Resistant Lines Research Articles

Distribution theories for genetic line of least resistance and evolvability measures.

Quantitative genetic theory on multivariate character evolution predicts that a population's response to directional selection is biased toward the major axis of the genetic covariance matrix G-the so-called genetic line of least resistance. Inferences on the genetic constraints in this sense have traditionally been made by measuring the angle of deviation of evolutionary trajectories from the major axis, or more recently by calculating the amount of genetic variance-the Hansen-Houle evolvability-available along the trajectories. However, there have not been clear practical guidelines on how these quantities can be interpreted, especially in a high-dimensional space. This study summarizes pertinent distribution theories for relevant quantities, pointing out that they can be written as ratios of quadratic forms in evolutionary trajectory vectors by taking G as a parameter. For example, a beta distribution with appropriate parameters can be used as a null distribution for squared cosine of the angle of deviation from a major axis or subspace. More general cases can be handled with the probability distribution of ratios of quadratic forms in normal variables. Apart from its use in hypothesis-testing, this latter approach could potentially be used as a heuristic tool for looking into various selection scenarios like directional and/or correlated selection as parameterized with mean and covariance of selection gradients.

Au/Doc/Quer@PDA/A10-3.2 Nanoparticles for targeted treatment of docetaxel-resistant prostate cancer

Docetaxel (Doc), as a first-line chemotherapy drug for prostate cancer (PC), often loses its therapeutic efficacy due to acquired resistance and lack of targeting specificity. Therefore, there is a need to develop a novel drug that can overcome Doc resistance and enhance its targeting ability to inhibit PC progression. In this study, we prepared Au/Doc/Quer@PDA/A10-3.2 nanoparticles (NPs) composite drug by encapsulating Doc and quercetin (Quer) within polydopamine (PDA)-coated Au NPs and further modifying them with RNA oligonucleotide aptamer A10-3.2. A10-3.2 was used for specific targeting of prostate-specific membrane antigen (PSMA)-positive PC cells (LNCaP). Quer was employed to reverse the resistance of Doc-resistant cell line (LNCaP/R) to Doc. Physical characterization using ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR) confirmed the successful preparation of Au/Doc/Quer@PDA/A10-3.2 NPs. Fluorescence imaging and flow cytometry experiments demonstrated the targeting ability of Au/Doc/Quer@PDA/A10-3.2 NPs towards PSMA-positive LNCaP/R cells. Cell proliferation, apoptosis, invasion, and migration experiments revealed that Quer reversed the resistance of LNCaP/R cells to Doc. Immunoblotting experiments further confirmed the mechanism behind sensitization of chemotherapy by Quer. Finally, we evaluated the therapeutic efficacy of Au/Doc/Quer@PDA/A10-3.2 NPs in a mouse model of PC. In conclusion, this study synthesized and validated a novel nano-composite drug (Au/Doc/Quer@PDA/A10-3.2 NPs) for combating Doc-resistant PC, which could potentially be applied in clinical treatment of PC.

KASP genotyping reveals disease resistance and yield enhancement in swarna introgression lines

Deployment of marker-assisted selection (MAS) in the molecular breeding programmes has transformed the crop breeding over the last decade, bringing speed and precision to the breeding programmes. The experiment was conducted during rainy (kharif) seasons of 2019, 2020, and 2021 at ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, Telangana for validation of the Kompetitive Allele Specific PCR (KASP) SNP markers for routine marker- assisted selection through genotyping and phenotyping of a set of breeding lines segregating for grain number, yield and resistance to bacterial blight and blast diseases. Initially, 150 introgression lines of Swarna possessing bacterial leaf blight (Xa21), blast (Pi54) and yield enhancing gene (Gn1a) at ICF4 generation were genotyped using trait specific KASP markers, and a set of 22 plants was identified to be homozygous for all the three genes, 68 plants were found positive for two genes possessing either Xa21 + Gn1a and Pi54 + Gn1a, rest were having different single gene. Simultaneously, plants which were triple gene positives were also subjected for stringent phenotypic screening for the targeted stresses, viz. bacterial blight and blast disease resistance and further evaluated for grain number and other key agronomic traits. All the three-gene positive plants exhibited high level of resistance to bacterial blight and blast diseases in comparison with positive checks and displayed higher grain number and yield than the recurrent parent, Swarna. The efficiency of SNP marker system provided by Intertek is accurate, cost effective and accurate with phenotype.

KASP genotyping reveals disease resistance and yield enhancement in swarna introgression lines

Deployment of marker-assisted selection (MAS) in the molecular breeding programmes has transformed the crop breeding over the last decade, bringing speed and precision to the breeding programmes. The experiment was conducted during rainy (kharif) seasons of 2019, 2020, and 2021 at ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, Telangana for validation of the Kompetitive Allele Specific PCR (KASP) SNP markers for routine marker- assisted selection through genotyping and phenotyping of a set of breeding lines segregating for grain number, yield and resistance to bacterial blight and blast diseases. Initially, 150 introgression lines of Swarna possessing bacterial leaf blight (Xa21), blast (Pi54) and yield enhancing gene (Gn1a) at ICF4 generation were genotyped using trait specific KASP markers, and a set of 22 plants was identified to be homozygous for all the three genes, 68 plants were found positive for two genes possessing either Xa21 + Gn1a and Pi54 + Gn1a, rest were having different single gene. Simultaneously, plants which were triple gene positives were also subjected for stringent phenotypic screening for the targeted stresses, viz. bacterial blight and blast disease resistance and further evaluated for grain number and other key agronomic traits. All the three-gene positive plants exhibited high level of resistance to bacterial blight and blast diseases in comparison with positive checks and displayed higher grain number and yield than the recurrent parent, Swarna. The efficiency of SNP marker system provided by Intertek is accurate, cost effective and accurate with phenotype.

Association between KRAS and PIK3CA Mutations and Progesterone Resistance in Endometriotic Epithelial Cell Line.

Although endometriosis is a benign disease, it is associated with cancer-related gene mutations, such as KRAS or PIK3CA. Endometriosis is associated with elevated levels of inflammatory factors that cause severe pain. In a previous study, we demonstrated that KRAS or PIK3CA mutations are associated with the activation of cell proliferation, migration, and invasion in a patient-derived immortalized endometriotic cell line, HMOsisEC10. In this study, we investigated the effects of these mutations on progesterone resistance. Since the HMOsisEC10 had suppressed progesterone receptor (PR) expression, we transduced PR-B to HMOsisEc10 cell lines including KRAS mutant and PIK3CA mutant cell lines. We conducted a migration assay, invasion assay, and MTT assay using dienogest and medroxyprogestrone acetate. All cell lines showed progesterone sensitivity with or without mutations. Regarding inflammatory factors, real-time quantitative RT-PCR revealed that the KRAS mutation cell line exhibited no suppression of Cox-2 and mPGES-1 on progesterone treatment, whereas IL-6, MCP-1, VEGF, and CYP19A1 were significantly suppressed by progesterone in both mutated cell lines. Our results suggest that KRAS mutation and PIK3CA mutation in endometriotic cells may not be associated with progesterone resistance in terms of aggressiveness. However, KRAS mutations may be associated with progesterone resistance in the context of pain.

Indole-3 acetic acid negatively regulates rose black spot disease resistance through antagonizing the salicylic acid signaling pathway via jasmonic acid.

IAA cooperates with JA to inhibit SA and negatively regulates rose black spot disease resistance. Black spot disease caused by the fungus Marssonina rosae is the most prevalent and severe ailment in rose cultivation, leading to the appearance of black spots on leaves and eventual leaf fall, significantly impacting the utilization of roses in gardens. Salicylic acid (SA) and jasmonic acid (JA) are pivotal hormones that collaborate with indole-3 acetic acid (IAA) in regulating plant defense responses; however, the detailed mechanisms underlying the induction of black spot disease resistance by IAA, JA, and SA remain unclear. In this study, transcript analysis was conducted on resistant (R13-54) and susceptible (R12-26) lines following M. rosae infection. In addition, the impact of exogenous interference with IAA on SA- and JA-mediated disease resistance was examined. The continuous accumulation of JA, in synergy with IAA, inhibited activation of the SA signaling pathway in the early infection stage, thereby negatively regulating the induction of effective resistance to black spot disease. IAA administration alleviated the inhibition of SA on JA to negatively regulate the resistance of susceptible strains by further enhancing the synthesis and accumulation of JA. However, IAA did not contribute to the negative regulation of black spot resistance when high levels of JA were inhibited. Virus-induced gene silencing of RcTIFY10A, an inhibitor of the JA signaling pathway, further suggested that IAA upregulation led to a decrease in disease resistance, a phenomenon not observed when the JA signal was inhibited. Collectively, these findings indicate that the IAA-mediated negative regulation of black spot disease resistance relies on activation of the JA signaling pathway.

Unraveling the Pathogenetic Mechanisms Underlying the Association between Specific Mitochondrial DNA Haplogroups and Parkinson's Disease.

Variants of mitochondrial DNA (mtDNA) have been identified as risk factors for the development of Parkinson's disease (PD). However, the underlying pathogenetic mechanisms remain unclear. Cybrid models carrying various genotypes of mtDNA variants were tested for resistance to PD-simulating MPP+ treatment. The most resistant line was selected for transcriptome profiling, revealing specific genes potentially influencing the resistant characteristic. We then conducted protein validation and molecular biological studies to validate the related pathways as the influential factor. Cybrids carrying the W3 mtDNA haplogroup demonstrated the most resistance to the MPP+ treatment. In the transcriptome study, PPP1R15A was identified, while further study noted elevated expressions of the coding protein GADD34 across all cybrids. In the study of GADD34-related mitochondrial unfolding protein response (mtUPR), we found that canonical mtUPR, launched by the phosphate eIF2a, is involved in the resistant characteristic of specific mtDNA to MPP+ treatment. Our study suggests that a lower expression of GADD34 in the late phase of mtUPR may prolong the mtUPR process, thereby benefitting protein homeostasis and facilitating cellular resistance to PD development. We herein demonstrate that GADD34 plays an important role in PD development and should be further investigated as a target for the development of therapies for PD.

Assessment of spring broad wheat varieties and lines for drought resistance in the conditions of the southern foreststeppe of Western Siberia using drought resistance indices

Assessment of spring broad wheat varieties and lines for drought resistance in the conditions of the southern foreststeppe of Western Siberia using drought resistance indices

Inhibition of NRF2 signaling overcomes acquired resistance to arsenic trioxide in FLT3-mutated Acute Myeloid Leukemia.

De novo acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) has been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro. We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML and molecular/pharmacological inhibition of NF-E2 related factor 2 (NRF2), a master regulator of antioxidant response improved the chemosensitivity to ATO and daunorubicin even in non FLT3-ITD mutated cell lines and primary samples.We examined the effects of molecular/pharmacological suppression of NRF2 on acquired ATO resistance in the FLT3-ITD mutant AML cell line (MV4-11-ATO-R). ATO-R cells showed increased NRF2expression, nuclear localization, and upregulation of bonafideNRF2 targets. Molecular inhibition of NRF2 in this resistant cell line improved ATO sensitivity in vitro. Digoxin treatment lowered p-AKT expression, abrogating nuclear NRF2 localization and sensitizing cells to ATO. However, digoxin and ATO did not sensitize non-ITD AML cell line THP1 with high NRF2 expression. Digoxin decreased leukemic burden and prolonged survival in MV4-11 ATO-R xenograft mice. We establish that altering NRF2 expression may reverse acquired ATO resistance in FLT3-ITD AML.

Evaluation of elite rice lines for resistance to Kenya blast fungus (Magnapothe Oryzae)

Blast resistance tends to often break down, these necessitate search-resistant genes. The screen house experiment was conducted in 2019 and 2020. A total of 56 rice genotypes, (Elite lines, monogenic lines, and local), were screened against ten Kenya isolates of Mangnaporthe oryzae. The establishment was by direct seeding of previously sprouted seeds. A completely Randomized Design (CRD) with two replicates was adopted. Inoculation was done 21 days after planting. Plants were maintained in the moist chamber (26–28 0C) for 48 hrs, then moved into an incubation chamber (25 ◦C ± 2). Disease assessment commenced 48 hours after inoculation until 21 days to full infection. Disease evaluations were performed according to the Standard Evaluation System of IRRI. Polymerase Chain Reactions (PCR) were carried out, blast resistant loci were identified using varied genetic markers that co-segregate with specific resistant loci Analysis was implemented in R version 3.3.2 (R Core Team, 2016). BLUPs were computed, in a mixed model to estimate the random effect of the genotypes. The data was subjected to analysis of variance and means separated by Tukey’s test at P < 0.05. Cluster analyses were performed in JMP software version 11.2 for Windows. Seven genotypes (IR12A311, IR10M210, IR74, 1R02A127, IR09A130, R66 and IR10N230) were more susceptible, IR13N152 and IR14F711 were more resistant to all Isolates. The monogenic lines IRBLsh-B and IRBLkh-K3 which carry blast-resistant genes, were susceptible to at least seven Kenya isolates. The local checks (Basmati 370, BAS 270 (pi9), BW196, and BAS370 (pi9) were highly susceptible. The genotype IR12F711 was consistently resistant to all Kenya Isolates.

Identification of new lines resistance to blackleg in an Argentinian rapeseed-breeding program

Rapeseed (Brassica napus L.) is the third most important oil crop worldwide and it has a high demand for its industrial characteristics that make it suitable for human consumption and production of biofuels. The main yield losses of the crop are produce by blackleg, caused by Leptosphaeria maculans (Desmaz.) Ces. and of Not. [Anamorph Phoma lingam (Tode ex Fr.) Desmaz]. In this context, 469 inbred lines (ILs) of the INTA-EEA Paraná breeding program were phenotypic characterized for its resistance to Blackleg by artificial inoculations with a local isolation. From the initial 469 ILs, a total of 59 (13%) were identified as phenotypically resistant and were characterized by molecular makers associated to resistant genes (Rlm1, Lepr3, Rpg3Dun, and Rlm4) with the aim to identify lines carrying different resistance genes to be selected as parental lines in the development of new resistant genotypes. Moreover, the resistant ILs were characterized for its agronomics performance, including yield; percentage of oil; and content of fatty acids, over two crop seasons (2018 and 2019). A Generalized Procrustes Analysis was conducted to correlate all the traits evaluated and identify elite lines. From the 59 resistants ILs, four lines were selected based on their high performances and multiple resistant gene combinations, as started lines to develop a pyramiding gene strategy. The selection of these elite lines to combine traditional breeding and gene-pyramiding based on MAS in our breeding program is a useful strategy to develop superior rapeseed genotypes.

Resistance response of yellow maize inbreds and F1 crosses against Philippine downy mildew [Peronosclerospora philippinensis (Weston) C.G. Shaw]

Maize is one of the world's most important staple crops, yet various pests and diseases impede its optimal production. Philippine maize downy mildew disease (PDM) caused by Peronosclerospora philippinensis (Weston) Shaw is one of the most devastating diseases in corn-growing areas in Asia. Breeding for resistant varieties is the most cost-effective and environmentally safe alternative for controlling the disease. In this study, we evaluated the resistance responses of yellow maize inbreds and hybrids against PDM to select promising genotypes for genetic analysis and varietal improvement. Resistance trials under greenhouse conditions were conducted to investigate the PDM resistance response of 14 selected CIMMYT lines (CMLs) and IPB Var 6 (susceptible check) to select potential parents prior to hybridization. CML 431 and CML 452 were identified to have significantly low disease incidence among the test genotypes. Subsequently, disease evaluation of the two inbred lines with putative DM resistance, four experimental hybrids derived from these inbred lines, and checks was performed in the field. Across three validation trials, inbred CML 431, and hybrid CML 452 × CML 431 showed degrees of resistance to PDM. This study's findings enabled the identification of resistant lines to downy mildew for the generation of mapping populations for genetic association studies, as well as the establishment of experimental units for transcriptomics research.

Evaluation of spring-type and winter-type Brassica napus germplasm for genetic diversity in response to flea beetle herbivory in typical and atypical planting windows

Current practices of flea beetle ( Phyllotreta spp.) control in Brassica napus L. rely heavily on seed treatments and due to growing concerns regarding the safety of such treatments on non-target and beneficial insect populations, genetic resistance would be beneficial for a more balanced integrated pest management strategy. However, none of the registered B. napus. canola varieties exhibit measurable resistance to flea beetle injury. To this end, an evaluation of 14 winter-type B. napus breeding lines and 15 spring-type B. napus breeding lines for resistance to flea beetle feedings was conducted, as it was found that at least one line in each family exhibited noticeably reduced flea beetle damage compared to sister lines in a breeding nursery. The study revealed natural genetic variation within B. napus for flea beetle antixenosis which could be used by dedicated breeders to develop canola varieties with higher levels of flea beetle resistance. Data indicated that host plant resistance did not vary between feeding by newly emerged adult flea beetles in the fall and the overwintered adults in the spring in either winter-type or spring-type canola, as well as for adult feeding preferences. This indicates that favourable genes identified in in either habitat-type could be used interchangeably to confer resistance even though the flea beetle life cycle is different for each planting period, while either overwintered adults or newly emerged adults can be used to evaluate feeding damage.

A cluster of putative resistance genes is associated with a dominant resistance to sunflower broomrape.

The HaOr5 resistance gene is located in a large genomic insertion containing putative resistance genes and provides resistance to O. cumana, preventing successful connection to the sunflower root vascular system. Orobanche cumana (sunflower broomrape) is a parasitic plant that is part of the Orobanchaceae family and specifically infests sunflower crops. This weed is an obligate parasitic plant that does not carry out photosynthetic activity or develop roots and is fully dependent on its host for its development. It produces thousands of dust-like seeds per plant. It possesses a high spreading ability and has been shown to quickly overcome resistance genes successively introduced by selection in cultivated sunflower varieties. The first part of its life cycle occurs underground. The connection to the sunflower vascular system is essential for parasitic plant survival and development. The HaOr5 gene provides resistance to sunflower broomrape race E by preventing the connection of O. cumana to the root vascular system. We mapped a single position of the HaOr5 gene by quantitative trait locus mapping using two segregating populations. The same location of the HaOr5 gene was identified by genome-wide association. Using a large population of thousands of F2 plants, we restricted the location of the HaOr5 gene to a genomic region of 193kb. By sequencing the whole genome of the resistant line harboring the major resistance gene HaOr5, we identified a large insertion of a complex genomic region containing a cluster of putative resistance genes.

Pilot protection based on the quasi-directional element of LCL-tuned half-wavelength transmission line

To create HWTL characteristics, the electrical distance of AC transmission lines is compensated to half the power frequency wavelength by utilizing tuned circuits. The LCL-tuned circuit exhibits low-pass filtering characteristics, blocking the transmission of high-frequency signals. Measurement devices were installed on both sides of the tuned circuit to assess its filtering characteristics. Based on these characteristics and by employing wavelet transformation to extract the high-frequency energy of the voltage signal on both sides of the tuned circuit as the directional protection criterion, a quasi-directional element was developed. It was determined that, if the high-frequency wavelet energy ratio exceeds one, the fault occurs in the forward direction of the tuned circuit, and if the ratio is less than one, the fault occurs in the reverse direction. Consequently, a pilot protection algorithm was formulated. The directional protection element was integrated with the tuned circuit differential protection to establish the LCL-tuned half-wavelength transmission line pilot protection scheme. This scheme mitigates the need for extensive data synchronization between both sides, and numerous simulations have demonstrated its effectiveness throughout the entire line length, regardless of transition resistance, fault type, noise and outgoing line.

Molecular mapping of the broad bean wilt virus 2 resistance locus bwvr in Capsicum annuum using BSR-seq

Key messageBulked segregant RNA seq of pools of pepper accessions that are susceptible or resistant to Broad bean wilt virus 2 identifies a gene that might confer resistance to this devastating pathogen.The single-stranded positive-sense RNA virus Broad bean wilt virus 2 (BBWV2) causes substantial damage to pepper (Capsicum annuum) cultivation. Here, we describe mapping the BBWV2 resistance locus bwvr using a F7:8 recombinant inbred line (RIL) population constructed by crossing the BBWV2-resistant pepper accession ‘SNU-C’ with the susceptible pepper accession ‘ECW30R.’ All F1 plants infected with the BBWV2 strain PAP1 were susceptible to the virus, and the RIL population showed a 1:1 ratio of resistance to susceptibility, indicating that this trait is controlled by a single recessive gene. To map bwvr, we performed bulked segregant RNA-seq (BSR-seq). We sequenced pools of resistant and susceptible lines from the RILs and aligned the reads to the high-quality ‘Dempsey’ reference genome to identify variants between the pools. This analysis identified 519,887 variants and selected the region from 245.9–250.8 Mb of the Dempsey reference genome as the quantitative trait locus region for bwvr. To finely map bwvr, we used newly designed high-resolution melting (HRM) and Kompetitive allele specific PCR (KASP) markers based on variants obtained from the BSR-seq reads and the PepperSNP16K array. Comparative analysis identified 11 SNU-C-specific SNPs within the bwvr locus. Using markers derived from these variants, we mapped the candidate bwvr locus to the region from 246.833–246.949 kb. SNU-C-specific variants clustered near DEM.v1.00035533 within the bwvr locus. DEM.v1.00035533 encodes the nitrate transporter NPF1.2 and contains a SNP within its 5′ untranslated region. The bwvr locus, which contains four genes including DEM.v1.00035533, could represent a valuable resource for global pepper breeding programs.

Honokiol induces apoptosis and autophagy in dexamethasone-resistant T-acute lymphoblastic leukemia CEM-C1 cells

ABSTRACT Objective: To study whether and, if so, how honokiol overcome dexamethasone resistance in DEX-resistant CEM-C1 cells. Methods: We investigated the effect of honokiol (0–20 µM) on cell proliferation, cell cycle, cell apoptosis and autophagy in DEX-resistant CEM-C1 cells and DEX-sensitive CEM-C7 cells. We also determined the role of c-Myc protein and mRNA in the occurrence of T-ALL associated dexamethasone resistance western blot and reverse transcription-qPCR (RT-qPCR) analysis. Results: Cell Counting Kit (CCK)−8 assay shows that DEX-resistant CEM-C1 cell lines were highly resistant to dexamethasone with IC50 of 364.1 ± 29.5 µM for 48 h treatment. However, upon treatment with dexamethasone in combination with 1.5 µM of honokiol for 48 h, the IC50 of CEM-C1 cells significantly decreased to 126.2 ± 12.3 µM, and the reversal fold was 2.88. Conversely, the IC50 of CEM-C7 cells was not changed combination of dexamethasone and honokiol as compared to that of CEM-C7 cells treated with dexamethasone alone. It has been shown that honokiol induced T-ALL cell growth inhibition by apoptosis and autophagy via downregulating cell cycle-regulated proteins (Cyclin E, CDK4, and Cyclin D1) and anti-apoptotic proteins BCL-2 and upregulating pro-apoptotic proteins Bax and led to PARP cleavage. Honokiol may overcome dexamethasone resistance in DEX-resistant CEM-C1 cell lines via the suppression of c-Myc mRNA expression. Conclusion: The combination of honokiol and DEX were better than DEX alone in DEX-resistant CEM-C1 cell lines. Honokiol may regulate T-ALL-related dexamethasone resistance by affecting c-Myc.

Molecular validation of Tomato Yellow Leaf curl Virus (TYLCV) (Ty-1/3, Ty-2 and Ty-3), Fusarium wilt (I-2) and Root- Knot Nematode (Mi-1) resistance in the breeding lines of tomato (Solanum lycopersicum L.)

Molecular validation of Tomato Yellow Leaf curl Virus (TYLCV) (Ty-1/3, Ty-2 and Ty-3), Fusarium wilt (I-2) and Root- Knot Nematode (Mi-1) resistance in the breeding lines of tomato (Solanum lycopersicum L.)

Developing a Novel Enzalutamide-Resistant Prostate Cancer Model via AR F877L Mutation in LNCaP Cells.

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.

Reversal Effect of Arctigenin on the Drug Resistance in Leukemia K562/A02 Cells and Its Mechanism

To study the effect of arctigenin(ARG) on adriamycin(ADM) resistance of leukemia cell line K562/A02 and the underlying mechanism. Human leukemia cell line K562 and ADM-resistant cell line K562/A02 were cultured and treated with 2.5-50 µmol/L ADM. Cell proliferation was measured using CCK-8 method, and half maximal inhibitory concentration (IC50) was calculated. K562/A02 cells were treated with different concentrations of ARG (1, 2, 4, 8, 16 mmol/L) to detect the effect of ARG on K562/A02 cells, and a suitable concentration (2 mmol/L) was selected for subsequent experiments. K562/A02 cells were treated with 2 mmol/L ARG and 5 µmol/L ADM, and cell apoptosis was detected by flow cytometry, the expression of P-gp, MRP, cleaved caspase-3, Bax, Bcl-2 proteins and the TLR4/NF-κB signaling pathway-related proteins were measured by Western blot. TLR4 overexpression plasmid was transfected into K562/A02 cells which were co-treated with ARG and ADM, then drug sensitivity and cell apoptosis were measured. The IC50 value of ADM on K562/A02 cells was 36.57 µmol/L, which was significantly higher than that on K562 cells (1.30 µmol/L). ARG with a concentration of ≤2 mmol/L did not have a significant effect on K562/A02 cells. 2 mmol/L ARG significantly reduced the IC50 of ADM on K562/A02 cells. In 5 µmol/L ADM-treated K562/A02 cells, compared with the control group, the apoptosis rate of K562/A02 cells in the ARG group was significantly increased, the expressions of cleaved caspase-3, Bax proteins were significantly upregulated, the expressions of P-gp, MRP, Bcl-2, TLR4, MyD88, and p-NF-κB proteins were significantly downregulated, and the differences were statistically significant (P < 0.05). After transfection with TLR4 overexpression plasmid, the sensitivity of ARG-treated K562/A02 cells to ADM was reduced (P < 0.05), the cell apoptosis was decreased, and the expressions of P-gp, MRP, Bcl-2 and TLR4/NF-κB signaling pathway-related proteins were significantly elevated, while the expressions of cleaved caspase-3 and Bax proteins were significantly decreased (all P < 0.05). ARG may reverse the resistance of human leukemia cell line K562/A02 to ADM by inhibiting TLR4/NF-κB signaling pathway.