Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma.

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.

Involvement of a MYB Transcription Factor in Anthocyanin Biosynthesis during Chinese Bayberry (Morella rubra) Fruit Ripening.

Anthocyanin is a class of water-soluble flavonoids found in Chinese bayberry (Morella rubra) that is not only responsible for the variety of colors visible in nature but also has numerous health-promoting benefits in humans. Through comparative transcriptomics, we isolated and identified a transcription factor (TF) of the R2R3-MYB type, MrMYB9, in order to explore the anthocyanin biosynthesis pathway in red and white Chinese bayberries. MrMYB9 transcript was positively correlated with anthocyanin level and anthocyanin biosynthetic gene expression during Chinese bayberry fruit maturation (R-values in the range 0.54-0.84, p < 0.05). Sequence analysis revealed that MrMYB9 shared a similar R2R3 domain with MYB activators of anthocyanin biosynthesis in other plants. MrMYB9 substantially transactivated promoters of anthocyanin biosynthesis-related EBGs (MrCHI, MrF3'H, and MrANS) and LBGs (MrUFGT) upon co-expression of the AtEGL3 gene. Our findings indicated that MrMYB9 may positively modulate anthocyanin accumulation in Chinese bayberry.

Role of miRNAs in regulation of SA-mediated upregulation of genes involved in folate and methionine metabolism in foxtail millet.

The effect of exogenous salicylic acid (SA) on folate metabolism and the related gene regulatory mechanisms is still unclear. In this study, the panicle of foxtail millet treated with different SA concentrations showed that 6 mM SA doubled the 5-methyltetrahydrofolate content compared to that of the control. An untargeted metabolomic analysis revealed that 275 metabolites were enriched in amino acid metabolic pathways. Significantly, the relative content of methionine (Met) after 6 mM SA treatment was 3.14 times higher than the control. Transcriptome analysis revealed that differentially expressed genes were mainly enriched in the folate and amino acid biosynthesis pathways (including Met, Cys, Pro, Ser et al.). The miRNA-mRNA interactions related to the folate and Met metabolic pathways were analyzed and several likely structural gene targets for miRNAs were identified, miRNA-seq analysis revealed that 33 and 51 miRNAs targeted 11 and 15 genes related to the folate and Met pathways, respectively. Eight key genes in the folate metabolism pathway were likely to be up-regulated by 14 new miRNAs and 20 new miRNAs up-regulated the 9 key genes in the Met metabolism pathway. The 6 miRNA-mRNA interactions related to the folate and Met metabolism pathways were verified by qRT-PCR, and consistent with the prediction. The results showed that DHFR1 gene expression level related to folate synthesis was directly up-regulated by Nov-m0139-3p with 3.8 times, but DHFR2 was down-regulated by Nov-m0731-5p with 0.62 times. The expression level of CYSC1 and APIP related to Met synthesis were up-regulated by Nov-m0461-5p and Nov-m0664-3p with 4.27 and 1.32 times, respectively. Our results suggested that exogenous SA could induce the folate and Met accumulated in the panicle of foxtail millet. The higher expression level of DHFR1, FTHFD, CYSC1 and APIP in the folate and Met metabolism pathway and their regulators, including Nov-m0139-3p, Nov-m0717-5p, Nov-m0461-5p and Nov-m0664-3p, could be responsible for these metabolites accumulation. This study lays the theoretical foundation for elucidating the post-transcription regulatory mechanisms of folate and Met metabolism.

Amino acid transporter (AAT) gene family in Tartary buckwheat (Fagopyrum tataricum L. Gaertn.): Characterization, expression analysis and functional prediction.

Tartary buckwheat (Fagopyrum tataricum L. Gaertn., TB) is an ancient minor crop and an important food source for humans to supplement nutrients such as flavonoids and essential amino acids. Amino acid transporters (AATs) play critical roles in plant growth and development through the transport of amino acids. In this study, 104 AATs were identified in TB genome and divided into 11 subfamilies by phylogenetic relationships. Tandem and segmental duplications promoted the expansion of FtAAT gene family, and the variations of gene sequence, protein structure and expression pattern were the main reasons for the functional differentiation of FtAATs. Based on RNA-seq and qRT-PCR, the expression patterns of FtAATs in different tissues and under different abiotic stresses were analyzed, and several candidate FtAATs that might affect grain development and response to abiotic stresses were identified, such as FtAAP12 and FtCAT7. Finally, combined with the previous studies, the expression patterns and phylogenetic relationships of AATs in multiple species, the functions of multiple high-confidence FtAAT genes were predicted, and the schematic diagram of FtAATs in TB was initially drawn. Overall, this work provided a framework for further functional analysis of FtAAT genes and important clues for the improvement of TB quality and stress resistance.

Heterologous Expression of SiFBP, a Folate-Binding Protein from Foxtail Millet, Confers Increased Folate Content and Altered Amino Acid Profiles with Nutritional Potential to Arabidopsis.

The mechanism underlying folate degradation in foxtail millet grains remains unclear. Here, we identified SiFBP (Setaria italica folate-binding protein) from foxtail millet. A phylogenetic tree revealed that FBPs have close genetic relationships among cereal crop species. Docking analysis and heterologous expression of SiFBP in yeast showed that it could bind folic acid (FA). The SiFBP localized to the plasma membrane in tobacco mesophyll cells by transient expression. In Arabidopsis, it was expressed specifically in the roots and germinating seeds. Overexpressing SiFBP in yeast and Arabidopsis significantly increased folate contents. Untargeted metabolome analysis revealed differentially accumulated metabolites between the transgenic lines (TLs) and wild type (WT); these metabolites were mainly enriched in the amino acid metabolism pathway. The relative contents of lysine and leucine, threonine, and l-methionine were significantly higher in the TLs than in WT. Genes related to the folate and lysine synthesis pathways were upregulated in the TLs. Thus, SiFBP can be used for biofortification of folate and important amino acids in crops via genetic engineering.

Folate metabolic profiling and expression of folate metabolism-related genes during panicle development in foxtail millet (Setaria italica (L.) P. Beauv).

BACKGROUND: Foxtail millet grain has higher folate content than other cereal crops. However, the folate metabolite content and the expression patterns of folate metabolite-related genes are unknown. RESULTS: Liquid chromatography-mass spectrometry was used to investigate 12 folate metabolites in a foxtail millet panicle. The content of total folate and derivatives gradually decreased during panicle development. Polyglutamate 5-formyl-tetrahydrofolate was the major form. Twenty-eight genes involved in the folate metabolic pathway were identified through bioinformatic analysis. These genes in Setaria italica, S. viridis and Zea mays showed genomic collinearity. Phylogenetic analysis revealed that the folate-related genes were closely related among the C4 plants compared to C3 plants. The gene expressions were then studied at three panicle development stages. The gene expression patterns were classified into two groups, namely SiADCL1 and SiGGH as two key enzymes, which are responsible for folate synthesis and degradation; their expression levels were highest at the early panicle development stage, up to 179.11- and 163.88-fold, respectively. Their expression levels had a similar downward trend during panicle development and were significantly positively correlated with the concentration of total folate and folate derivatives. However, SiSHMT3 expression levels were significantly negatively correlated with total folate concentration. CONCLUSION: Besides being the major determinants of folate and folate derivatives accumulation, SiADCL1 and SiGGH expression levels are key limiting factors in the foxtail millet panicle. Therefore, SiADCL1 and SiGGH expression levels can be targeted in genetic modification studies to improve folate content in foxtail millet seeds in the future. © 2021 Society of Chemical Industry.

Total Protein Content, Amino Acid Composition and Eating-Quality Evaluation of Foxtail Millet (Setaria italica (L.) P. Beauv).

Foxtail millet has attracted substantial attention in recent years because of its excellent properties as a cereal crop with high nutritional value. Although the cultivation area of foxtail millet keeps growing, the fundamental research into the nutritional and eating qualities of foxtail millet germplasm collections is limited. In this study, we performed a survey of protein content, amino acid composition and eating quality among a germplasm collection of foxtail millet accessions grown in different environments. Our results revealed 21 accessions with stable protein content under different environments. The correlation analysis further revealed that the protein content of the grains was affected by environmental and genotypic interactions. The further amino acid composition analyses suggested that higher protein content accessions have a better essential amino acid index, providing more nutritional value for human beings and animal feedstock. Moreover, the flavor-related amino acid content and other eating-quality trait analyses were also performed. The subordinative analysis suggested that B331 could be the best accession with high protein content and superior eating quality. Taken together, this study provides essential nutritional and eating-quality data on our germplasm collection of foxtail millets, and provides a core genetic resource from which to breed elite foxtail millet varieties in the future.

Mouse Organ-Specific Proteins and Functions.

Organ-specific proteins (OSPs) possess great medical potential both in clinics and in biomedical research. Applications of them-such as alanine transaminase, aspartate transaminase, and troponins-in clinics have raised certain concerns of their organ specificity. The dynamics and diversity of protein expression in heterogeneous human populations are well known, yet their effects on OSPs are less addressed. Here, we used mice as a model and implemented a breadth study to examine the panorgan proteome for potential variations in organ specificity in different genetic backgrounds. Using reasonable resources, we generated panorgan proteomes of four in-bred mouse strains. The results revealed a large diversity that was more profound among OSPs than among proteomes overall. We defined a robustness score to quantify such variation and derived three sets of OSPs with different stringencies. In the meantime, we found that the enriched biological functions of OSPs are also organ-specific and are sensitive and useful to assess the quality of OSPs. We hope our breadth study can open doors to explore the molecular diversity and dynamics of organ specificity at the protein level.

Clinical effect of TESSYS technique under spinal endoscopy combined with drug therapy in patients with lumbar disc herniation and its effect on quality of life and serum inflammatory factors: results of a randomized trial.

BACKGROUND: This study was carried out based on the background that lumbar disc herniation seriously affects patients' quality of life but its clinical treatment effect remains unsatisfactory. METHODS: In total, 140 patients with lumbar disc herniation are randomly divided into a single operation group (SO) and a combined treatment group (CT). Among them, patients in the SO group received single treatment of TESSYS technique under spinal endoscopy, while patients in the CT group received combined drug therapy including coenzyme A, adenosine triphosphate, 10% glucose injection, 10% potassium chloride, vitamin B6, vitamin B12, dexamethasone, 20% mannitol and traditional Chinese medicine on the basis of the SO group. The clinical effect, Japanese Orthopaedic Association (JOA) score, visual analogue scale (VAS), recurrence rate, levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and quality of life score were compared between the two groups. RESULTS: The treatment effectiveness rate of the CT group was markedly better than that in the SO group (P<0.01). At 3- and 6-month postoperatively, the JOA scores, VAS scores, World Health Organization Quality of Life Brief Questionnaire (WHOQOL-BREF) and the levels of IL-1ß, IL-6, and TNF-α in the CT group were significantly better than those in the SO group (P<0.05). During the 6-12-month follow-up, the recurrence rate and WHOQOL-BREF scores in the SO group and CT group was no statistical difference (P>0.05). CONCLUSIONS: TESSYS technique under spinal endoscopy combined with drug therapy in the treatment of lumbar disc herniation has a significant clinical effect. Therefore, it is worthy of clinical popularization. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100049153.

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

[Diagnosis and treatment of the fourth branchial fissure: a clinical report of 1 case and literature review].

The patient repeatedly suffer from pain in the left side neck for 4 years and had 1-2 recurrence per year. We used neck ultrasound and neck CT examination to find an abnormal soft tissue lumps exist in the patient's left neck root to the trachea esophageal ditch. Diagnostic analysis combines embryogenesis and anatomy, and the diagnosis results are infection with the fourth branchial fissure. The lesion site was completely excisioned with full hemp surgery, the incision showed I type healing after surgery, and there was no recurrence after six months of follow-up.

Macropinocytosis in Cancer-Associated Fibroblasts Is Dependent on CaMKK2/ARHGEF2 Signaling and Functions to Support Tumor and Stromal Cell Fitness.

Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is also operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAF). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca2+ and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has a dual function in CAFs-it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. SIGNIFICANCE: Glutamine deprivation drives stromal macropinocytosis to support CAF cell fitness and provide amino acids that sustain PDAC cell survival. Selective disruption of macropinocytosis in CAFs suppresses PDAC tumor growth.This article is highlighted in the In This Issue feature, p. 1601.

Glutamine depletion regulates Slug to promote EMT and metastasis in pancreatic cancer.

Tumor cells rely on glutamine to fulfill their metabolic demands and sustain proliferation. The elevated consumption of glutamine can lead to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression. Here, we show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, we demonstrate that glutamine deficiency regulates EMT through the up-regulation of the EMT master regulator Slug, a process that is dependent on both MEK/ERK signaling and ATF4. We find that Slug is required in PDAC cells for glutamine deprivation-induced EMT, cell motility, and nutrient stress survival. Importantly, we decipher that Slug is associated with nutrient stress in PDAC tumors and is required for metastasis. These results delineate a novel role for Slug in the nutrient stress response and provide insight into how nutrient depletion might influence PDAC progression.

Golgi Acidification by NHE7 Regulates Cytosolic pH Homeostasis in Pancreatic Cancer Cells.

Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium-hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown in vivo leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. SIGNIFICANCE: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a "proton sink" for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth.See related commentary by Ward and DeNicola, p. 768.This article is highlighted in the In This Issue feature, p. 747.

EGFR-Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis.

Macropinocytosis has emerged as an important nutrient-scavenging pathway that supports tumor cell fitness. By internalizing extracellular protein and targeting it for lysosomal degradation, this endocytic pathway functions as an amino acid supply route, permitting tumor cell growth and survival despite the nutrient-poor conditions of the tumor microenvironment. Here, we provide evidence that a subset of pancreatic ductal adenocarcinoma (PDAC) tumors are wired to integrate contextual metabolic inputs to regulate macropinocytosis, dialing up or down this uptake pathway depending on nutrient availability. We find that regional depletion of amino acids coincides with increased levels of macropinocytosis and that the scarcity of glutamine uniquely drives this process. Mechanistically, this stimulation of macropinocytosis depends on the nutrient stress-induced potentiation of epidermal growth factor receptor signaling that, through the activation of Pak, controls the extent of macropinocytosis in these cells. These results provide a mechanistic understanding of how nutritional cues can control protein scavenging in PDAC tumors.

Macropinocytosis in Cancer: A Complex Signaling Network.

Macropinocytosis is an important nutrient-scavenging pathway in numerous cancer types, including pancreatic, lung, prostate, and bladder. This Forum highlights recent work identifying the key regulators of macropinocytosis that support tumor cell fitness in different contexts, providing a unique framework for strategies to target macropinocytosis in the treatment of cancer.

Synthesis and biological evaluation of genistein-O-alkylamine derivatives as potential multifunctional anti-Alzheimer agents.

A series of genistein derivatives were synthesized and evaluated as multifunctional anti-Alzheimer agents. The results showed that these derivatives had significant acetylcholinesterase (AChE) inhibitory activity; compound 5a exhibited the strongest inhibition to AChE with an IC50 value (0.034 µM) much lower than that of rivastigmine (6.53 µM). A Lineweaver-Burk plot and molecular modeling study showed that compound 5a targeted both the catalytic active site and the peripheral anionic site of AChE. These compounds also showed potent peroxy scavenging activity and metal-chelating ability. The compounds did not show obvious effect on HepG2 and PC12 cell viability at the concentration of 100 µM. Therefore, these genistein derivatives can be utilized as multifunctional agents for the treatment of AD.

Competing endogenous RNA analysis reveals the regulatory potency of circRNA_036186 in HNSCC.

This study aimed to characterize circular RNA (circRNA) expression profiles and biological functions in head and neck squamous cell carcinoma (HNSCC). Differentially expressed circRNAs were screened using an Arraystar Human CircRNA Array and verified by reverse transcription-quantitative polymerase chain reaction. Multiple bioinformatics methods and a hypergeometric test were employed to predict the interactions between RNAs and the functional circRNA­microRNA (miRNA)-mRNA axes in HNSCC. As a result, 287 circRNAs and 1,053 mRNAs were determined to be differentially expressed in HNSCC compared with the adjacent tissue. In addition, the expression levels of circRNA_036186 and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ polypeptide (14­3­3ζ) were identified to be significantly different. A competing endogenous RNA (ceRNA) network was constructed, consisting of 5 circRNAs, 385 miRNAs and 96 mRNAs. Furthermore, we predicted that miR­193b­3p exerts a significant effect on 14­3­3ζ, and was significantly associated with the Hippo signaling pathway in HNSCC. On the whole, these findings suggest that circRNA_036186 likely regulates 14­3­3ζ expression by functioning as a ceRNA in the development and progression of HNSCC.

HER2-Driven Breast Tumorigenesis Relies upon Interactions of the Estrogen Receptor with Coactivator MED1.

Studies of the estrogen receptor (ER) coactivator protein Mediator subunit 1 (MED1) have revealed its specific roles in pubertal mammary gland development and potential contributions to breast tumorigenesis, based on coamplification of MED1 and HER2 in certain breast cancers. In this study, we generated a mouse model of mammary tumorigenesis harboring the MMTV-HER2 oncogene and mutation of MED1 to evaluate its role in HER2-driven tumorigenesis. MED1 mutation in its ER-interacting LxxLL motifs was sufficient to delay tumor onset and to impair tumor growth, metastasis, and cancer stem-like cell formation in this model. Mechanistic investigations revealed that MED1 acted directly to regulate ER signaling through the downstream IGF1 pathway but not the AREG pathway. Our findings show that MED1 is critical for HER2-driven breast tumorigenesis, suggesting its candidacy as a disease-selective therapeutic target.Significance: These findings identify an estrogen receptor-binding protein as a critical mediator of HER2-driven breast tumorigenesis, suggesting its candidacy as a disease-selective therapeutic target. Cancer Res; 78(2); 422-35. ©2017 AACR.

Study on the interaction of anthracenyl-methyl homospermidine conjugate (ANTMHspd) with DNA by spectroscopic methods.

The interaction between anthracenyl-methyl homospermidine conjugate (ANTMHspd) with herring sperm DNA was investigated by UV/vis absorption, fluorescent spectra, circular dichroism (CD) spectroscopy and 1HNMR under physiological conditions (pH=7.4). The observed hypochromism effect and fluorescence quenching of ANTMHspd by DNA, and the displacement of EB from DNA-EB system by ANTMHspd suggested that ANTMHspd might interact with DNA by the combined mode of intercalation and groove binding. Further fluorescent tests at different temperatures revealed that the quenching mechanism was a static type. The quenching constant, binding constant and thermodynamic parameter obtained from fluorescence showed that the type of interaction force included mainly hydrogen bonding and van der Waals, which promoted the binding process. The CD test revealed that ANTMHspd could cause the B to A-like conformational change while ANTMHspd is not a typical DNA intercalator. The 1H NMR tests showed that ANTMHspd partially intercalated DNA. The effect of NaCl and KI on ANTMHspd-DNA interaction provided additional evidences of intercalation. Molecular docking simulation was carried out and the docking model in silico suggested that the binding modes of ANTMHspd and DNA were groove binding and intercalation, with the anthracene moiety inserted in DNA base pairs and the polyamine chain embedded in the DNA groove.