 values in SVA than in ADM, while iminoctadine tris-albesilate had lower EC50 value in ADM. For fluazinam, the EC50 values of SVA and ADM were 1.63 and 2.40 μg/ml, respectively. As EC50 values of five fungicides according to the spore growth phase of A. dauci KACC 42997, the efficacy of each fungicide as chlorothalonil, dithianon, and folpet decreased when treated after spore germination rather than when treated with spores before germination. However, iminoctadine tris-albesilate was more effective when treated after spores germinated than when treated before treatment. The excellent effect of fluazinam on the pathogen was maintained until A. dauci KACC 42997 was cultured in potato dextrose broth for 6 hr and the germ tube grew beyond the size of the spore. However, when treated with iminoctadine tris-albesilate and fluazinam after culturing for 12 hr, as the EC50 values of the two fungicides increased to 8.87 and 20.65 μg/ml, their efficacies decreased. The results of this study show that the treatment time of the fungicide should be determined by considering the effect of the fungicide on the spore growth phase of pathogens.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Tracing holocene paleoenvironmental changes along the northern Tyrrhenian coast (Cornia and Pecora coastal plains, Tuscany, Italy): data from geochemical and sedimentological proxies

In this paper, sedimentological and geochemical data from six cores are presented and discussed in order to better understand the Holocene palaeoenvironmental evolution of the coastal plains of the Pecora and Cornia Rivers (southern Tuscany, Italy). Overall, the stratigraphies show a general upward trend in the evolution of the sedimentary succession of lagoonal environments (low Zr/Rb, high EC values) connected or partially connected to the sea (low EC values), to a seaward progradation of marshy-swampy environments (Mn/Ti, high TOC values) and finally to continental environments with the progradation of distal alluvial plains (high Zr/Rb, low EC values) influenced by runoff phenomena or local water stagnation. This trend is closely related to the increasing input of sediments from inland due to soil erosion processes and, especially in the last two centuries, land reclamation works. In the Cornia coastal plain, the chronologies indicate that lagoonal environments were already present before ca. 8.2ka BC and started receding seaward at around 3.7ka BC. On the other hand, chronologies from ca. 0.5ka BC to ca. 0.6ka AD point to a persistence of lagoonal environments only in areas very close to those already identified as such in historical cartography. In the Pecora coastal plain, the chronologies show that the lagoon environments had already disappeared before ca. 3.3ka BC, whilst the peripheral environments were characterised by abundant vegetation. The latter gradually degraded in favour of the development of shallow to very deep, unvegetated marshlands, which persisted until ca. 1ka AD.

Aureobasidium pullulans formulations: evaluation of the effectiveness against grey mould of table grape

Antagonism against Botrytis cinerea is often carried out using yeast as direct antagonists. Aureobasidium pullulans strain AP1 was tested in two different formulations: wettable powder (WP) and oil dispersion (OD). By in vitro assays, the viability of the strain cells was constantly evaluated for seven months and the OD formulation ensured the highest cells viability. The efficacy of the formulations was assayed by evaluating the production of volatile and non-volatile metabolites. Results showed that the formulation affected the non-volatile less than the volatile metabolites. Both AP1 WP and AP1 OD non-volatile metabolites displayed almost 50% of mycelial pathogen inhibition. Comparing the two products, the lowest EC50 value (518.15 mg L− 1) was detected for the AP1 OD formulation that was thus chosen for postharvest in vivo assays. The preventative treatments (200, 400, 800 mg L− 1) were active in reducing the pathogen incidence on table grape on average by 52%. Instead, in the curative application assay, the highest concentration (800 mg L− 1) reduced grey mold incidence by 86%. The present study reported the potential of two new formulations to use against the postharvest grey mold of table grape for a possible further commercial product development.

Determination of the critical aggregation concentration in water of Gum Arabic functionalized with curcumin oxidation products by micro-scale thermophoresis approach

Gum Arabic underwent enzymatic modification with curcumin oxidation products, prompting self-assembly in water at lower concentrations than native gum Arabic, which was fully soluble. The resulting particles displayed a narrow size distribution, suggestive of a micellization mechanism akin to Critical Micellization Concentration (CMC) in surfactants or Critical Aggregation Concentration (CAC) in polymers. Accurately determining CAC is vital for utilizing polymers in molecule encapsulation, but precise measurement is challenging, requiring multiple techniques. Initially, CAC was probed via turbidity measurements, dynamic light scattering (DLS), and isothermal calorimetric titration (ITC), yielding a range of 0.0015 to 0.01 %. Micro-scale thermophoresis (MST) was then employed for the first time to define CAC more precisely, facilitated by the intrinsic fluorescence of modified gum Arabic. Using MST, CAC was pinpointed at 0.001 % (w/v), a novel approach. Furthermore, MST revealed a low EC50 value of 0.007 % (w/t) for self-assembly, signifying uniformity among GAC sub-units and assembly stability upon dilution.

Synthesis and Antifungal Activity of Coumarin Derivatives Containing Hydrazone Moiety.

Plant disease control mainly relies on pesticides. In this study, a series of coumarin derivatives containing hydrazone moiety were designed and synthesized. The synthesized compounds were characterized and used to evaluate the antifungal activity against four pathogens, Botrytis cinerea, Alternaria solani, Fusarium oxysporum, and Alternaria alternata. The results showed that the inhibition rate of some compounds at 100 μg/mL in 96 hours reached around 70 % against A. alternata, higher than that of the positive control. The corresponding EC50 values were found at around 30 μg/mL. Finally, the compound 3 b was screened out with the lowest EC50 value (19.49 μg/mL). The analysis of SEM and TEM confirmed that the compound 3 b can obviously damage the morphological structure of hyphae, resulting in the depletion of the cells by the destruction of morphological matrix and leakage of contents. RNA sequencing showed that compounds 3 b mainly affected the pentose phosphate pathway, which caused to destroy the layer of mitochondrial structure. Molecular docking showed that compounds 3 b fitted the binding pocket of yeast transketolase and interacted with lysine at the hydrazone structure. Our results suggested that the introduction of hydrazone was an effective strategy for the design of novel bioactive compounds.

Revisiting the intron hypothesis of QoI resistance in Phyllosticta ampelicida, the causal agent of grape black rot, and other Phyllosticta species

AbstractChemical control of grape black rot, caused by Phyllosticta ampelicida, relies mainly on the use of demethylation inhibitors (DMIs) and quinone outside inhibitors (QoIs). The effectiveness of QoI fungicides is influenced by alternative respiration activity, and the exon/intron structure and point mutations in the target protein's gene, the cytochrome b (cytb) gene. Our study aims to investigate the QoI fungicide sensitivity of 48 P. ampelicida isolates in vitro by measuring EC50 and the molecular characteristics of the cytb gene and its mRNA in P. ampelicida and other Phyllosticta species. Mycelial growth tests revealed that the P. ampelicida isolates were sensitive to both azoxystrobin and trifloxystrobin; baseline EC50 values were 0.029 and 0.022 μg/mL, respectively. Addition of salicylhydroxamic acid (SHAM) resulted in lower EC50 values (0.024 and 0.017 μg/mL, respectively). None of the typical point mutations conferring resistance to QoIs in some fungi were detected. A group I intron was present right after the 143rd codon in the cytb gene in four of the six Phyllosticta species examined. The sequence and exon/intron structure of the cytb gene of P. ampelicida isolated from Vitis vinifera is studied in detail and published here. Our results indicate a low risk of QoI resistance development via the G143A mutation in P. ampelicida.

Robenidine derivatives as potential antischistosomal drug candidates

Schistosomiasis caused by Schistosoma spp. is a disease that causes a considerable health burden to millions of people worldwide. The limited availability of effective drugs on the market and the increased risk of resistance development due to extensive usage, highlight the urgent need for new antischistosomal drugs. Recent studies have shown that robenidine derivatives, containing an aminoguanidine core, exhibit promising activities against Plasmodium falciparum, motivating further investigation into their efficacy against Schistosoma mansoni, due to their similar habitat and the resulting related cellular mechanisms like the heme detoxification pathway. The conducted phenotypic screening of robenidine and 80 derivatives against newly transformed schistosomula and adult Schistosoma mansoni yielded 11 candidates with low EC50 values for newly transformed schistosomula (1.12–4.63 μM) and adults (2.78–9.47 μM). The structure-activity relationship revealed that electron-withdrawing groups at the phenyl moiety, as well as the presence of methyl groups adjacent to the guanidine moiety, enhanced the activity of derivatives against both stages of Schistosoma mansoni. The two compounds 2,2′-Bis[(3-cyano-4-fluorophenyl)methylene] carbonimidic Dihydrazide Hydrochloride (1) and 2,2′-Bis[(4-difluoromethoxyphenyl) ethylidene] carbonimidic Dihydrazide Hydrochloride (19), were selected for an in vivo study in Schistosoma mansoni-infected mice based on their potency, cytotoxicity, pharmacokinetic-, and physicochemical properties, but failed to reduce the worm burden significantly (worm burden reduction <20%). Thus, robenidine derivatives require further refinements to obtain higher antischistosomal specificity and in vivo activity.

Multiple resistance of Colletotrichum truncatum from soybean to QoI and MBC fungicides in Brazil

AbstractColletotrichum truncatum, the predominant fungal species associated with soybean anthracnose, is responsible for significant losses in this crop. Chemical control via fungicide application is the most effective strategy for the control of soybean foliar diseases. However, the increasing incidence of anthracnose in some regions of Brazil indicates that current chemical control is not effective against anthracnose. In this study, we evaluated the fungicide sensitivity of C. truncatum genetic lineages to the fungicides azoxystrobin, thiophanate‐methyl, difenoconazole, and fludioxonil using isolates representing two important regions of soybean production in Brazil. We characterized the molecular resistance to the quinone‐outside inhibitors (QoI), methyl benzimidazole carbamates (MBC), and demethylation inhibitor (DMI) fungicide groups based on amino acid sequences of the cytochrome b (cytb), β‐tubulin gene (β‐tub), and P450 sterol 14a‐demethylases (CYP51) genes. Multiple resistance of C. truncatum isolates to QoI and MBC was observed associated with mutation points in the β‐tub (E198A and F200Y) and cytb (G143A). Alternatively, low EC50 values were found for fludioxonil and difenoconazole indicating high efficacy. Analysis of C. truncatum genomes revealed two potential DMI targets, CYP51A and CYP51B, and higher genetic variability in the CYP51A gene. A positive correlation was found between genetic differentiation of C. truncatum populations and fungicide sensitivity (Student's t‐test &lt;0.001). To our knowledge, this is the first report of multiple resistance to QoI and MBC fungicides in C. truncatum in Brazil.

Structural refinement, dielectric and ferroelectric properties of Cr modified Ba0·90Sr0·10TiO3 ceramics

In this study, we investigated the impact of Chromium substitution on the structural, dielectric and ferroelectric characteristics of Ba0·90Sr0·10Ti1-3x/4CrxO3 (x = 0, 0.005, 0.010, 0.015 and 0.020) ceramics fabricated by solid state reaction method. Rietveld refinement of X-ray diffraction pattern confirmed tetragonal perovskite structure for all the samples. Based on the crystal structure and the lattice parameter obtained through XRD measurement, the Madelung energy of each sample was computed. SEM images revealed a reduction in grain size with rising Cr concentration. TEM, HRTEM, SAED and EDS analysis was carried out for BST-Cr3 sample. Diffused rings observed in Selective Area Electron Diffraction (SAED) patterns indicate the crystallinity of the sample. EDS analysis indicated the presence of Ba, Sr, Ti, O and Cr elements. In the temperature range of 30 °C–150 °C, dielectric measurements (dielectric constant ‘ε’ and tangent loss ‘tanδ’) have been made at different frequencies (1, 10, 50, 100 and 1000 kHz). The samples displayed a diffused phase transition, with noticeable reduction in Curie temperature ‘Tc’ and tangent loss ‘tanδ’ with the substitution of Cr. Polarization versus electric field loops were recorded at different electric fields (5, 10, 15, 20 and 25 kV/cm) and improved values of remnant polarization ‘Pr’ and coercive field ‘Ec’ were observed. A lower Ec value facilitates easier domain movement within the unit cell, leading to enhanced ferroelectric properties. Temperature dependent P-E loops indicated that as the temperature increased, the material became softer accompanied by simultaneous reduction in Pr and Ec values.

Abstract LB273: Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes

Abstract Introduction: Inhibition of poly(ADP-ribose) polymerase (PARP) induces synthetic lethality in cells exhibiting defects in homologous repair (HR) pathways, such as BRCA1 and BRCA2-mutated breast or ovarian cancers, and 4 different PARP inhibitors (PARPi) are currently approved by the FDA. While HR-deficient cancers usually respond well to single agent PARPi, HR-proficient cancers are generally less sensitive to the treatment. Acquired resistance to PARPi due to activating mutations in BRCA genes are also not uncommon. In this work, we have examined strategies for enhancing PARPi activity in a HR-proficient context and identified histone deacetylases (HDACs) as targets for pharmacological synthetic lethality in the context of PARPi. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 were determined using CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by immunoblotting and immunofluorescence using gamma-H2AX antibodies, and comet assays for confirmation of double-strand breaks. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module. In vivo metastasis formations were examined using the pulmonary metastasis assay (PuMA). Results and discussion: Combinations of PARP and HDAC inhibitors in HR-proficient tumor cells demonstrated that HDACi could enhance the efficacy of PARPi in vitro. This was associated with increased acute and sustained DNA-damage distinct cell cycle profiles showing activity in both S- and G2/M-phases. Importantly, dual single-molecule PARP-HDAC inhibitors showed potent inhibition of PARP1 and PARP2 activity and PAR synthesis, comparable to olaparib, as well as inhibition of HDAC with IC50 values in the low µM range. Dual PARP-HDAC inhibition decreased survival of HR-proficient cells in both 2D cultures and 3D spheroid assays with lower EC50 values than olaparib or vorinostat alone. Importantly, lung metastasis formation was significantly impaired by dual PARP-HDAC inhibition. Conclusion: Combination of PARP inhibition and HDAC inhibition showed increased efficacy compared to single agent PARP inhibition in HR-proficient cells. Development of bifunctional inhibitors may provide a novel therapeutic opportunity for tumors with limited response rates to single agent PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Fariba Ghaidi, Mona Marzban, Hans Adomat, Xiaoqi Chen, John Langlands, Dennis Brown, Jeffrey Bacha, Colin Collins, Poul H. Sorensen, Wang Shen, Mads Daugaard. Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB273.

Abstract 5801: Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody

Abstract The formulation of novel chemotherapeutics capable of precisely targeting and damaging tumor cells, while preserving the integrity of healthy tissues, represents a principal challenge and is recognized as one of the biggest achievements in cancer treatment in the last few decades. Antibody-drug conjugates (ADCs) hold significant potential as an effective cancer treatment, by targeting and delivering cytotoxic drugs exclusively to tumor cells that express distinct antigens associated with malignancy. Due to the necessity for specific surface antigens, therapies with ADCs are limited to specific tumor subtypes. In this study, we show the potential of a lupus-derived, cell-penetrating, anti-DNA antibody for delivering of cytotoxic payload to tumors in vivo. We utilized a fully humanized variant, named as V66, which was chemically linked to the Topoisomerase I inhibitor, Exatecan. The primary mechanism involved in V66 cellular penetration is mediated by the Equilibrative Nucleoside Transporter ENT2. The latter shows elevated expression in a substantial subset of cancer types in comparison to healthy tissues, broadening the range of applications for ADCs in tumor therapy. Using labeled V66, we demonstrated specific tumor delivery in vivo, with minimal impact on healthy tissues. In vitro experiments validated the nuclear localization of V66-Exatecan, where it triggers the activation of the DNA damage repair pathway (DDR). As a consequence of the profound DNA damage, exposure to V66-Exatecan leads to cell death, exhibiting low nanomolar EC50 values across a diverse range of cell lines. Furthermore, V66-Exatecan treatment presents synthetic lethality with BRCA2 deficiency, evidenced by a substantial increase in sensitivity. In vivo efficacy studies conducted in BRCA2 deficient tumors revealed a remarkable effectiveness of V66-Exatecan, leading to almost complete tumor regression and substantial improvement in the probability of survival. In addition to the lack of significant weight changes observed throughout the treatment, further toxicological studies conducted in serum and tissues demonstrated the absence of short- and long-term toxicity, with optimal liver and kidney function and the absence of damage to other tissues. Likewise, bone marrow cellularity remained unaffected. Collectively, these studies indicate that V66-Exatecan achieves targeted treatment of tumor cells, where it induces DNA damage leading to cell death. The in vivo application of V66-Exatecan leads to tumor regression without triggering general toxicity that is otherwise associated with Topoisomerase I inhibitors. This work provides the basis to advance a novel nuclear-penetrating ADC for delivering cytotoxic payloads. Citation Format: Zaira Ianniello, Elias Quijano, Peter M. Glazer. Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5801.