Herbicide Bioassay Using a Multi-Well Plate and Plant Spectral Image Analysis.

A spectral image analysis has the potential to replace traditional approaches for assessing plant responses to different types of stresses, including herbicides, through non-destructive and high-throughput screening (HTS). Therefore, this study was conducted to develop a rapid bioassay method using a multi-well plate and spectral image analysis for the diagnosis of herbicide activity and modes of action. Crabgrass (Digitaria ciliaris), as a model weed, was cultivated in multi-well plates and subsequently treated with six herbicides (paraquat, tiafenacil, penoxsulam, isoxaflutole, glufosinate, and glyphosate) with different modes of action when the crabgrass reached the 1-leaf stage, using only a quarter of the recommended dose. To detect the plant's response to herbicides, plant spectral images were acquired after herbicide treatment using RGB, infrared (IR) thermal, and chlorophyll fluorescence (CF) sensors and analyzed for diagnosing herbicide efficacy and modes of action. A principal component analysis (PCA), using all spectral data, successfully distinguished herbicides and clustered depending on their modes of action. The performed experiments showed that the multi-well plate assay combined with a spectral image analysis can be successfully applied for herbicide bioassays. In addition, the use of spectral image sensors, especially CF images, would facilitate HTS by enabling the rapid observation of herbicide responses at as early as 3 h after herbicide treatment.

Induction of Fatty Acid Oxidation Underlies DNA Damage-Induced Cell Death and Ameliorates Obesity-Driven Chemoresistance.

The DNA damage response is essential for preserving genome integrity and eliminating damaged cells. Although cellular metabolism plays a central role in cell fate decision between proliferation, survival, or death, the metabolic response to DNA damage remains largely obscure. Here, this work shows that DNA damage induces fatty acid oxidation (FAO), which is required for DNA damage-induced cell death. Mechanistically, FAO induction increases cellular acetyl-CoA levels and promotes N-alpha-acetylation of caspase-2, leading to cell death. Whereas chemotherapy increases FAO related genes through peroxisome proliferator-activated receptor α (PPARα), accelerated hypoxia-inducible factor-1α stabilization by tumor cells in obese mice impedes the upregulation of FAO, which contributes to its chemoresistance. Finally, this work finds that improving FAO by PPARα activation ameliorates obesity-driven chemoresistance and enhances the outcomes of chemotherapy in obese mice. These findings reveal the shift toward FAO induction is an important metabolic response to DNA damage and may provide effective therapeutic strategies for cancer patients with obesity.

Fatty acid oxidation regulates cellular senescence by modulating the autophagy-SIRT1 axis.

Senescence, a cellular process through which damaged or dysfunctional cells suppress the cell cycle, contributes to aging or age-related functional decline. Cell metabolism has been closely correlated with aging processes, and it has been widely recognized that metabolic changes underlie the cellular alterations that occur with aging. Here, we report that fatty acid oxidation (FAO) serves as a critical regulator of cellular senescence and uncover the underlying mechanism by which FAO inhibition induces senescence. Pharmacological or genetic ablation of FAO results in a p53-dependent induction of cellular senescence in human fibroblasts, whereas enhancing FAO suppresses replicative senescence. We found that FAO inhibition promotes cellular senescence through acetyl-CoA, independent of energy depletion. Mechanistically, increased formation of autophagosomes following FAO inhibition leads to a reduction in SIRT1 protein levels, thereby contributing to senescence induction. Finally, we found that inhibition of autophagy or enforced expression of SIRT1 can rescue the induction of senescence as a result of FAO inhibition. Collectively, our study reveals a distinctive role for the FAO-autophagy-SIRT1 axis in the regulation of cellular senescence. [BMB Reports 2023; 56(12): 651-656].

YAP governs cellular adaptation to perturbation of glutamine metabolism by regulating ATF4-mediated stress response.

Proliferating cells have metabolic dependence on glutamine to fuel anabolic pathways and to refill the mitochondrial carbon pool. The Hippo pathway is essential for coordinating cell survival and growth with nutrient availability, but no molecular connection to glutamine deprivation has been reported. Here, we identify a non-canonical role of YAP, a key effector of the Hippo pathway, in cellular adaptation to perturbation of glutamine metabolism. Whereas YAP is inhibited by nutrient scarcity, enabling cells to restrain proliferation and to maintain energy homeostasis, glutamine shortage induces a rapid YAP dephosphorylation and activation. Upon glutaminolysis inhibition, an increased reactive oxygen species production inhibits LATS kinase via RhoA, leading to YAP dephosphorylation. Activated YAP promotes transcriptional induction of ATF4 to induce the expression of genes involved in amino acid homeostasis, including Sestrin2. We found that YAP-mediated Sestrin2 induction is crucial for cell viability during glutamine deprivation by suppressing mTORC1. Thus, a critical relationship between YAP, ATF4, and mTORC1 is uncovered by our findings. Finally, our data indicate that targeting the Hippo-YAP pathway in combination with glutaminolysis inhibition may provide potential therapeutic approaches to treat tumors.

Fatty acid oxidation facilitates DNA double-strand break repair by promoting PARP1 acetylation.

DNA repair is a tightly coordinated stress response to DNA damage, which is critical for preserving genome integrity. Accruing evidence suggests that metabolic pathways have been correlated with cellular response to DNA damage. Here, we show that fatty acid oxidation (FAO) is a crucial regulator of DNA double-strand break repair, particularly homologous recombination repair. Mechanistically, FAO contributes to DNA repair by activating poly(ADP-ribose) polymerase 1 (PARP1), an enzyme that detects DNA breaks and promotes DNA repair pathway. Upon DNA damage, FAO facilitates PARP1 acetylation by providing acetyl-CoA, which is required for proper PARP1 activity. Indeed, cells reconstituted with PARP1 acetylation mutants display impaired DNA repair and enhanced sensitivity to DNA damage. Consequently, FAO inhibition reduces PARP1 activity, leading to increased genomic instability and decreased cell viability upon DNA damage. Finally, our data indicate that FAO serves as an important participant of cellular response to DNA damage, supporting DNA repair and genome stability.

Fatty acid oxidation supports melanoma cell migration through autophagy regulation.

Metastasis is one of the most malignant characteristics of cancer cells, in which metabolic reprogramming is crucial for promoting and sustaining multi-steps of metastasis, including invasion, migration and infiltration. Recently, it has been shown that melanoma cells undergo a metabolic switching toward the upregulation of fatty acid oxidation (FAO) during metastasis. However, the underlying mechanisms by which FAO contributes to metastasis of melanoma cells remain obscure. Here, we report that FAO contributes to melanoma cell migration and invasion by regulating the formation of autophagosomes. Pharmacological or genetic inhibition of FAO impairs migration of melanoma cells, which seems not to be linked to energy production or redox homeostasis. Importantly, we reveal that acetyl-CoA production by FAO contributes to melanoma cell migration through autophagy regulation. Mechanistically, FAO inhibition results in increased autophagosome formation, which suppresses migration and invasion properties of melanoma cells. Our results underscore the crucial role of FAO in melanoma cell migration and support the potential therapeutic relevance of modulating cellular acetyl-CoA levels to inhibit cancer metastasis.

The impact of cancer cachexia on gut microbiota composition and short-chain fatty acid metabolism in a murine model.

This study investigates the relationship between cancer cachexia and the gut microbiota, focusing on the influence of cancer on microbial composition. Lewis lung cancer cell allografts were used to induce cachexia in mice, and body and muscle weight changes were monitored. Fecal samples were collected for targeted metabolomic analysis for short chain fatty acids and microbiome analysis. The cachexia group exhibited lower alpha diversity and distinct beta diversity in gut microbiota, compared to the control group. Differential abundance analysis revealed higher Bifidobacterium and Romboutsia, but lower Streptococcus abundance in the cachexia group. Additionally, lower proportions of acetate and butyrate were observed in the cachexia group. The study observed that the impact of cancer cachexia on gut microbiota and their generated metabolites was significant, indicating a host-to-gut microbiota axis. [BMB Reports 2023; 56(7): 404-409].

Suppression of fatty acid oxidation supports pancreatic cancer growth and survival under hypoxic conditions through autophagy induction.

Hypoxia, one of the key features of solid tumors, induces autophagy, which acts as an important adaptive mechanism for tumor progression under hypoxic environment. Cellular metabolic reprogramming has been correlated with hypoxia, but the molecular connection to the induction of autophagy remains obscure. Here, we show that suppression of fatty acid oxidation (FAO) by hypoxia induces autophagy in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for their growth and survival. Reduced cellular acetyl-CoA levels caused by FAO inhibition decreases LC3 acetylation, resulting in autophagosome formation. Importantly, PDAC cells are significantly dependent on this metabolic reprogramming, as improving FAO leads to a reduction in hypoxia-induced autophagy and an increase in cell death after chemotherapy. Thus, our study supports that suppression of FAO is an important metabolic response to hypoxia and indicates that targeting this pathway in PDAC may be an effective therapeutic approach.

Antifungal and carboxylesterase-producing bacteria applied into corn silage still affected the fermented total mixed ration.

OBJECTIVE: This study investigated the effects of corn silage as a source of microbial inoculant containing antifungal and carboxylesterase-producing bacteria on fermentation, aerobic stability, and nutrient digestibility of fermented total mixed ration (FTMR) with different energy levels. METHODS: Corn silage was used as a bacterial source by ensiling for 72 d with an inoculant mixture of Lactobacillus brevis 5M2 and L. buchneri 6M1 at a 1:1 ratio. The corn silage without or with inoculant (CON vs MIX) was mixed with the other ingredients to formulate for low and high energy diets (LOW vs HIGH) for Hanwoo steers. All diets were ensiled into 20 L mini silo (5 kg) for 40 d in quadruplicate. RESULTS: The MIX diets had lower (p<0.05) acid detergent fiber with higher (p<0.05) in vitro digestibilities of dry matter and neutral detergent fiber compared to the CON diets. In terms of fermentation characteristics, the MIX diets had higher (p<0.05) acetate than the CON diets. The MIX diets had extended (p<0.05) lactic acid bacteria growth at 4 to 7 d of aerobic exposure and showed lower (p<0.05) yeast growth at 7 d of aerobic exposure than the CON diets. In terms of rumen fermentation, the MIX diets had higher (p<0.05) total fermentable fraction and total volatile fatty acid, with lower (p<0.05) pH than those of CON diets. The interaction (p = 0.036) between inoculant and diet level was only found in the immediately fermentable fraction, which inoculant was only effective on LOW diets. CONCLUSION: Application of corn silage with inoculant on FTMR presented an antifungal effect by inhibiting yeast at aerobic exposure and a carboxylesterase effect by improving nutrient digestibility. It also indicated that fermented feedstuffs could be used as microbial source for FTMR. Generally, the interaction between inoculant and diet level had less effect on this FTMR study.

Machine learning-derived gut microbiome signature predicts fatty liver disease in the presence of insulin resistance.

A simple predictive biomarker for fatty liver disease is required for individuals with insulin resistance. Here, we developed a supervised machine learning-based classifier for fatty liver disease using fecal 16S rDNA sequencing data. Based on the Kangbuk Samsung Hospital cohort (n = 777), we generated a random forest classifier to predict fatty liver diseases in individuals with or without insulin resistance (n = 166 and n = 611, respectively). The model performance was evaluated based on metrics, including accuracy, area under receiver operating curve (AUROC), kappa, and F1-score. The developed classifier for fatty liver diseases performed better in individuals with insulin resistance (AUROC = 0.77). We further optimized the classifiers using genetic algorithm. The improved classifier for insulin resistance, consisting of ten microbial genera, presented an advanced classification (AUROC = 0.93), whereas the improved classifier for insulin-sensitive individuals failed to distinguish participants with fatty liver diseases from the healthy. The classifier for individuals with insulin resistance was comparable or superior to previous methods predicting fatty liver diseases (accuracy = 0.83, kappa = 0.50, F1-score = 0.89), such as the fatty liver index. We identified the ten genera as a core set from the human gut microbiome, which could be a diagnostic biomarker of fatty liver diseases for insulin resistant individuals. Collectively, these findings indicate that the machine learning classifier for fatty liver diseases in the presence of insulin resistance is comparable or superior to commonly used methods.

RNA binding protein HuD mediates the crosstalk between ß cells and islet endothelial cells by the regulation of Endostatin and Serpin E1 expression.

RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma ßTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3'-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3'UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of ßTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between ß cells and islet endothelial cells, and the results showed that HuD downregulation in ßTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between ß cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.

Targeting Cdc20 for cancer therapy.

The Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase, and two co-activators, Cdc20 and Cdh1, enable the ubiquitin-dependent proteasomal degradation of various critical cell cycle regulators and govern cell division in a timely and precise manner. Dysregulated cell cycle events cause uncontrolled cell proliferation, leading to tumorigenesis. Studies have shown that Cdh1 has tumor suppressive activities while Cdc20 has an oncogenic property, suggesting that Cdc20 is an emerging therapeutic target for cancer treatment. Therefore, in this review, we discussed recent findings about the essential roles of APC/C-Cdc20 in cell cycle regulation. Furthermore, we briefly summarized that the regulation of Cdc20 expression levels is strictly controlled to order cell cycle events appropriately. Finally, given the function of Cdc20 as an oncogene, therapeutic interventions targeting Cdc20 activity may be beneficial in cancer treatment.

Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.

The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.

Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype.

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic ß-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3'-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.

Use of Disease-modifying Antirheumatic Drugs After Cancer Diagnosis in Rheumatoid Arthritis Patients.

Objective: There is no recommendation for the use of disease-modifying antirheumatic drugs (DMARDs) in patients with rheumatoid arthritis (RA) who developed cancer. We examined changes in the DMARDs prescription patterns associated with cancer diagnosis in RA patients. Methods: We reviewed the medical records of 2,161 RA patients who visited rheumatology clinic between January 2008 and February 2017 and found 40 patients who developed cancer during RA treatment. In these patients, we examined DMARDs prescription patterns before and right after cancer diagnosis and at recent outpatient clinic visits. Results: Before cancer diagnosis, methotrexate (MTX)-combined conventional synthetic DMARDs (csDMARDs) were most commonly prescribed (22, 55.0%) and biological DMARDs (biologics) in nine patients (22.5%). For cancer treatment, 19 patients received chemotherapy (including adjuvant chemotherapy) and 21 patients had surgery only. Right after cancer diagnosis, changes in the DMARDs prescription patterns were similar in discontinuation (13, 32.5%), switching (14, 35.0%), and maintenance (13, 32.5%). DMARDs were discontinued more frequently in the chemotherapy group (9/19, 47.4%) than the surgery only group (4/2, 19.0%) (p<0.05). Among the 13 patients who discontinued DMARDs, nine (69.2%) resumed DMARDs after a median of 5.5 months (interquartile range [IQR] 2.9, 18.3) due to arthritis flare. At a median of 4.6 years (IQR 3.3, 6.7) after cancer diagnosis, 25 patients were evaluated at recent outpatient clinic visits. Four patients received no DMARD, three MTX monotherapies, 11 csDMARDs combination therapies, and seven biologics. Conclusion: A significant number of RA patients who developed cancer during RA treatment were still receiving DMARDs including biologics after cancer diagnosis.

Improvement of conception rate on Hanwoo; The key hormones and novel estrus detector.

Two field experiments were conducted to improve the conception rate of Hanwoo cow. The first experiment aimed to investigate the physiological condition of Hanwoo cows on estrus, including metabolic profiles and body condition score (BCS). The second experiment investigated the effect of a novel estrus detector on the artificial insemination (AI) conception rate for Hanwoo cows. For the first experiment, 80 Hanwoo cows (2.5 ± 0.10 of parity), approximately one month before estrus, were housed in 16 pens and offered the experimental diets twice daily with free water access. The BCS were recorded, and blood was collected from the jugular veins just before AI. The collected blood was used to measure physiological conditions, such as metabolite and hormone levels. For the second experiment, each cow was equipped with a neck-mounted estrus detector collar, which had a sensor connected through the internet. Approximately one month before estrus, three hundred sixty Hanwoo cows (2.4 ± 0.21 of parity) were assigned into groups with or without W-Tag collar treatments. The animals were managed the same as in the first experiment. The pregnancy rate reached 55% in the first experiment. The concentration of luteinizing hormone (LH) was higher (p < 0.012; 1.56 vs. 1.08 ng/mL) in cows that were not pregnant (NPG) than in cows that were pregnant (PG) after AI. The BCS and other concentrations of metabolites and hormones in the blood were not different in both NPG and PG cows. The ranges of estrogen, LH, and follicle-stimulating hormone for PG cows were 11.9 to 39.0 pg/mL, < 0.25 to 1.98 ng/mL, and < 0.50 to 0.82 ng/mL, respectively. In the second experiment, cows with the estrus detector had lower days open (p < 0.001; 78.1 vs. 84.8 d), insemination frequency (p < 0.001; 1.26 vs. 2.52), and return of estrus (p < 0.001; 70.9 vs. 79.1 d) than those in cows without the estrus detector. In conclusion, the present study indicated that lower LH concentration just before AI potentially increased the pregnancy rate of Hanwoo cows. Furthermore, the application of estrus detectors to Hanwoo cows could improve the conception success rate for AI.

Mitochondrial glutamine metabolism regulates sensitivity of cancer cells after chemotherapy via amphiregulin.

The DNA damage response is essential for sustaining genomic stability and preventing tumorigenesis. However, the fundamental question about the cellular metabolic response to DNA damage remains largely unknown, impeding the development of metabolic interventions that might prevent or treat cancer. Recently, it has been reported that there is a link between cell metabolism and DNA damage response, by repression of glutamine (Gln) entry into mitochondria to support cell cycle arrest and DNA repair. Here, we show that mitochondrial Gln metabolism is a crucial regulator of DNA damage-induced cell death. Mechanistically, inhibition of glutaminase (GLS), the first enzyme for Gln anaplerosis, sensitizes cancer cells to DNA damage by inducing amphiregulin (AREG) that promotes apoptotic cell death. GLS inhibition increases reactive oxygen species production, leading to transcriptional activation of AREG through Max-like protein X (MLX) transcription factor. Moreover, suppression of mitochondrial Gln metabolism results in markedly increased cell death after chemotherapy in vitro and in vivo. The essentiality of this molecular pathway in DNA damage-induced cell death may provide novel metabolic interventions for cancer therapy.

SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.

SWItch/Sucrose Non-Fermentable (SWI/SNF) is a multiprotein complex essential for the regulation of eukaryotic gene expression. SWI/SNF complex genes are genetically altered in over 20% of human malignancies, but the aberrant regulation of the SWI/SNF subunit genes and subsequent dysfunction caused by abnormal expression of subunit gene in cancer, remain poorly understood. Among the SWI/SNF subunit genes, SMARCA4, SMARCC1, and SMARCA2 were identified to be overexpressed in human hepatocellular carcinoma (HCC). Modulation of SMARCA4, SMARCC1, and SMARCA2 inhibited in vitro tumorigenesis of HCC cells. However, SMARCA4-targeting elicited remarkable inhibition in an in vivo Ras-transgenic mouse HCC model (Ras-Tg), and high expression levels of SMARCA4 significantly associated with poor prognosis in HCC patients. Furthermore, most HCC patients (72-86%) showed SMARCA4 overexpression compared to healthy controls. To identify SMARCA4-specific active enhancers, mapping, and analysis of chromatin state in liver cancer cells were performed. Integrative analysis of SMARCA4-regulated genes and active chromatin enhancers suggested 37 genes that are strongly activated by SMARCA4 in HCC. Through chromatin immunoprecipitation-qPCR and luciferase assays, we demonstrated that SMARCA4 activates Interleukin-1 receptor-associated kinase 1 (IRAK1) expression through IRAK1 active enhancer in HCC. We then showed that transcriptional activation of IRAK1 induces oncoprotein Gankyrin and aldo-keto reductase family 1 member B10 (AKR1B10) in HCC. The regulatory mechanism of the SMARCA4-IRAK1-Gankyrin, AKR1B10 axis was further demonstrated in HCC cells and in vivo Ras-Tg mice. Our results suggest that aberrant overexpression of SMARCA4 causes SWI/SNF to promote IRAK1 enhancer to activate oncoprotein Gankyrin and AKR1B10, thereby contributing to hepatocarcinogenesis.

Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease.

Brachyury is an embryonic nuclear transcription factor required for mesoderm formation and differentiation. Here, we introduced an mCherry reporter into the C-terminus of Brachyury in the human pluripotent stem cell line SNUhES3 using the CRISPR/Cas9 nuclease approach. Successful gene editing was verified by DNA sequencing. SNUhES3-Brachyury-mCherry cells expressed pluripotent stem cell markers, exhibited a normal karyotype, and could generate all three germ layers. This cell line expressed the red fluorescence protein mCherry upon the induction of mesoderm differentiation. This reporter cell line could be used to monitor mesodermal population enrichment during mesodermal differentiation.