Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Liquid- liquid (Cyclohexanone: Cyclohexanol) separation using augmented tight nanofiltration membrane: A sustainable approach.

Organic solvent nanofiltration (OSN) is an incipient technology in the field of organic liquid-liquid separation. The incomplete separations and complexity involved in these, forces many organic liquids to be released as effluents and the adverse effects of these on environment is enormous and irreparable. The work prominences on the complete separation of industrially significant cyclohexanone: cyclohexanol (keto-alcohol oil) and heptane: toluene mixtures. The separations of these above-mentioned organic liquid mixtures were carried out using the fabricated Lewis acid modified graphitic carbon nitride (Cu2O@g-C3N4) incorporated polyvinylidene difluoride (PVDF) composite membranes. These fabricated membranes showed a separation factor of 18.16 and flux of 1.62 Lm-2h-1 for cyclohexanone: cyclohexanol mixture and separation of heptane and toluene mixture (with heptane flux of 1.52 Lm-2h-1) showed a separation factor of 9.9. The selectivity and productivity are based on the polarity and size of the organic liquids. The role of Cu2O@g-C3N4 is influencing the pore size distribution, increased divergence from solubility parameters, polarity, solvent uptake and porosity of the composite membranes. The developed composite membranes are thus envisioned to be apt for a wide range of liquid-liquid separations due to its implicit nature.

Metabolism of the 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor, Mesotrione, in Multiple-Herbicide-Resistant Palmer amaranth (Amaranthus palmeri).

Metabolic resistance to the maize-selective, HPPD-inhibiting herbicide, mesotrione, occurs via Phase I ring hydroxylation in resistant waterhemp and Palmer amaranth; however, mesotrione detoxification pathways post-Phase I are unknown. This research aims to (1) evaluate Palmer amaranth populations for mesotrione resistance via survivorship, foliar injury, and aboveground biomass, (2) determine mesotrione metabolism rates in Palmer amaranth populations during a time course, and (3) identify mesotrione metabolites including and beyond Phase I oxidation. The Palmer amaranth populations, SYNR1 and SYNR2, exhibited higher survival rates (100%), aboveground biomass (c.a. 50%), and lower injury (25-30%) following mesotrione treatment than other populations studied. These two populations also metabolized mesotrione 2-fold faster than sensitive populations, PPI1 and PPI2, and rapidly formed 4-OH-mesotrione. Additionally, SYNR1 and SYNR2 formed 5-OH-mesotrione, which is not produced in high abundance in waterhemp or naturally tolerant maize. Metabolite features derived from 4/5-OH-mesotrione and potential Phase II mesotrione-conjugates were detected and characterized by liquid chromatography-mass spectrometry (LCMS).

A mixture of mesotrione and atrazine harms adults and larvae of the predatory wasp Polistes satan.

Herbicides have been intensively used for weed control, raising concerns about their potentially adverse effects on non-target organisms. Research on the effects of these common agrochemicals on beneficial insects and the ecosystem services they provide (e.g., predation and pollination) is scarce. Therefore, we tested whether a commercial formulation comprising a mixture of mesotrione and atrazine was detrimental to adult females and larvae of the Neotropical predatory social wasp Polistes satan, which is an effective natural enemy of crop pests. Wasps were individually fed syrups contaminated with different concentrations of the herbicide above and below the maximum label rate (MLR = 12 mL/L). Survival was assessed. The locomotor activity, immune response, and midgut morphology of adults as well as the immune response of the larvae were also studied. Herbicide concentrations far above the MLR (12, 40, and 100 times) caused adult mortality, whereas lower concentrations (0.5, 1, and 6 times) did not. Herbicide exposure at 0.5 to 12 times the MLR increased adult activity. Adult exposure at 0.1 or 0.5 times the MLR did not affect melanotic encapsulation of foreign bodies but led to changes in the morphology of the midgut epithelium and peritrophic matrix. In larvae, the ingestion of herbicide at 0.1 or 0.2 times the MLR (corresponding to 9.6 and 19.2 ng of herbicide per individual) did not cause mortality but decreased their melanization-encapsulation response. Increased locomotor activity in herbicide-exposed adults can affect their foraging activity. The altered midgut morphology of adults coupled with the decreased immune response in larvae caused by herbicide exposure at realistic concentrations can increase the susceptibility of wasps to infections. Therefore, herbicides are toxic to predatory wasps.

A novel isophorone-based fluorescent probe for recognizing Al3+ and its bioimaging in plants.

Aluminium ions (Al3+) are widely present in industries and daily life and are closely related to human health and environmental protection. Therefore, it is crucial to detect their concentration. In this paper, a convenient and reliable small molecule fluorescent probe based on a dicyanoisophorone Schiff base and 2-pyridinecarbohydrazide has been developed. The probe is capable of selectively detecting Al3+ with the advantages of near-infrared emission (maximum emission wavelength of 625 nm), good selectivity, high sensitivity (detection limit of 2.18 × 10-7 M) and fast response time (15 s). It has good potential for rapid detection and visual tracking of Al3+ in aqueous solutions and plant bodies.

Metabolism of Tembotrione, a Triketone Herbicide, confers Differential Sensitivity in Winter Wheat (Triticum aestivum).

Tembotrione is a triketone herbicide widely used for broad-spectrum weed control in corn but not registered for use in wheat. A wide collection of spring, winter, and EMS-derived mutant lines of wheat was evaluated for their response to tembotrione treatment. Two winter wheat (WW) genotypes (WW-1 and WW-2) were found to be least sensitive to this herbicide, surviving >6 times the field recommended dose (92 g ai ha-1) compared to the most sensitive genotype (WW-24). Further, HPLC analysis using [14C] tembotrione suggested that both WW-1 and WW-2 metabolized tembotrione rapidly to nontoxic metabolites. Pretreatment with a P450 inhibitor (malathion) followed by tembotrione application increased the sensitivity of WW-1 and WW-2 genotypes to this herbicide, suggesting likely involvement of P450 enzymes in metabolizing tembotrione similar to corn. Overall, our results suggest that the genotypes WW-1 and WW-2 can potentially be used to develop tembotrione-resistant wheat varieties.

A novel isophorone-based NIR fluorescent and colormetric probe for Al3+ sensing and its application for living cells and plants imaging.

In this paper, an isophorone-based NIR fluorescent and colormetric probe BDDH for Al3+ was synthesized and characterized, it showed highly selectivity and sensitivity through significant fluorescence enhancement and visible color change towards Al3+. The job plot confirmed that the binding ratio of BDDH with Al3+ was 1:1. Furthermore, the limit of detection (LOD) of Al3+ was determined to be 4.01 × 10-8 M. Moreover, BDDH was successfully applicated in identification of Al3+ in the different water samples, cell imaging in alive MCF-7 cells and plant imaging in soybean roots.

Discovery of Novel Pyrazole Acyl Thiourea Skeleton Analogue as Potential Herbicide Candidates.

To discover novel transketolase (TKL, EC 2.2.1.1) inhibitors with potential herbicidal applications, a series of pyrazole acyl thiourea derivatives were designed based on a previously obtained pyrazolamide acyl lead compound, employing a scaffold hopping strategy. The compounds were synthesized, their structures were characterized, and they were evaluated for herbicidal activities. The results indicate that 7a exhibited exceptional herbicidal activity against Digitaria sanguinalis and Amaranthus retroflexus at a dosage of 90 g ai/ha, using the foliar spray method in a greenhouse. This performance is comparable to that of commercial products, such as nicosulfuron and mesotrione. Moreover, 7a showed moderate growth inhibitory activity against the young root and stem of A. retroflexus at 200 mg/L in the small cup method, similar to that of nicosulfuron and mesotrione. Subsequent mode-of-action verification experiments revealed that 7a and 7e inhibited Setaria viridis TKL (SvTKL) enzyme activity, with IC50 values of 0.740 and 0.474 mg/L, respectively. Furthermore, they exhibited inhibitory effects on the Brassica napus acetohydroxyacid synthase enzyme activity. Molecular docking predicted potential interactions between these (7a and 7e) and SvTKL. A greenhouse experiment demonstrated that 7a exhibited favorable crop safety at 150 g ai/ha. Therefore, 7a is a promising herbicidal candidate that is worthy of further development.

The cathodically pretreated boron-doped diamond electrode as an environmentally friendly electrochemical tool for the detection and monitoring of mesotrione in food samples.

Excessive pesticide use can harm human health, making it essential to develop new techniques to monitor hazardous pesticides in food. Our study focuses on detecting mesotrione (MST) using an unmodified boron-doped diamond (BDD) electrode. This was the first application of cathodically pretreated BDD electrode for the detection of MST, based on its oxidation at a high potential value of +1.4 V. We theoretically examined the oxidation mechanism of MST trough the utilization of density functional theory (DFT) methodology. The utilized DPV method achieved a detection limit of 0.45 µM and showed satisfactory selectivity. The practical application of this method was demonstrated by examining corn-based food products. To ensure practical application of the method, MST was deliberately added to the samples to evaluate the accuracy of the proposed method. The effectiveness of the method was confirmed by using HPLC method. This environmentally-friendly approach can establish a solid foundation for future use in food analysis.

Lachnuoic Acids A-F: Ambuic Acid Congeners from a Saprotrophic Lachnum Species.

Chemical prospection of an extract derived from a saprotrophic fungus Lachnum sp. IW157 resulted in the isolation and characterization of six unprecedentedly reported ambuic acid analogues named lachnuoic acids A-F (1-6). Chemical structures of 1-6 were determined based on comprehensive 1D and 2D NMR spectroscopic analyses together with HR-ESI-MS spectrometry. The relative configurations of 1-3 were defined by ROESY spectroscopic analyses while their absolute configurations were unambiguously determined by Mosher's esters method. All isolated compounds were subjected to cytotoxic, antimicrobial, antibiofilm and nematicidal activity assays where only lachnuoic acid A (1) revealed potent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis at MIC values of 16.6 and 8.3 µg/mL, respectively.

A novel Zn2+-coordination fluorescence probe for sensing HPPD inhibitors and its application in environmental media and biological imaging.

Mesotrione, topramezone, tembotrione, and sulcotrione are four types of 4-hydroxyphenylpyruvate dioxidase (HPPD) inhibitor herbicides that are extensively employed in agricultural practices, but their usage also leads to environmental pollution and poses risks to human health. A probe (E)-1-((2-(pyridin-2-yl) hydrazineylidene) methyl) naphthalen-2-ol (CHMN) based on chelation enhancement (CHEF) effect synthesized. CHMN was first chelated with Zn2+ to form a probe system with green, which can be further used to detect mesotrione, topramezone, tembotrione and sulcotrione in complicated environment. CHMN-Zn2+ detection of four pesticides was accurate, with an excellent linear relationship between 0 and 100 µM. The detection limits were LODmesotrione = 7.79 µM, LODtopramezone = 1.91 µM, LODtembotrione = 1.38 µM and LODsulcotrione = 2.43 µM. The detection time is 1 min, and it is successfully applied in real water sample and bioimaging. This work can provide a novel method for studying the migration and behavior of environmental pollutants.

Anticancer behaviour of 2,2'-(pyridin-2-ylmethylene)bis(5,5-dimethylcyclohexane-1,3-dione)-based palladium(II) complex and its DNA, BSA binding propensity and DFT study.

Herein, we report the synthesis and biological evaluation of [Pd(L)(OH2)Cl] complex (where L = 2,2'-(pyridin-2-ylmethylene)bis(5,5-dimethylcyclohexane-1,3-dione) as a novel promising anticancer candidate. The complex was characterized by single-crystal X-ray diffraction and other various spectroscopic techniques. Besides, the optimized structure was determined through DFT calculations revealing that the coordination geometry of [Pd(L)(OH2)Cl] complex is square planar. The binding propensity of [Pd(L)(OH2)Cl] complex with DNA and BSA was assessed by the spectrophotometric method. The antimicrobial profile of the ligand and its [Pd(L)(OH2)Cl] complex was screened against clinically important bacterial strains. [Pd(L)(OH2)Cl] complex showed promising activity against these microorganisms. Pd(L)(OH2)Cl] complex exhibited a potent antiproliferative potential compared to its ligand against different human cancer cells (A549, HCT116, MDA-MB-231, and HepG2) with less toxic effect against normal cells (WI-38). Additionally, [Pd(L)(OH2)Cl] complex exerted its anticancer effects against the most responsive cells (HCT116 cells; IC50 = 11 ± 1 µM) through suppressing their colony-forming capabilities and triggering apoptosis and cell cycle arrest at S phase. Quantitative PCR analysis revealed a remarkable upregulation of the mRNA expression level of p53 and caspase-3 by 4.8- and 5.9-fold, respectively, relative to control. Remarkable binding properties and non-covalent interactions between L and its [Pd(L)(OH2)Cl] complex with the binding sites of different receptors including CDK2, MurE ligase, DNA, and BSA were established using molecular docking. Based on our results, [Pd(L)(OH2)Cl] complex is an intriguing candidate for future investigations as a potential anticancer drug for the treatment of colon cancer.

Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa.

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to ß-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying ß-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL.

Paenibacillus lacisoli sp. nov., a mesotrione-degrading strain isolated from lakeside soil.

A Gram-stain-positive, facultatively anaerobic, rod-shaped bacterium, designated JX-17T, was isolated from a soil sample collected in Jiangxi Province, PR China. Growth was observed at 15-48 °C (optimum 37 °C), at pH 5.0-9.0 (optimum pH 7.0) and with 0-6.0% (w/v) NaCl (optimum 1.0%). Strain JX-17T could degrade approximately 50% of 50 mg/L mesotrione within 2 days of incubation, but could not use mesotrione as sole carbon source for growth. Strain JX-17T showed less than 95.3% 16S rRNA gene sequence similarity with type strains of the genus Paenibacillus. In the phylogenetic tree based on 16S rRNA gene and genome sequences, strain JX-17T formed a distinct lineage within the genus Paenibacillus. The ANI values between JX-17T and the most closely related type strains P. lentus CMG1240T and P. farraposensis UY79T were 70.1% and 71.4%, respectively, and the dDDH values between them were 19.0% and 23.3%, respectively. The major cellular fatty acids were anteiso-C15:0, iso-C16:0, anteiso-C17:0 and C16:0, the predominant respiratory quinone was MK-7, the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid, an aminophospholipid and a phosphatidylinositol. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid, and the DNA G + C content was 50.1 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, strain JX-17T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus lacisoli sp. nov is proposed, with strain JX-17T (= GDMCC 1.3962T = KCTC 43568T) as the type strain.

Evaluation of fumigant toxicity of cyclohexanone to 5 species of insect pests.

Cyclohexanone is a major precursor for nylon production and is also used as a pesticide solvent. In this study, cyclohexanone was evaluated as a fumigant against rice weevil adults, confused flour beetle adults, western flower thrips larvae and adults, spotted wing drosophila adults, and subterranean termite workers. Cyclohexanone fumigation was effective against all 5 insects, and there were considerable variations in susceptibility to cyclohexanone fumigation among the 5 species. At 20 °C, complete control of spotted wing drosophila adults was achieved in 1-h fumigation with 25 µl/l of cyclohexanone and complete control of eastern subterranean termite workers was achieved in 3-h fumigations with 50 µl/l dose of cyclohexanone. Stored-product insects confused flour beetle, and rice weevil adults were more tolerant to cyclohexanone fumigation. Fumigations of 24 h with 75 µl/l dose of cyclohexanone caused 100% mortality of rice weevil adults and 98% mortality of confused flower beetle adults. Even at a 100 µl/l dose, the 24-h fumigations did not achieve 100% mortality of confused flour beetle adults. At 5 °C, complete control of western flower thrips was achieved in 3- and 6-h fumigations with 100 and 50 µl/l doses of cyclohexanone, respectively. Cyclohexanone vapor concentrations were measured using cyclohexanone detector tubes. Vapor concentrations were far below the expected saturation concentration indicating that most cyclohexanone did not exist as vapor in fumigation chambers. The results of effective control of all 5 insect species suggest that cyclohexanone has the potential to be used as a fumigant for postharvest pest control.

A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

A Berberine Bridge Enzyme-like Oxidase Mediates the Cage-like Acresorbicillinol C Biosynthesis.

Oxosorbicillinol and cage-like acresorbicillinol C are bioactive sorbicillinoids produced by Acremonium chrysogenum. We found that a berberine bridge enzyme-like oxidase AcsorD was responsible for their biosynthesis by gene deletion and heterologous expression. AcsorD catalyzed oxidation of sorbicillinol to form oxosorbicillinol in in vitro assays, which was successively condensed with sorbicillinol to form acresorbicillinol C spontaneously. Finally, site-directed mutation revealed that Tyr525 was the key residue in the catalysis of the oxidation reaction and unlocking cage-like acresorbicillinol C production.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.