Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells.

The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium Myxococcus xanthus harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the M. xanthus genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

LncRNA HEIH modulates the proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating the miR-193a-5p/CDK8 axis.

Background: Hepatocellular carcinoma (HCC), a malignant tumor with a high mortality rate, is a serious problem worldwide. This research sought to examine how long non-coding RNA (lncRNA) high expression in hepatocellular carcinoma (HEIH) affects the development and progression of HCC. Methods: The expression of HEIH in HCC patients and HCC cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, HEIH was knocked down, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, wound-healing and transwell assays were conducted to evaluate the effects of HEIH on the proliferation, migration, and invasion of the HCC cells, respectively. A xenografted mice model was constructed to investigate the function of HEIH on HCC tumorigenesis in vivo. The interactions among HEIH, microRNA (miR)-193a-5p and cyclin-dependent kinase 8 (CDK8) were also investigated by dual luciferase reporter (DLR) gene and RNA immunoprecipitation (RIP) assays. Results: HEIH was highly expressed in HCC tissues, and was correlated with advanced TNM stage and the absence of vascular invasion. The in vitro experiments showed that silencing HEIH restrained the viability, migration, and invasion of HCC cells, and hampered xenograft tumor growth in vivo. Additionally, HEIH was shown to bind directly to microRNA 193a-5p (miR-193a-5p) and facilitate the expression of the target gene CDK8 in the HCC cells. CDK8 overexpression and miR-193a-5p silencing attenuated the effects of si-HEIH-induced inhibition on the proliferation, migration, and invasion of HCC cells. Conclusions: Silencing HEIH restrained the proliferation, migration, and invasion of HCC cells via the miR-193a-5p/CDK8 axis.

Responsiveness to long days for flowering is reduced in Arabidopsis by yearly variation in growing season temperatures.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

MDFI is a novel biomarker for poor prognosis in LUAD.

Background: Approximately 80% of lung cancers are non-small cell lung cancers (NSCLC). Lung adenocarcinoma (LUAD) is the main subtype of NSCLC. The incidence and mortality of lung cancer are also increasing yearly. Myogenic differentiation family inhibitor (MDFI) as a transcription factor, its role in lung cancer has not yet been clarified. Methods: LUAD data were downloaded from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO), analyzed and plotted using the R language. Associations between Clinical information and MDFI expression were assessed using logistic regression analyses to explore the effects of MDFI on LUAD. Two sets of tissue microarrays (TMAs) further confirmed the overexpression of MDFI in LUAD and its impact on prognosis. In addition, we examined the correlation between MDFI and immune infiltration. To investigate the effect of MDFI on the biological behavior of LUAD tumor cells by GSEA and GO/KEGG analysis. The survival status and somatic mutational characteristics of patients according to MDFI levels were depicted and analyzed. Results: Expression of high MDFI in LUAD tissues via analyzing TCGA dataset (P <0.001). Kaplan-Meier survival analysis indicated a poor prognosis for those patients with LUAD who had upregulated MDFI expression levels (P <0.001). This was also verified by two groups of TMAs (P=0.024). Using logistic statistics analysis, MDFI was identified as an independent predictive factor and was associated with poor prognosis in LUAD (P <0.001, P =0.021). Assessment of clinical characteristics, tumor mutation burden (TMB), and tumor microenvironment (TME) between high- and low-expression score groups showed lower TMB, richer immune cell infiltration, and better prognosis in the low-risk group. Conclusion: This study showed that MDFI was overexpressed in LUAD and was significantly associated with poor prognosis, indicating that MDFI may be used as a potential novel biomarker for the diagnosis and prognosis of LUAD. MDFI is associated with immune infiltration of LUAD and it is reasonable to speculate that it plays an important role in tumor proliferation and spread. In view of the significant differences in MDFI expression between different biological activities, LUAD patients with MDFI overexpression may obtain more precise treatment strategies in the clinic.

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (-2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Aberrant Expression of BUB1B Contributes to the Progression of Thyroid Carcinoma and Predicts Poor Outcomes for Patients.

Objective: This study aimed to clarify the function and potential mechanism of BUB1B in THCA. Methods: Expression of BUB1B in THCA was firstly determined, and its important prognostic value was then demonstrated. The potential mechanism was initially predicted by KEGG analysis. To explore the specific function of BUB1B in THCA, we used lentivirus infection to knock down the BUB1B, and then performed flow cytometry, colony formation, transwell, and wound-healing assays. Related protein expression was detected through western blotting. Additionally, we predicted the BUB1B-regulated pathways involved in THCA by GSEA analysis. Results: BUB1B expression was highly increased in THCA tissues relative to normal controls. We further found that BUB1B was essential for tumor cell proliferation, and BUB1B high expression predicted a shorter PFS time of THCA patients. More importantly, Cox regression determined the BUB1B as an independent prognostic factor for PFS in THCA. BUB1B was initially found to participate in the cell cycle pathway from KEGG analysis. Unexpectedly, we did not detect the disturbing effect on the cell cycle distribution of THCA cells with BUB1B knockdown. But, BUB1B knockdown inhibited the proliferation, invasion, and migration of THCA cells, as well as increased apoptotic cells, and the results were further confirmed by western blotting. Through GSEA analysis, we predicted a positive correlation between BUB1B and metastasis-related pathways such as mTOR and NF-kappa B signaling pathways. Conclusions: Present study identified BUB1B as a promising clinical prognostic factor in THCA, as well as a potential novel therapeutic target for cancer treatment.

First-line treatment options for advanced non-small cell lung cancer patients with PD-L1 ≥ 50%: a systematic review and network meta-analysis.

INTRODUCTION: Single-agent immune checkpoint inhibitors (ICIs) like pembrolizumab or atezolizumab have been approved as first-line monotherapy for advanced non-small cell lung cancer (NSCLC) patients with PD-L1 ≥ 50%. However, emerging evidences have showed that ICI combinations (chemoimmunotherapy or dual-agent ICIs) argue to offer a higher response rate. In this network meta-analysis, we aimed to evaluate the efficacy and toxicity of first-line single-agent ICIs versus ICI combinations for advanced NSCLC patients with PD-L1 ≥ 50%. METHODS: PubMed, Embase, Cochrane Library and the Clinicaltrials.gov were systematically searched to extract eligible literature until December 2020. Outcomes included overall survival (OS), progression free survival (PFS), objective response rate (ORR) and treatment related adverse events (TRAEs) of grades 3-5. RESULTS: Fourteen studies with 3448 patients were included. The results showed that chemotherapy plus ICIs significantly improved PFS and ORR compared to chemotherapy, and sinti-chemo (HR: 0.31, 95% CI: 0.20-0.49) and pembro-chemo (OR: 4.2, 95% CI: 2.6-6.7) ranked first. In terms of OS, cemiplimab provided the best benefit versus chemotherapy (HR: 0.57, 95% CI: 0.43-0.77), followed by atezolizumab and pembro-chemo. In the subgroup analysis of histological type, pembro-chemo and sinti-chemo showed the best benefit of PFS in squamous and nonsquamous NSCLC, respectively, while there was no significant difference between ICI combinations with single-agent ICIs in OS. Moreover, the addition of chemotherapy to ICIs elevated toxicity compared to chemotherapy. CONCLUSION: The study suggested that chemotherapy plus ICIs might improve PFS and ORR than single-agent ICIs for advanced NSCLC patients with PD-L1 ≥ 50%. However, it did not lead to OS benefit.

Favorable function of Ectonucleoside triphosphate diphosphohydrolase 1 high expression in thyroid carcinoma.

BACKGROUND: Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) has been proved to play a vital role in human cancers. Nevertheless, the exact role of ENTPD1 in thyroid carcinoma (THCA) remained unclear. This study aimed to evaluate its prognostic value and reveal the potential regulatory mechanism in THCA. RESULTS: (1) Higher expression of ENTPD1 was found in THCA tissue compared with normal tissue (all P < 0.05). ENTPD1 expression was associated with age, sub-type and clinical stage of THCA patients (all P < 0.05). Immunohistochemistry showed its higher expression in patients with early stage. (2) ENTPD1 high expression was associated with favorable overall survival of THCA patients (all P < 0.05), especially for male patients and those with advanced stage, B-cells and Natural killer T-cells decreased (all P < 0.05). (3) Pathway analysis indicated that ENTPD1 mainly participated in metabolic process and negatively regulated metabolism-related pathway such as butanoate metabolism, pyruvate metabolism and fatty acid metabolism ((all P < 0.05). (4) ENTPD1 appeared genetic alteration in THCA, and the main mutation type of ENTPD1 was missense substitution (15.89%). (5) A weak correlation between ENTPD1 expression and methylation was found (P < 0.001). Methylation of ENTPD1 in THCA was lower than in normal group (P < 0.001), but it did not correlate with any clinical phenotypes of THCA patients. CONCLUSIONS: ENTPD1 was highly expressed in THCA, and ENTPD1 high expression contributed to the prognosis of THCA patients. The present study inferred that ENTPD1 might serve as a metabolism-related gene and play a critical role in THCA through regulating metabolic pathways.

Favorable Prognostic Impact of Cathepsin H (CTSH) High Expression in Thyroid Carcinoma.

BACKGROUND: Presently, no study reported the function of cathepsin H (CTSH) in thyroid carcinoma (THCA). The aim of present study was to initially explore the factors affecting CTSH expression, and association between CTSH expression and survival rate in THCA. METHODS: We explored mRNA expression of CTSH in normal and BRCA tissues, and evaluated prognostic impact of CTSH expression on the overall survival of THCA patients. Then, related factors influencing CTSH mRNA expression in THCA were analyzed. Functional enrichment analysis was performed to reveal the potential function of CTSH involved in THCA. We also constructed PPI network among co-expressed genes of CTSH to determine hub genes, followed by association analysis on hub genes with CTSH. RESULTS: (1) CTSH mRNA was highly expressed in THCA compared with normal group (P<0.001). High expression of CTSH was conducive to the overall survival of THCA patients (P=0.0027). CTSH was then determined as an independent prognostic factor in THCA (P=0.024). (2) The mRNA expression of CTSH was statistically related to patient's histological type, N stage, T stage, tumor stage and sample type (all P<0.001). CTSH copy number variation and methylation also influenced its mRNA expression (all P<0.001). (3) Pathway analysis indicated that CTSH mainly participated in cancer-related pathways, such as hedgehog signaling pathway, cytokine-cytokine receptor interaction and JAK-STAT signaling pathway (all P<0.05). (4) The top 10 co-expressed genes in whole PPI network showed significant correlation with CTSH expression (all P<0.001). CONCLUSION: CTSH higher expression was observed in THCA, which caused a good prognosis of patients. CTSH expression might be regulated by multiple factors including clinical characteristic, methylation, copy number and other genes. This study demonstrated the clinical significance of CTSH in THCA, as well as revealed the potential pathway associated with CTSH involved in thyroid cancer.

Expression and Prognostic Value Identification of Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2) in Brain Low-Grade Glioma.

OBJECTIVE: This study aimed to reveal the potential function of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and emphasized its importance in brain low-grade glioma (LGG). METHODS: We firstly explored the differential expression of MTHFD2 mRNA in LGG and normal tissues, followed by correlation analysis of MTHFD2 mRNA expression with patient's clinical characteristics. MTHFD2 protein expression in LGG and subcellular location were also evaluated. Then, survival analysis was performed to reveal the influence of MTHFD2 expression on the overall survival of patients, and Cox regression analysis was applied to predict the prognostic factor for overall survival of LGG. Finally, we performed functional analysis to reveal potential MTHFD2-associated pathways involved in LGG. RESULTS: We found that MTHFD2 was highly expressed in LGG patients (P<0.05), and MTHFD2 expression was related to patient's age and IDH mutation status (all P<0.05). MTHFD2 protein was mainly localized to the mitochondria. Survival analysis showed that high expression of MTHFD2 desirably improved the prognosis of LGG patients (P<0.001), especially for those patients with age ≥45 years (P<0.05). But independent prognostic role of MTHFD2 in LGG was not observed. Pathway enrichment analysis indicated that MTHFD2 high expression significantly and positively participated in the pathway of one carbon pool by folate (all P<0.05). CONCLUSION: High expression of MTHFD2 was observed in LGG, which was favorable for the overall survival of LGG patients. Our results assumed that MTHFD2 high expression might play a pivotal role in LGG through positively regulating pathway of one carbon pool by folate.

Identification and Validation of Biglycan as Prognosis and Therapy Markers for Patients with Stomach Adenocarcinoma.

OBJECTIVE: Previous studies have confirmed the biglycan (BGN) as a core gene in stomach adenocarcinoma (STAD). Present study aimed at conducting further investigations to reveal the potential function of BGN in STAD. METHODS: The mRNA and protein expressions of BGN in STAD were firstly evaluated, followed by immune infiltration analyses. The influence of BGN expression on the overall survival of STAD patients was subsequently analyzed, and a restrict survival analysis was performed as well. The protein-protein interaction (PPI) network analysis on the co-expressed genes with BGN was finally adopted to obtain the most important module in the whole network, and significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway associated with hub genes within the main module was further predicted. RESULTS: (1) We verified the mRNA high expression of BGN in STAD (all P<0.05), and higher expression was observed in patients with stage 4 (P<0.001) and grade 3 (P<0.001). The BGN protein was mainly localized to the golgi apparatus, and protein expression displayed an individual difference. (2) Immune infiltration analysis showed the strongest correlation between BGN expression and abundance of natural killer cell (P<0.001), Transforming Growth Factor beta 1 (TGFB1) (P<0.001), TNF Receptor Superfamily Member 4 (TNFRSF4) (P<0.001) and C-X-C Motif Chemokine Ligand 12 (CXCL12) (P<0.001) in STAD. BGN expression was also correlated to immune subtypes (P=0.0347) and molecular subtypes (P=0.0263) in STAD. (3) High expression of BGN shortened the overall survival time of STAD patients (all P<0.01). The influence of BGN expression on the prognosis was statistically affected by several clinical phenotypes and cohorts of patients. Cox regression showed that BGN can be considered as a prognostic predictor of STAD (P<0.05). (4) Pathway analysis indicated that BGN possibly participated in ECM-receptor interaction, focal adhesion, human papillomavirus infection and PI3K-Akt signaling pathway (all P<0.001). CONCLUSION: BGN was highly expressed in STAD, implying a poor prognosis of patients. Relevant signal pathways associated with BGN were distinguished as well. BGN could be used as a potential therapeutic biomarker for STAD.

Prognostic Value and Significant Pathway Exploration Associated with TOP2A Involved in Papillary Thyroid Cancer.

BACKGROUND: Topoisomerase 2-alpha (TOP2A) has been identified as a hub gene that played an important role in the initiation and progression of thyroid carcinoma (THCA). However, the exact function of TOP2A in papillary thyroid cancer (PTC) remained elusive. The current study aimed to evaluate the TOP2A expression, prognosis significance and key signaling pathways involved in PTC. METHODS: We firstly evaluated the expression of TOP2A in PTC via UALCAN, cBioportal, HPA and LinkdedOmics databases. Genetic alteration of TOP2A in PTC was then explored in cBioportal. Prognostic impacts of TOP2A expression on disease-free survival (DFS) of PTC patients were subsequently evaluated using Kaplan-Meier plotter and Gepia databases. Taking gender, age, cancer stage, T, N and M stages into consideration, we compared survival difference between TOP2A high and low expression groups. KEGG pathway analysis in WebGestalt and GSEA analysis were further performed to reveal the potential TOP2A-associated signaling pathways involved in PTC. Finally, the upstream microRNAs of TOP2A were assessed using DIANA, TargetScan, miRDB and miRWALK database, followed by mechanism exploration of upstream microRNAs. RESULTS: 1) The mRNA and protein of TOP2A were highly expressed in PTC tissue compared with normal thyroid tissue. TOP2A expression was associated with patient's age, N stage and cancer stage (all P<0.05). TOP2A protein was mainly localized to nucleoplasm. 2) Most of samples occurred the missense substitution, and mutation site was located at K1199E. Nucleotide mutations were mainly presented as G>A (35.29%). 3) TOP2A high expression significantly influenced the DFS of PTC patients (P=0.015). Restricted survival analysis showed that TOP2A high expression caused poorer DFS of female patients (P=0.003) and those with age <60 years old (P=0.002), early clinical stage (P=0.012), N0 stage (P=0.002) or M0 stage (P=0.040). 4) Pathway analysis suggested that TOP2A positively participated in the cell cycle, oocyte meiosis and p53 signaling pathways (all P<0.05) involved in thyroid cancer. CONCLUSION: The expression of TOP2A was higher in PTC tissue, which resulted in a worse DFS of patients with PTC. TOP2A might act as an effective therapeutic target for PTC treatment.

Establishment of the technology of cambial meristematic cells (CMCs) culture from shoots and high expression of FmPHV (PHAVOLUTA) functions in identification and differentiation of CMCs and promoting the shoot regeneration by hypocotyl in Fraxinus mandshurica.

In Fraxinus mandshurica, we successfully isolated and identified the loose, uniform and creamy-white cambial meristematic cells (CMCs) from newborn shoots, and established a culture technology for induction, proliferation and differentiation of CMCs. In this technology, higher induction rate (83.0%, 0.57-fold to the control) was obtained by an effective pretreatment after 28-day induction culture, CMCs can be better proliferation cultured than common calli and maintain same growth states after several times of cultures and 3.3% CMCs primarily realized differentiation. Gene expressions in the differentiated CMCs revealed that, low expression of FmWOX5 (regulator in establishment of competence for shoot formation, 0.09-fold to the control) and high expressions of FmWOX4 (cambium stem cell regulator, 16.7-fold to the control) and 9 key genes in shoot regeneration (2.4-fold-72.1-fold to the control) function in CMCs differentiation. In addition to the function of high expression of PHAVOLUTA (FmPHV) in CMCs differentiation (5.4-fold-157.3-fold to undifferentiated CMCs), functions of high expression of FmPHV in CMCs identification (22.4-fold to common calli) and generating more shoots (2.3-fold to the control) by significantly changing expressions of key regulators in HD-Zip Class III related shoot regeneration networks in positive transgenic plants through the hypocotyl transforming system in F. mandshurica, were further revealed. These works were of profound significance in providing the culture technology of CMCs from newborn shoots in F. mandshurica for the first time and revealing the positive functions of FmPHV in CMCs identification and differentiation in F. mandshurica and promoting the shoot regeneration by hypocotyls.

Our emerging understanding of the roles of long non-coding RNAs in normal liver function, disease, and malignancy.

Long non-coding RNAs (lncRNAs) are important biological mediators that regulate numerous cellular processes. New experimental evidence suggests that lncRNAs play essential roles in liver development, normal liver physiology, fibrosis, and malignancy, including hepatocellular carcinoma and cholangiocarcinoma. In this review, we summarise our current understanding of the function of lncRNAs in the liver in both health and disease, as well as discuss approaches that could be used to target these non-coding transcripts for therapeutic purposes.

Identification of APEX2 as an oncogene in liver cancer.

BACKGROUND: DNA damage is one of the critical contributors to the occurrence and development of some cancers. APEX1 and APEX2 are the most important molecules in the DNA damage, and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma (LIHC). However, the expression of APEX2 and its functional mechanisms in LIHC are still unclear. AIM: To examine the expression of APEX2 and the potential mechanism network in LIHC. METHODS: We conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool. GEO datasets, including GSE14520, GSE22058, and GSE64041, were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues. Then, we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis. We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression, showing the potential mechanisms of APEX2 in LIHC. After that, we conducted Pearson correlation analysis using GEPIA2. Next, we performed quantitative polymerase chain reaction (qPCR) assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines, including HepG2, Huh7, SMMC7721, and HCCLM3. APEX2 in HCCLM3 cells was knocked down using small interfering RNA. The role of APEX2 in cell viability was confirmed using CCK-8. Dual-luciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC. RESULTS: APEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA, BRCA, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, READ, and STAD. APEX2 overexpression in LIHC was validated using GSE14520, GSE22058, and GSE64041 datasets. The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate, even in the AJCC T1 patients. High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis. The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology. APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication. The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling. Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC. APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells. Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells. Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC. CONCLUSION: APEX2 is overexpressed in LIHC, and the higher APEX2 level is associated with a worse prognosis in overall survival. APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication. APEX2 expression is positively correlated with the pro-oncogenic pathways. Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways, suggesting that APEX2 is an oncogene in LIHC, which could be a potential pharmaceutic target in the anti-tumor therapy.

The diverse and unanticipated roles of histone deacetylase 9 in coordinating plant development and environmental acclimation.

Plants tightly control gene transcription to adapt to environmental conditions and steer growth and development. Different types of epigenetic modifications are instrumental in these processes. In recent years, an important role for the chromatin-modifying RPD3/HDA1 class I HDAC HISTONE DEACETYLASE 9 (HDA9) emerged in the regulation of a multitude of plant traits and responses. HDACs are widely considered transcriptional repressors and are typically part of multiprotein complexes containing co-repressors, DNA, and histone-binding proteins. By catalyzing the removal of acetyl groups from lysine residues of histone protein tails, HDA9 negatively controls gene expression in many cases, in concert with interacting proteins such as POWERDRESS (PWR), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15), WRKY53, ELONGATED HYPOCOTYL 5 (HY5), ABA INSENSITIVE 4 (ABI4), and EARLY FLOWERING 3 (ELF3). However, HDA9 activity has also been directly linked to transcriptional activation. In addition, following the recent breakthrough discovery of mutual negative feedback regulation between HDA9 and its interacting WRKY-domain transcription factor WRKY53, swift progress in gaining understanding of the biology of HDA9 is expected. In this review, we summarize knowledge on this intriguing versatile-and long under-rated-protein and propose novel leads to further unravel HDA9-governed molecular networks underlying plant development and environmental biology.

Improved production of recombinant Rhizomucor miehei lipase by coexpressing protein folding chaperones in Pichia pastoris, which triggered ER stress.

Rhizomucor miehei lipase (RML) is a biocatalyst that widely used in laboratory and industrial. Previously, RML with a 70-amino acid propeptide (pRML) was cloned and expressed in P. pastoris. Recombinant strains with (strain containing 4-copy prml) and without ER stress (strain containing 2-copy prml) were obtained. However, the effective expression of pRML in P. pastoris by coexpressing ER-related elements in pRML-produced strain with or without ER stress has not been reported to date. In this study, an efficient way to produce functional pRML was explored in P. pastoris. The coexpression of protein folding chaperones, including PDI and ERO1, in different strains with or without ER stress, was investigated. PDI overexpression only increased pRML production in 4-copy strain from 705 U/mL to 1430 U/mL because it alleviated the protein folded stress, increased the protein concentration from 0.56  mg/mL to 0.65 mg/mL, and improved enzyme-specific activity from 1238 U/mg to 2186 U/mg. However, PDI coexpression could not improve pRML production in the 2-copy strain because it increased protein folded stress, while ERO1 coexpression in the two strains all had a negative effect on pRML expression. We also investigated the effect of the propeptide on the substrate specificity and the condition for pRML enzyme powder preparation. Results showed that the relative activity exceeded 80% when the substrates C8-C10 were detected at 35°C and pH 6, and C8-C12 at 45°C and pH 8. The optimal enzyme powder preparation pH was 7, and the maximum recovery rate for pRML was 73.19%.

Feedback regulation between phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 and transforming growth factor ß1 and prognostic value in gastric cancer.

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (PREX1) was reported to be overexpressed in some cancers and involved in cancer development, but its expression and significance in gastric cancer remain unclear. AIM: To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer, especially to evaluate the potential mechanism of PREX1 in gastric cancer. METHODS: Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer. The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal. The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor (TGF) ß1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics. Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFß1 on the expression of PREX1. Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFß1 pathway. Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells. RESULTS: PREX1 was overexpressed in the gastric tumors, and the expression levels were positively associated with the development of gastric cancer. Also, the high expression of PREX1 revealed poor prognosis, especially for those advanced and specific intestinal gastric cancer patients. PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFß1-related mediators. Furthermore, TGFß1 could induce the expression of PREX1 at both the protein and mRNA level. Also, PREX1 could activate the TGFß1 pathway. The induced PREX1 could increase the migration and invasion activity of gastric cancer cells. CONCLUSION: PREX1 is overexpressed in gastric cancer, and the high level of PREX1 predicts poor prognosis. PREX1 is closely associated with TGFß signaling and promotes the metastasis of gastric cancer cells.

Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells.

Background & Aims: Oropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas. Methods: We generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo. Results: Tumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment. Conclusions: The single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.