Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

Novel quantitative trait loci from an interspecific Brassica rapa derivative improve pod shatter resistance in Brassica napus.

Pod shatter is a trait of agricultural relevance that ensures plants dehisce seeds in their native environment and has been subjected to domestication and selection for non-shattering types in several broadacre crops. However, pod shattering causes a significant yield reduction in canola (Brassica napus L.) crops. An interspecific breeding line BC95042 derived from a B. rapa/B. napus cross showed improved pod shatter resistance (up to 12-fold than a shatter-prone B. napus variety). To uncover the genetic basis and improve pod shatter resistance in new varieties, we analysed F2 and F2:3 derived populations from the cross between BC95042 and an advanced breeding line, BC95041, and genotyped with 15,498 DArTseq markers. Through genome scan, interval and inclusive composite interval mapping analyses, we identified seven quantitative trait loci (QTLs) associated with pod rupture energy, a measure for pod shatter resistance or pod strength, and they locate on A02, A03, A05, A09 and C01 chromosomes. Both parental lines contributed alleles for pod shatter resistance. We identified five pairs of significant epistatic QTLs for additive x additive, additive dominance and dominance x dominance interactions between A01/C01, A03/A07, A07/C03, A03/C03, and C01/C02 chromosomes for rupture energy. QTL effects on A03/A07 and A01/C01 were in the repulsion phase. Comparative mapping identified several candidate genes (AG, ABI3, ARF3, BP1, CEL6, FIL, FUL, GA2OX2, IND, LATE, LEUNIG, MAGL15, RPL, QRT2, RGA, SPT and TCP10) underlying main QTL and epistatic QTL interactions for pod shatter resistance. Three QTLs detected on A02, A03, and A09 were near the FUL (FRUITFULL) homologues BnaA03g39820D and BnaA09g05500D. Focusing on the FUL, we investigated putative motifs, sequence variants and the evolutionary rate of its homologues in 373 resequenced B. napus accessions of interest. BnaA09g05500D is subjected to purifying selection as it had a low Ka/Ks ratio compared to other FUL homologues in B. napus. This study provides a valuable resource for genetic improvement for yield through an understanding of the genetic mechanism controlling pod shatter resistance in Brassica species.

Characterization and Potential Function Analysis of the SRS Gene Family in Brassica napus.

SRS (SHI-related sequence) transcription factors play a crucial role in plant growth, development, and abiotic stress response. Although Brassica napus (B. napus) is one of the most important oil crops in the world, the role of SRS genes in B. napus (BnSRS) has not been well investigated. Therefore, we employed a bioinformatics approach to identify BnSRS genes from genomic data and investigated their characteristics, functions, and expression patterns, to gain a better understanding of how this gene family is involved in plant development and growth. The results revealed that there were 34 BnSRS gene family members in the genomic sequence of B. napus, unevenly distributed throughout the sequence. Based on the phylogenetic analysis, these BnSRS genes could be divided into four subgroups, with each group sharing comparable conserved motifs and gene structure. Analysis of the upstream promoter region showed that BnSRS genes may regulate hormone responses, biotic and abiotic stress response, growth, and development in B. napus. The protein-protein interaction analysis revealed the involvement of BnSRS genes in various biological processes and metabolic pathways. Our analysis of BnSRS gene expression showed that 23 BnSRS genes in the callus tissue exhibited a dominant expression pattern, suggesting their critical involvement in cell dedifferentiation, cell division, and tissue development. In addition, association analysis between genotype and agronomic traits revealed that BnSRS genes may be linked to some important agronomic traits in B. napus, suggesting that BnSRS genes were widely involved in the regulation of important agronomic traits (including C16.0, C18.0, C18.1, C18.2 C18.3, C20.1, C22.1, GLU, protein, TSW, and FFT). In this study, we predicted the evolutionary relationships and potential functions of BnSRS gene family members, providing a basis for the development of BnSRS gene functions which could facilitate targeted functional studies and genetic improvement for elite breeding in B. napus.

Contribution of PGAP3 co-amplified and co-overexpressed with ERBB2 at 17q12 involved poor prognosis in gastric cancer.

The locus at 17q12 erb-b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co-amplification and co-overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co-overexpression and clinical significance of the co-amplified genes, and to evaluate the impact of the co-amplified genes on the malignancy of GC. Co-amplification of PGAP3 and ERBB2 accompanied with co-overexpression was observed in a haploid chromosome 17 of NCI-N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co-overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI-N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI-N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co-overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co-amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way.

Genome-wide characterization of ubiquitin-conjugating enzyme gene family explores its genetic effects on the oil content and yield of Brassica napus.

Ubiquitin-conjugating enzyme (UBC) is a critical part of the ubiquitin-proteasome pathway and plays crucial roles in growth, development and abiotic stress response in plants. Although UBC genes have been detected in several plant species, characterization of this gene family at the whole-genome level has not been conducted in Brassica napus. In the present study, 200 putative BnUBCs were identified in B. napus, which were clustered into 18 subgroups based on phylogenetic analysis. BnUBCs within each subgroup showed relatively conserved gene architectures and motifs. Moreover, the gene expression patterns in various tissues as well as the identification of cis-acting regulatory elements in BnUBC promoters suggested further investigation of their potential functions in plant growth and development. Furthermore, three BnUBCs were predicted as candidate genes for regulating agronomic traits related to oil content and yield through association mapping. In conclusion, this study provided a wealth of information on the UBC family in B. napus and revealed their effects on oil content and yield, which will aid future functional research and genetic breeding of B. napus.

The prognostic and immunological role of MYB: from bladder cancer validation to pan-cancer analysis.

BACKGROUND: MYB proto-oncogene is verified as a transcription factor. Although emerging evidence showed that MYB plays a critical part in tumor progression and immunity, a systematic pan-cancer analysis of MYB still remains to be performed for determining whether MYB could serve as a biomarker for cancer screening, prognosis prediction and accurate therapy design in various human cancers. METHODS: In the present study, we performed qRT-PCR, wound healing assay and transwell assay to validate the expression level and biological function of MYB in bladder cancer. Then, we utilized several open-source databases including UCSC Xena database, TCGA, GTEx, etc. Online tools was used to process the raw data from UCSC Xena database. RESULTS: We found that the expression level of MYB is significantly higher in bladder cancer cell lines than urothelial cells. Further experiments confirmed that overexpression of MYB enhanced the ability of migration in bladder cancer. Next, we found that the expression level of MYB is significantly higher in most cancers. Meanwhile, MYB expression was positively or negatively related with the prognosis in different cancer types. In addition, MYB expression is significantly related to immune score and immune cells in most cancer types. Moreover, MYB act as an immunotherapy biomarker superior to several traditional immunotherapy biomarkers. Finally, deep deletion was the most frequent genetic alteration of MYB. CONCLUSION: MYB may serve as a powerful biomarker for tumor screening, prognostic, individualized treatment strategy in a broad range of malignancies.

Role of LONP2 in head and neck squamous cell carcinoma.

Lon Peptidase 2, Peroxisomal (LONP2) is a peptidase in peroxisomes that selectively degrades oxidatively damaged proteins in cells and is upregulated in several cancers. In this study, we found both the mRNA and protein level of LONP2 was upregulated in head and neck squamous cell carcinoma (HNSCC) samples. Ectopic overexpressed LONP2 enhanced HNSCC cell growth and migration. Nevertheless, LONP2 silencing suppressed tumor growth in vitro and in vivo. It was also indicated that knockdown of LONP2 also impaired the cell cycle progression and improved apoptosis rate of HNSCC cells. The results of RNA sequencing showed that plastin 3 (PLS3) was heavily downregulated after LONP2 silencing. PLS3 is an actin-bundling protein that functions as an oncogene in several cancers. Furthermore, knockdown of PLS3 inhibited the cell proliferation induced by LONP2 overexpression. In conclusion, we identified an oncogenic effect of LONP2 on cell proliferation and migration in HNSCC via positively regulating the expression of PLS3.

Genome-Wide Characterization of Trehalose-6-Phosphate Synthase Gene Family of Brassica napus and Potential Links with Agronomic Traits.

Trehalose and trehalose-6 phosphate played important roles in floral organ development, embryonic development, cell morphogenesis, and signal transduction under abiotic stress. However, little is known about the trehalose-6-phosphate synthase (TPS) gene family in Brassica napus. In this study, in total, 26 TPS genes in B. napus (BnTPS genes) were identified and classified into two groups. In each group, the BnTPS genes showed relatively conserved gene structures. The protein-protein interaction (PPI) network and enrichment analysis indicated that BnTPS genes were involved in the glycolysis/gluconeogenesis, fructose and mannose metabolism, galactose metabolism, pentose phosphate pathway, carbohydrate transmembrane transport, trehalose-phosphatase activity, etc. The expression of BnTPS genes varied greatly across different tissues, while most of the BnTPS genes showed a considerable improvement in expression under different abiotic stresses, indicating that BnTPS genes were significantly responsive to the abiotic treatments. In addition, the association mapping analysis revealed that eight BnTPS genes were potential regulators of particular agronomic traits. Among them, the gene BnTPS23 was significantly associated with the primary flowering time (PFT), full flowering time (FFT1), and final flowering time (FFT2), suggesting that BnTPS genes may play an important role in regulating key agronomic traits in B. napus. In summary, our research provides a better understanding of BnTPS genes, facilitates the breeding of superior B. napus varieties, and paves the way for future functional studies.

Genome-wide characterization of ovate family protein gene family associated with number of seeds per silique in Brassica napus.

Ovate family proteins (OFPs) were firstly identified in tomato as proteins controlling the pear shape of the fruit. Subsequent studies have successively proved that OFPs are a class of negative regulators of plant development, and are involved in the regulation of complex traits in different plants. However, there has been no report about the functions of OFPs in rapeseed growth to date. Here, we identified the OFPs in rapeseed at the genomic level. As a result, a total of 67 members were obtained. We then analyzed the evolution from Arabidopsis thaliana to Brassica napus, illustrated their phylogenetic and syntenic relationships, and compared the gene structure and conserved domains between different copies. We also analyzed their expression patterns in rapeseed, and found significant differences in the expression of different members and in different tissues. Additionally, we performed a GWAS for the number of seeds per silique (NSPS) in a rapeseed population consisting of 204 natural accessions, and identified a new gene BnOFP13_2 significantly associated with NSPS, which was identified as a novel function of OFPs. Haplotype analysis revealed that the accessions with haplotype 3 had a higher NSPS than other accessions, suggesting that BnOFP13_2 is associated with NSPS. Transcript profiling during the five stages of silique development demonstrated that BnOFP13_2 negatively regulates NSPS. These findings provide evidence for functional diversity of OFP gene family and important implications for oilseed rape breeding.

From Cognitive MR-Targeted Fusion Prostate Biopsy to Radical Prostatectomy: Incidence and Predictors of Gleason Grade Group Upgrading in a Chinese Cohort.

Purpose: To access the incidence and predictors of Gleason grade group upgrading from cognitive MR-targeted fusion prostate biopsy to radical prostatectomy in a Chinese cohort. Materials and Methods: We included 199 patients in our institution between January 2016 and June 2021. Multivariable logistic regression model and nomograms were utilized to analyze the collected data. Results: The concordance rate of biopsy Gleason grade group and radical prostatectomy was 50.3% (100 in 199). Upgrading occurred in 80 (40.2%) patients and 37 (68.5%) patients have an upgrading Gleason grade group when the biopsy Gleason grade group was 1. Multivariable logistic regression models were established to analyze the incidence and predictors of Gleason grade group upgrading from cognitive MR-targeted fusion prostate biopsy to radical prostatectomy. Biopsy Gleason grade group, prostate volume, and patient year were confirmed to be individual predictors of upgrading. Based on the logistic regression models, nomograms for predicting probability of prostate Gleason grade group upgrading were generated. Conclusions: We established a logistic regression model to predict the accuracy of prostate biopsy GG and provide the probability of upgrading. Clinicians should be more cautious when deciding the treatment strategy especially for prostate cancer biopsy GG1 patients. Future studies should expand the sample size and include more variables to improve the accuracy of predicting upgrading and prostate cancer early screening program is urgently needed in our city in China.

Correction to "Technical and Economic Assessment of a High-Quality Syngas Production Process Integrating Oxygen Gasification and Water Electrolysis: The Chinese Case".

[This corrects the article DOI: 10.1021/acsomega.1c03489.].

Awareness about laryngopharyngeal reflux disease among Chinese otolaryngologists: a nationwide survey.

OBJECTIVES: This study aimed to investigate the status of the current knowledge about laryngopharyngeal reflux disease (LPRD) among Chinese otolaryngologists. DESIGN: Multi-centre cross-sectional survey. SETTING: 220 medical centres in different regions of China. PARTICIPANTS: A total of 2254 otolaryngologists from 220 medical centres in China who were successfully on-site surveyed between November 2019 and December 2020. MAIN OUTCOME MEASURES: Awareness about LPRD included knowledge about risk factors, symptoms, laryngoscope signs, related diseases, current diagnostic methods and treatments. RESULTS: The percentage of participants who had heard of LPRD was 96.4%, with academic conferences as the most common source of information (73.3%). The most commonly known risk factor, symptom, laryngoscope sign, related disease, diagnostic method and treatment were alcohol consumption (44.0%), pharyngeal foreign body sensation (66.9%), hyperaemia (52.4%), pharyngolaryngitis (54.8%), pH monitoring (47.6%) and medication (82.1%), respectively. Only 28.3% of all participants knew that 24 h pH or multichannel intraluminal impedance pH monitoring was the most accurate diagnostic test. As many as 73.1% of all participants knew that proton pump inhibitors were the first-line treatment drugs. An analysis of the overall status of awareness using a scoring system suggested that otolaryngologists were better aware owing to more access, working at 3A hospitals, and postgraduate or above educational background (all p<0.05). CONCLUSION: Although the majority of Chinese otolaryngologists had heard of LPRD, their overall awareness about the disease was not encouraging. More efforts are needed to increase the knowledge about LPRD among this group of physicians. TRIAL REGISTRATION NUMBER: ChiCTR1900025581.

Roles and mechanisms of the m6A reader YTHDC1 in biological processes and diseases.

N6-methyladenosine (m6A) is a key area in Epigenetics and has been increasingly focused these years. In the m6A process, readers recognize the m6A modification on mRNAs or noncoding RNAs and mediate different downstream events. Emerging studies have shown that YTHDC1, an important m6A reader, plays a key role in many biological functions and disease progression, especially cancers. Here we summarized the current mechanisms of YTHDC1 in biological functions and diseases and offered guidance for future researches to provide potential strategy for clinical diagnose and therapy.

The promoter methylation drives down-regulation mode of HIC1 in gastric cancer, its molecular characteristics and downstream functional pathways.

Gastric cancer is a common malignant tumor of the gastrointestinal tract with a high incidence and mortality rate. Previous results have suggested that the HIC1 gene might be a tumor suppressor candidate in gastric cancer. However, several critical points need to be elucidated: (1) The correlation of HIC1 promoter methylation with its specific expression level in gastric cancer; (2) The molecular characterization of HIC1 promoter methylation; (3) The possible mechanism by which HIC1 performs its inhibitory role in gastric cancer. To address these questions, we retrieved data from TCGA database to analyze HIC1 promoter methylation levels and transcript expression data, and performed targeted region bisulfite sequencing on three stable HIC1 down-regulated cell lines and normal control cell lines, and performed whole transcriptome and metabolite assays in HIC1 knockout cell lines by CRISPR-Cas9 technique. Results demonstrated that HIC1 promoter hypermethylation might be a crucial driving force leading to its down-regulation in HIC1 expression in gastric cancer. This implicated that promoter CG methylation of HIC1 might play a major role in the development of gastric carcinogenesis. Besides, HIC1 may suppress gastric cancer progression by maintaining the normal cellular metabolism, and inhibiting the mTOR signaling pathway activity.

Integrating GWAS, linkage mapping and gene expression analyses reveal the genetic control of first branch height in Brassica napus L.

Rapeseed (Brassica napus L.) is a crucial oil crop cultivated worldwide. First branch height, an essential component of rapeseed plant architecture, has an important effect on yield and mechanized harvesting; however, the underlying genetic mechanism remains unclear. In this study, based on the 60K single nucleotide polymorphism array and a recombinant inbred lines population derived from M083 and 888-5, a total of 19 QTLs were detected in five environments, distributed on linkage groups A02, A09, A10, C06, and C07, which explained phenotypic variation ranging from 4.87 to 29.87%. Furthermore, 26 significant SNPs were discovered on Chr.A02 by genome-wide association study in a diversity panel of 324 re-sequencing accessions. The major QTL of the first branch height trait was co-located on Chr.A02 by integrating linkage mapping and association mapping. Eleven candidate genes were screened via allelic variation analysis, inter-subgenomic synteny analysis, and differential expression of genes in parental shoot apical meristem tissues. Among these genes, BnaA02g13010D, which encodes a TCP transcription factor, was confirmed as the target gene according to gene function annotation, haplotype analysis, and full-length gene sequencing, which revealed that TATA insertion/deletion in the promoter region was closely linked to significantly phenotypic differences BnaA02.TCP1 M083 overexpression resulted in decreased branch height and increased branch number in Arabidopsis. These results provide a genetic basis for first branch height and the ideal architecture of B. napus.

Fruit and Vegetable Consumption and the Risk of Prostate Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: Emerging researches has evaluated whether fruit and vegetable consumption reduce the risk of prostate cancer. However, the conclusions of published articles remained confusing. Thus, we conducted an updated systematic review and meta-analysis to confirm the relationship of fruit and vegetable consumption and the risk of prostate cancer. METHOD: We searched PubMed, EMBASE, Web of Science and Chinese National Knowledge Infrastructure (CNKI) up to September 1, 2020. We finally included 17 cohort studies related to fruit or vegetable intake after rigid quality assessment and checking references of the retrieved articles and relevant reviews. Newcastle-Ottawa scale was adopted to assess the quality of studies and random effect model with RR and 95% CI were used to assess the risk. RESULTS: No significant relationship was found between fruit consumption (RR = 1.00, 95% CI = 0.94-1.05) and vegetable consumption (RR = 0.98, 95% CI = 0.94-1.02) and the risk of prostate cancer. No significant heterogeneity or publication bias was identified. CONCLUSION: Our updated meta-analysis demonstrated that fruit and vegetable consumption can barely reduce the risk of prostate cancer with several limitations. Further clinical and basic researches are eagerly awaited to confirm our results and clarify the potential biological mechanisms.

Technical and Economic Assessment of a High-Quality Syngas Production Process Integrating Oxygen Gasification and Water Electrolysis: The Chinese Case.

Natural gas shortage, waste treatment, and renewable energy utilization and storage are the common issues in China. This research aims to evaluate a simple and reliable biomass and power-to-syngas process for high-quality syngas production integrating oxygen gasification and water electrolysis. The technical and economic evaluations are successively performed based on the simulation data. The single-stage and two-stage gasifiers are integrated and compared. The results show that the process with a two-stage gasifier has advantages in both technical and cost performances over that with a single-stage gasifier. Then, the optimal values of the operating variables are determined by investigating their effects on syngas composition and yield and the overall energy and exergy efficiencies. Then, by setting appropriate amounts of water for electrolysis and methanation pressures, the process can generate two typical products: thermochemical synthetic biogas (TSB) or synthetic natural gas (SNG). The overall energy and exergy efficiencies of TSB can reach 74.1 and 69.5%, respectively, while those of SNG can achieve 64.8 and 64.1%, respectively. The unit product cost of TSB is about one third of that of SNG, indicating that TSB makes a profit while SNG results in loss. A sensitivity analysis of the cost indicates that the constraints for TSB are much looser than those for SNG to gain profit. Additionally, this study proposes two indicators to characterize the electricity-storage capacities of biomass and syngas. The indicators show that SNG has big advantages over TSB in the specific capacity of electricity storage, and the capacities of SNG and TSB are approximately 52.0 and 4.7 times of the specific energy density of the lithium-ion battery pack in 2019, respectively. The work could be used as a reference for the countries lacking natural gas and valuable to guide the development of a two-stage gasifier, reduction in equipment cost, and evaluation of electricity-storage performance using biofuels.