Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor

Scientific Reports ◽

10.1038/s41598-021-85486-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yu-Cheng Chang ◽

Yi-Ching Chiu ◽

Nai-Wen Tsao ◽

Yuan-Lin Chou ◽

Choon-Meng Tan ◽

...

Keyword(s):

Transcriptome Analysis ◽

Natural Antioxidants ◽

Biosynthesis Pathway ◽

Comparative Transcriptome ◽

Amaranthus Tricolor ◽

Enzymatic Assays ◽

Comparative Transcriptome Analysis ◽

Elevated Expression ◽

Green Coloration

AbstractAmaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

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Clinicopathologic features of TDO2 overexpression in renal cell carcinoma

BMC Cancer ◽

10.1186/s12885-021-08477-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Quoc Thang Pham ◽

Daiki Taniyama ◽

Yohei Sekino ◽

Shintaro Akabane ◽

Takashi Babasaki ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

In Silico Analysis ◽

Immunohistochemical Analysis ◽

Rna Seq ◽

Anoikis Resistance ◽

Silico Analysis ◽

Proliferation And Invasion

Abstract Background Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme catabolizing tryptophan. Several lines of evidence revealed that overexpression of TDO2 is involved in anoikis resistance, spheroid formation, proliferation, and invasion and correlates with poor prognosis in some cancers. The aim of this research was to uncover the expression and biofunction of TDO2 in renal cell carcinoma (RCC). Methods To show the expression of TDO2 in RCC, we performed qRT-PCR and immunohistochemistry in integration with TCGA data analysis. The interaction of TDO2 with PD-L1, CD44, PTEN, and TDO2 expression was evaluated. We explored proliferation, colony formation, and invasion in RCC cells line affected by knockdown of TDO2. Results RNA-Seq and immunohistochemical analysis showed that TDO2 expression was upregulated in RCC tissues and was associated with advanced disease and poor survival of RCC patients. Furthermore, TDO2 was co-expressed with PD-L1 and CD44. In silico analysis and in vitro knockout of PTEN in RCC cell lines revealed the ability of PTEN to regulate the expression of TDO2. Knockdown of TDO2 suppressed the proliferation and invasion of RCC cells. Conclusion Our results suggest that TDO2 might have an important role in disease progression and could be a promising marker for targeted therapy in RCC. (199 words)

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Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22042006 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2006

Author(s):

Mi Jin Kim ◽

Jinhong Park ◽

Jinho Kim ◽

Ji-Young Kim ◽

Mi-Jin An ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Transcriptome Analysis ◽

Caspase 3 ◽

Apoptotic Cell ◽

Expression Patterns ◽

Nitrogen Compound ◽

Apoptotic Cell Death ◽

Mercury Chloride ◽

Rna Seq

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

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Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

Scientific Reports ◽

10.1038/s41598-021-88392-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Risa Okada ◽

Shin-ichiro Fujita ◽

Riku Suzuki ◽

Takuto Hayashi ◽

Hirona Tsubouchi ◽

...

Keyword(s):

Gene Expression ◽

Muscle Mass ◽

Muscle Atrophy ◽

Transcriptome Analysis ◽

Fiber Type ◽

Expression Profiles ◽

Space Station ◽

Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

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Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽

10.1186/s12915-021-01053-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Milda Mickutė ◽

Kotryna Kvederavičiūtė ◽

Aleksandr Osipenko ◽

Raminta Mineikaitė ◽

Saulius Klimašauskas ◽

...

Keyword(s):

Rna Sequencing ◽

Regulatory Networks ◽

Library Preparation ◽

Rna Seq ◽

Basic Principles ◽

Cofactor Binding ◽

Sequencing Library ◽

Sequencing Library Preparation ◽

Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

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The BET Inhibitor JQ1 Augments the Antitumor Efficacy of Gemcitabine in Preclinical Models of Pancreatic Cancer

Cancers ◽

10.3390/cancers13143470 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3470

Author(s):

Aubrey L. Miller ◽

Patrick L. Garcia ◽

Samuel C. Fehling ◽

Tracy L. Gamblin ◽

Rebecca B. Vance ◽

...

Keyword(s):

Pancreatic Cancer ◽

Dna Damage ◽

Cholesterol Biosynthesis ◽

Antitumor Efficacy ◽

Rna Seq ◽

Bet Inhibitor ◽

Bet Inhibitors ◽

Xenograft Models

Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models (P < 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.

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