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Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure. For complete details on the use and execution of this protocol, please refer to Grandi et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1 H/ 13 C methyl protonated proteins from 100 ml cultures in M9 ++ /D 2 O medium induced at high (OD 600  ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2 H/ 15 N/ 13 C and 1 H/ 13 C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D 2 O medium, while using three-fold less α-ketoisovaleric (1,2,3,4- 13 C 4 ; 3,4',4',4'-d 4 ) and α-ketobutyric ( 13 C 4 ; 3,3-d 2 ) acid precursors.

The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.

Course-based undergraduate research experiences (CUREs) have been shown to increase student retention and learning in the biological sciences. Most CURES cover only one aspect of gene regulation, such as transcriptional control. Here we present a new inquiry-based lab that engages understanding of gene expression from multiple perspectives. Students carry out a forward genetic screen to identify regulators of the stationary phase master regulator RpoS in the model organism Escherichia coli and then use a series of reporter fusions to determine if the regulation is at the level of transcription or the post-transcription level. This easy-to-implement course has been run both as a 9-week long project and a condensed 5-6 week version in three different schools and types of courses. A majority of the genes found in the screen are novel, thus giving students the opportunity to contribute to original findings to the field. Assessments of this CURE show student gains in learning in many knowledge areas. In addition, attitudinal surveys suggest the students are enthusiastic about the screen and their learning about gene regulation. In summary, this lab would be an appropriate addition to an intermediate or advanced level Molecular Biology, Genetics, or Microbiology curriculum. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):449-458, 2017., (© 2017 The International Union of Biochemistry and Molecular Biology.)

This graduate-level DNA methods laboratory course is designed to model a discovery-based research project and engages students in both traditional DNA analysis methods and modern recombinant DNA cloning techniques. In the first part of the course, students clone the Drosophila ortholog of a human disease gene of their choosing using Gateway ® cloning. In the second part of the course, students examine the expression of their gene of interest in human cell lines by reverse transcription PCR and learn how to analyze data from quantitative reverse transcription PCR (qRT-PCR) experiments. The adaptability of the Gateway ® cloning system is ideally suited for students to design and create different types of expression constructs to achieve a particular experimental goal (e.g., protein purification, expression in cell culture, and/or subcellular localization), and the genes chosen can be aligned to the research interests of the instructor and/or ongoing research in a department. Student evaluations indicate that the course fostered a genuine excitement for research and in depth knowledge of both the techniques performed and the theory behind them. Our long-term goal is to incorporate this DNA methods laboratory as the foundation for an integrated laboratory sequence for the Master of Science degree program in Molecular and Cellular Biology at Quinnipiac University, where students use the reagents and concepts they developed in this course in subsequent laboratory courses, including a protein methods and cell culture laboratory. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):351-359, 2017., (© 2017 The International Union of Biochemistry and Molecular Biology.)

The flow of genetic information from DNA to RNA to protein, otherwise known as the "central dogma" of biology, is one of the most basic and overarching concepts in the biological sciences. Nevertheless, numerous studies have reported student misconceptions at the undergraduate level of this fundamental process of gene expression. This study reports on the efficacy of a model system for teaching gene expression in the undergraduate laboratory. A student-centered investigation of Tgfb1 gene expression in two murine melanoma cell lines was used to emphasize not only the process of gene expression but also various research methods for studying this phenomenon. Traditional RT-PCR, quantitative real-time RT-PCR, and flow cytometry-based in situ hybridization assays were employed to study expression of this immunosuppressive cytokine gene in the highly tumorigenic B16-F1 melanoma cell line and the poorly tumorigenic D5.1G4 melanoma cell line, both at the population and single-cell levels. A pre- and post-laboratory assessment instrument demonstrated the utility of this model system in enhancing student learning both of content related to gene expression and of research methods and data analysis skills. The pedagogical approach described in this study is therefore an effective way to improve the teaching and learning of gene expression at the undergraduate level. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):397-404, 2016., (© 2016 The International Union of Biochemistry and Molecular Biology.)

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor., (© 2015 The International Union of Biochemistry and Molecular Biology.)

SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative CT method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events. It requires only 4-5 hr of dedicated laboratory time and focuses on use of the primary literature, data analysis, and interpretation. Importantly, this exercise provides a mechanism to introduce the concept of differential gene expression and provides a starting point for development of more complex guided or open inquiry projects for students moving into upper level molecular biology, immunology, and biochemistry course work., (© 2015 The International Union of Biochemistry and Molecular Biology.)

ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5' sequence coding for its secretion signal peptide (chiA74Δsp) into a cold shock expression vector (pColdI) for production of the enzyme in Escherichia coli BL21-Rosetta 2. As a result, the N-terminal end of ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6× histidine tag for purification of recombinant 6×His tagged-ChiA74Δsp (rChiA74, ∼74kDa). Along with a protein of ∼74kDa, we co-purified its ∼55kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40°C. Most divalent cations (e.g. Ba(+2), Ca(+2), Mn(+2), Mg(+2), Zn(+2) and Cu(+2)) at concentration of 10mM reduced chitinase activity by ∼30%, and Hg(+2) (10mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11±0.01nmol/min, 2.15μM±0.45 and 3.81s(-1), respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The high incidence of fragility fractures in cortico-cancellous bone locations, plus the fact that individual skeletal sites exhibit different responsiveness to load and disease, emphasizes the need to document separately gene expression in cortical and cancellous bone. A further confounding factor is marrow contamination since its high cellularity may effect gene expression measurements. We isolated RNA from cortical and cancellous bone of intact mouse tibiae, and also after marrow removal by flushing or centrifugation. RNA isolated from cancellous bone by each method was sufficient for gene expression analysis. Centrifugation removed contaminating cells more efficiently than flushing, as indexed by histology and decreased expression of Icam4, a highly expressed erythroid gene. In contrast, centrifuged cortical bone had 12- and 13- fold higher expression of the bone-related genes Col1a1 and Bglap, while levels in marrow-free cancellous bone were 30- and 31-fold higher when compared to bone where marrow was left intact. Furthermore, cortical bone had higher expression of Col1a1 and Bglap than cancellous bone. Thus, RNA isolated by this novel approach can reveal site-specific changes in gene expression in cortical and cancellous bone sites., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of β-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.

The time-evolution of continuous-time discrete-state biochemical processes is governed by the Chemical Master Equation (CME), which describes the probability of the molecular counts of each chemical species. As the corresponding number of discrete states is, for most processes, large, a direct numerical simulation of the CME is in general infeasible. In this paper we introduce the method of conditional moments (MCM), a novel approximation method for the solution of the CME. The MCM employs a discrete stochastic description for low-copy number species and a moment-based description for medium/high-copy number species. The moments of the medium/high-copy number species are conditioned on the state of the low abundance species, which allows us to capture complex correlation structures arising, e.g., for multi-attractor and oscillatory systems. We prove that the MCM provides a generalization of previous approximations of the CME based on hybrid modeling and moment-based methods. Furthermore, it improves upon these existing methods, as we illustrate using a model for the dynamics of stochastic single-gene expression. This application example shows that due to the more general structure, the MCM allows for the approximation of multi-modal distributions.

We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the FLOWERING LOCUS C gene, a key regulator of floral timing in Arabidopsis thaliana plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate students in biochemistry or molecular biology courses. The project provides students with hands-on experience with RT-qPCR, the current "gold standard" for gene expression analysis, including detailed data analysis using the common 2-ΔΔCT method. Moreover, it provides a convenient starting point for many inquiry-driven projects addressing diverse questions concerning ecological biochemistry, naturally occurring genetic variation, developmental biology, and the regulation of gene expression in nature., (Copyright © 2013 Wiley Periodicals, Inc.)

Globular adiponectin (GAD) as the active domain of adiponectin is a promising candidate for anti-diabetic drug development. The recombinant production of GAD in Escherichia coli, however, is difficult because it is mainly expressed as inclusion bodies which need to be refolded to regain function. In this study we developed a novel method for refolding of GAD with a high efficiency by using polyethylene glycol (PEG) conjugation. An artificially designed DNA sequence encoding for GAD was synthesized and inserted into the pET28a vector to construct an expression plasmid which was thereafter transformed into E. coli BL21 (DE3) host cells for heterologous expression. After bacterial cell culture employing auto-induction medium, the inclusion bodies were collected, washed and dissolved in guanidine hydrochloride before PEG conjugation. Then the PEG-conjugated GAD was refolded by dialysis and purified by two steps of chromatography. The refolded conjugate showed a marked glucose-lowering activity in mice, demonstrating that it had been successfully refolded. As a convenient method, PEGylation-aided refolding could also be tested on other proteins to explore its suitability.

To be secreted or transported to their target compartments, newly synthesized proteins leave the endoplasmic reticulum to reach the Golgi apparatus, where they are processed and sorted toward their final destinations along the secretory pathway. It is now clear that many Golgi-intersecting and non-intersecting pathways exist in cells to carry out proper transport, modification, and addressing. To analyze and visualize the intracellular trafficking of any secretory protein, we developed the retention using selective hooks (RUSH) system. This assay allows the simultaneous release of a pool of particular secretory proteins from the endoplasmic reticulum and the monitoring of their anterograde trafficking. The use of the RUSH system is detailed in these protocols, from sub-cloning the sequence coding for the protein of interest into RUSH plasmids to visualization of its trafficking.

Diphosphoinositol polyphosphates, also known as inositol pyrophosphates, are a family of water soluble inositol phosphates that possess diphosphate or pyrophosphate moieties. In the presence of divalent cations such as Mg(2+), the "high energy" beta phosphate can be transferred from the inositol pyrophosphates, InsP(7) and InsP(8), to prephosphorylated serine residues on proteins, to form pyrophosphoserine. This chapter provides detailed methods to identify proteins that are substrates for pyrophosphorylation by InsP(7), conduct phosphorylation assays on purified protein, and detect protein pyrophosphorylation.

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods--cell-mediated dissolution of type-1 collagen fibrils, direct and reverse zymography, enzyme capture based on alpha2-macroglobulin and TIMP-1 and -2, and demonstration of cryptic thiol groups in metalloproteinase precursors--that are used to characterize the functions of matrix metalloproteinases and their inhibitors., (Copyright 2008 by John Wiley & Sons, Inc.)

Construction of a chemical system capable of replication and evolution, fed only by small molecule nutrients, is now conceivable. This could be achieved by stepwise integration of decades of work on the reconstitution of DNA, RNA and protein syntheses from pure components. Such a minimal cell project would initially define the components sufficient for each subsystem, allow detailed kinetic analyses and lead to improved in vitro methods for synthesis of biopolymers, therapeutics and biosensors. Completion would yield a functionally and structurally understood self-replicating biosystem. Safety concerns for synthetic life will be alleviated by extreme dependence on elaborate laboratory reagents and conditions for viability. Our proposed minimal genome is 113 kbp long and contains 151 genes. We detail building blocks already in place and major hurdles to overcome for completion.

Motivation: Metabolic networks combine metabolism and regulation. These complex networks are difficult to understand and visualize due to the amount and diverse types of information that need to be represented. For example, pathway information gives indications of interactions. Experimental data, such as transcriptomics, proteomics and metabolomics data, give snapshots of the system state. Stereoscopic virtual environments provide a true three-dimensional representation of metabolic networks, which can be intuitively manipulated, and may help to manage the data complexity., Results: MetNet3D, a 3D virtual reality system, allows a user to explore gene expression and metabolic pathway data simultaneously. Normalized gene expression data are processed in R and visualized as a 3D plot. Users can find a particular gene of interest or a cluster of genes that behave similarly and see how these genes function in metabolic networks from MetNetDB, a database of Arabidopsis metabolic networks, using animated network graphs. Interactive virtual reality, with its enhanced ability to display more information, makes such integration more effective by abstracting key relationships., Availability: MetNet3D and some sample datasets are available at http://www.vrac.iastate.edu/research/sites/metnet/Download/Download.htm., Supplementary Information: Color snapshots and movies are available at http://www.vrac.iastate.edu/research/sites/metnet/Bioinformatics/SupplementaryInformation.htm.

Biochemistry in the context of a living cell or organism is complicated by many variables such as supramolecular organization, cytoplasmic viscosity, and substrate heterogeneity. While these variables are easily excluded or avoided in reconstituted systems, they must be dealt with in cellular environments. New developments have allowed researchers to begin probing the inner workings of the cell to gain new insight into cell function and metabolism. Advances in cellular imaging and in small molecule-controlled gene expression, signal transduction and cell surface modification are discussed in this review. These techniques have permitted the study of molecular components within the context of living cells.

Molecular imaging can reveal in vivo analysis and quantification of biochemical reactions. To enable cell-surface imaging of receptors, novel ligands have been developed which can be radiolabeled or imaged by bioluminescence. Specific examples include somatostatin receptors, estrogen and progesterone receptors, receptors involved in adhesion and externalization of phosphatidyl serine as an indicator of apoptosis. Central nervous system imaging can be carried out using ligands for receptors including dopamine, serotonin and Gamma amino butyric acid (GABA). In addition, tumor and metabolic imaging can be carried out with the Na-K ATPase pump using the tracer thallium-201 for SPECT or F-18 FDG for PET imaging. Finally, novel receptors or endogenous metabolic pathways can be analyzed combining cell-gene therapy to create specific tracer targets in cells that can be studied by molecular imaging. The challenge of molecular imaging is to first identify key pathways that are unique for a specific disease processes, such as atherosclerosis, cancer, CNS disorders, immunologic and arthritis disorders and next to devise a high-affinity specific small molecular ligand that can be adapted to be a radiolabeled tracer to study this pathway. Advances in genomics and proteomics combine with new peptide-chemistry approaches should provide a large number of targets and tracers in the near future to achieve these imaging objectives., (Copyright 2002 Wiley-Liss, Inc.)

Analysis of organisms using techniques that provide high spatial and temporal resolution is of increasing interest in many disciplines of biomedical research. Although the examination of animal tissues has been the main focus to date, the recent development and improvement of methods for the sampling and handling of single cells and for their biochemical analysis now provide tools for investigating plant as well as animal cells.

Cells regulate gene expression through various RNA regulatory mechanisms, and this regulation often becomes less efficient with age, contributing to accelerated aging and various age-related diseases. Nonsense-mediated mRNA decay (NMD), a well-characterized RNA surveillance mechanism, degrades aberrant mRNAs with premature termination codons (PTCs) to prevent the synthesis of truncated proteins. While the role of NMD in cancer and developmental and genetic diseases is well documented, its implications in human aging remain largely unexplored. This study reveals a significant decline in the levels of the protein UPF1, a key player in NMD, during cellular senescence. Additionally, NMD substrates accumulate in senescent cells, along with decreased levels of cap-binding protein 80/20 (CBP80/20)-dependent translation (CT) factors and reduced binding to active polysomes, indicating reduced efficiency of NMD. Moreover, knockdown of UPF1 in proliferating WI-38 cells induces senescence, as evidenced by increased senescence-associated β-galactosidase activity, alterations in senescence-associated molecular markers, increased endogenous γ-H2AX levels, and reduced cell proliferation. These findings suggest that the decline in UPF1 levels during cellular senescence accelerates the senescent phenotype by impairing NMD activity and the consequent accumulation of abnormal mRNA. Our study highlights the decline of UPF1, a key component of nonsense-mediated mRNA decay (NMD), during cellular senescence and its impact on NMD efficiency, leading to the accumulation of aberrant mRNAs. [ABSTRACT FROM AUTHOR]

Objectives: Back pain subsequent to intervertebral disc injury is a common clinical problem. Previous work examining early molecular changes post injury mainly used a candidate marker approach. In this study, gene expression in the injured and intact mouse tail intervertebral discs was determined with a nonbiased whole transcriptome approach. Design: Mouse tail intervertebral disc injury was induced by a needle puncture. Whole murine transcriptome was determined by RNASeq. Transcriptomes of injured intervertebral discs were compared with those of intact controls by bioinformatic methods. Results: Among the 18,078 murine genes examined, 592 genes were differentially expressed (P.adj < 0.01). Novel genes upregulated in injured compared with intact intervertebral discs included Chl1, Lum , etc. Ontology study of upregulated genes revealed that leukocyte migration was the most enriched biological process, and network analysis showed that Tnfa had the most protein-protein interactions. Novel downregulated genes in the injured intervertebral discs included 4833412C05Rik , Myoc , etc. The most enriched downregulated pathways were related to cytoskeletal organization. Conclusions: Novel genes highly regulated after disc injury were identified with an unbiased approach; they may serve as biomarkers of injury and response to treatments in future experiments. Enriched biological pathways and molecules with high numbers of connections may be targets for treatments after injury. [ABSTRACT FROM AUTHOR]

Background: The Hezuo (HZ) pig, a famous indigenous breed in China, is characterized by precocious puberty compared with foreign-introduced pig breeds. Sexual maturation is a complex physiological process, and in recent years, circular RNAs (circRNAs), a new class of noncoding RNAs with endogenous regulatory functions, have been shown to play important roles in regulating sexual maturation. However, the dynamic expression and regulatory mechanism of circRNAs during sexual maturation in HZ pigs remain unclear. In this study, we performed RNA sequencing and bioinformatics analysis to reveal circRNA expression patterns in the testes of HZ boars at 30 days (sexual immaturity; Ha) and 120 days (sexual maturity; Hb), with Landrace (LC) boars of the same age (La and Lb) as controls. Subsequently, an abundant circ_005678 (circPAN3) transcribed from the PAN3 gene, was functionally investigated by RT-qPCR, Western Blot, CCK-8, and flow cytometry. Results: We identified 31,134 circRNAs in 12 samples, and 2,562, 2,401, 749, and 831 differentially expressed (DE) circRNAs were identified in the Ha-vs-Hb, La-vs-Lb, Ha-vs-La, and Hb-vs-Lb groups, respectively. The results of functional enrichment analyses indicated that these source genes of the DE circRNAs were involved mainly in testicular development and spermatogenesis. Furthermore, we constructed a circRNA-miRNA-mRNA interaction network and functionally analyzed the target genes. GO functional annotation of the target genes suggested that they were mainly involved in biological processes such as gland development, cell proliferation, and reproduction. KEGG pathway analysis further revealed that these genes were enriched mainly in signaling pathways involved in testicular development and spermatogenesis, including the PI3K-Akt and MAPK signaling pathways. Cellular assays revealed that circPAN3 promoted proliferation and inhibited apoptosis in immature Sertoli cells, whereas opposite changes were observed by circPAN3 knockdown. Conclusions: This study revealed the dynamic expression profiles and regulatory mechanisms of circRNAs during sexual maturation in HZ pigs. Further functional studies demonstrated that circPAN3 promoted the proliferation and inhibited the apoptosis of immature Sertoli cells, suggesting that circPAN3 may be closely related to the characteristics of precocious puberty in HZ boars. These findings provide a new perspective for exploring the regulatory mechanism of circRNAs in precocious puberty in HZ pigs. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer patients frequently develop a syndrome named cachexia that causes severe muscle loss and frailty, eventually representing the cause of death. Muscle atrophy and muscle weakness are characterized by massive degradation of endogenous proteins, potentially consequent to excessive disposal of mitochondria through the selective autophagic process of mitophagy. This study explored whether selectively silencing BNIP3, a mitophagy-related protein upregulated in the muscle of both mouse and human cancer hosts, could help in preventing muscle loss. Two distinct methodological silencing approaches were tested, either by electroporation of a plasmid or via adenoviral particle injection. Although the first method was ineffective in tumor-bearing mice, the adenovirus-based approach significantly reduced BNIP3 levels and moderately increased muscle fiber size, suggesting partial prevention of muscle atrophy. BNIP3 silencing also maintained mitochondrial mass without disrupting oxidative balance, highlighting BNIP3's central role in cancer cachexia and suggesting that targeting BNIP3 may help in supporting muscle health in cancer patients. Background and Aims: Cancer cachexia is a complex syndrome affecting most cancer patients and is directly responsible for about 20% of cancer-related deaths. Previous studies showed muscle proteolysis hyper-activation and mitophagy induction in tumor-bearing animals. While basal mitophagy is required for maintaining muscle mass and quality, excessive mitophagy promotes uncontrolled protein degradation, muscle loss and impaired function. BNIP3, a key mitophagy-related protein, is significantly increased in the muscles of both mice and human cancer hosts. This study aimed to define the potential of mitigating mitophagy via BNIP3 downregulation in preserving mitochondrial integrity, counteracting skeletal muscle loss in experimental cancer cachexia. Methods: Two in vivo gene delivery methods were performed to knock down muscle BNIP3: electroporation of a BNIP3-specific shRNA expression vector or adenovirus injection. Results: The electroporation effectively reduced muscle BNIP3 in healthy mice but was ineffective in C26 tumor-bearing mice. In contrast, adenovirus-mediated BNIP3 knockdown successfully decreased BNIP3 levels also in tumor hosts. Although BNIP3 knockdown did not impact overall on body or muscle mass, it improved muscle fiber size in C26-bearing miceh2, suggesting partial prevention of muscle atrophy. Mitochondrial respiratory chain complexes (OxPhos) and TOM20 protein levels were consistently rescued, indicating improvements in mitochondrial mass, while H2O2 levels were unchanged among the groups, suggesting that BNIP3 downregulation does not impair the endogenous control of oxidative balance. Conclusions: These findings suggest that a fine balance between mitochondrial disposal and biogenesis is fundamental for preserving muscle homeostasis and highlight a potential role for BNIP3 modulation against cancer-induced muscle wasting. [ABSTRACT FROM AUTHOR]

Background: Chronic pain poses a clinical challenge due to its associated costly disability and treatment needs. Determining how pain transitions from acute to chronic is crucial for effective management. Upregulation of the chemokine C-X-C motif ligand 12 (CXCL12) in nociceptive pathway is associated with chronic pain. Our previous study has reported that elevated plasma CXCL12 mediates intracerebral neuroinflammation and the comorbidity of cognitive impairment in neuropathic pain, but whether it is also involved in the pathogenesis of pathologic pain has not been investigated. Methods: Intravenous or intrathecal injection (i.v. or i.t.) of recombinant mouse CXCL12, neutralizing antibody (anti-CXCL12) or AMD3100 [an antagonist of its receptor C-X-C chemokine receptor type 4 (CXCR4)] was used to investigate the role of CXCL12 signaling pathway in pain chronicity. Two behavioral tests were used to examine pain changes. ELISA, immunofluorescence staining, Western blot, quantitative Real Time-PCR and Cytokine array were applied to detect the expressions of different molecules. Results: We found that increased plasma CXCL12 was positively correlated with pain severity in both chronic pain patients and neuropathic pain model in mice with spared nerve injury (SNI). Neutralizing plasma CXCL12 mitigated SNI-induced hyperalgesia. A single i.v. injection of CXCL12 induced prolonged mechanical hyperalgesia and activation of the nociceptive pathway. Multiple intravenous CXCL12 caused persistent hypersensitivity, enhanced structural plasticity of nociceptors and up-regulation of the CXCL12/CXCR4 axis in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). However, intrathecal blocking of CXCL12/CXCR4 pathway by CXCL12 antibody or CXCR4 antagonist AMD3100 significantly alleviated CXCL12-induced pain hypersensitivity and pathological changes. Conclusions: Our study provides strong evidence that a sustained increase in plasma CXCL12 contributes to neuropathic pain through a positive feedback loop that enhances nociceptor plasticity, and suggests that targeting CXCL12/CXCR4 axis in plasma or nociceptive pathways has potential value in regulating pain chronicity. Highlights: 1) Plasma CXCL12 in neuropathic pain states were positively correlated with pain severity. 2) A sustained increase in plasma CXCL12 contributes to pain chronicity. 3) Elevated plasma CXCL12 enhances spinal synaptic structural plasticity of pain through positive feedback. [ABSTRACT FROM AUTHOR]

Simple Summary: CR6 interacting factor 1 (CRIF1) plays an essential role in the regulation of mitochondrial function. We provide important details to demonstrate that CRIF1 deletion-induced mitochondrial damage has an antitumor effect via TIGAR induction in BT549 breast cancer cells. CRIF1 deficiency results in OXPHOS dysfunction, which has been investigated as a potential therapeutic target for the treatment of cancer. Background/Objectives: Mitochondrial oxidative phosphorylation (OXPHOS) has been exploited as a therapeutic target in cancer treatments because of its crucial role in tumorigenesis. CR6-interacting factor 1 (CRIF1), a mitochondrial ribosomal subunit protein, is essential for the regulation of mitochondrial OXPHOS capacity. However, the mechanism of CRIF1 in triple-negative breast cancer (TNBC) cells remains unclear. Methods/Results: We showed that the downregulation of CRIF1 reduced cell proliferation in the TNBC cell lines MDA-MB-468, MDA-MB-231, and, especially, BT549. In addition, wound scratch and Transwell assays showed that CRIF1 deficiency inhibited the migration and invasion of BT549 cells. CRIF1 downregulation resulted in the suppression of mitochondrial bioenergetics in BT549 cells, specifically affecting the inhibition of OXPHOS complexes I and II. This was evidenced by a decrease in the mitochondrial oxygen consumption rate and the depolarization of the mitochondrial membrane potential. Damage to mitochondria resulted in a lower adenosine triphosphate level and an elevated production of mitochondrial reactive oxygen species. In addition, CRIF1 deficiency decreased hypoxia-inducible factor 1α accumulation, NADPH synthesis, and TP53-induced glycolysis and apoptosis regulator (TIGAR) expression in BT549 cells. These events contributed to G0/G1-phase cell cycle inhibition and the upregulation of the cell cycle protein markers p53, p21, and p16. Transfection with a TIGAR overexpression plasmid reversed these effects and prevented CRIF1 downregulation-induced proliferation and migration reduction. Conclusions: These results indicate that blocking mitochondrial OXPHOS synthesis via CRIF1 may have a therapeutic antitumor effect in BT549 TNBC cells. [ABSTRACT FROM AUTHOR]

Simple Summary: A tumor's extracellular matrix (ECM) serves as a vital connection and support network, orchestrating events that regulate tumor activity. Versican (VCAN), a proteoglycan found within the ECM, plays a crucial role in invasion and metastasis. Like the ECM, VCAN can undergo cleavage by metalloproteinases, such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). This proteolytic process produces a bioactive fragment known as versikine (VKINE). Remarkably, this fragment exhibits immunomodulatory activity in certain cancer types. Consequently, the proteolysis of VCAN and associated factors may be valuable indicators of tumor progression. Background: The present study investigates VKINE, a bioactive proteolytic fragment of the proteoglycan VCAN, as a novel and significant element in the tumor extracellular matrix (ECM). Although VKINE has been recognized for its immunomodulatory potential in certain tumor types, its impact on ECM degradation and prognostic implications remains poorly understood. Objectives: This study aimed to evaluate VCAN proteolysis and its association with ADAMTS enzymes involved in extracellular matrix remodeling in spontaneous canine mammary gland cancer. Methods: The expression levels of VKINE, ADAMTS enzymes, and collagen fibers were comparatively analyzed in situ and in invasive areas of carcinoma in mixed tumor (CMT) and carcinosarcoma (CSS) with different prognoses. Results: VKINE was notably expressed in the stroma adjacent to the invasion areas in CMT, whereas ADAMTS-15 was identified as the enzyme associated with VCAN proteolysis. Inverse correlations were observed between type III collagen and VCAN expression in in situ areas. Conclusions: Our findings suggest that VKINE and ADAMTS-15 play crucial roles in the tumor microenvironment, influencing invasiveness and type III collagen deposition. This study contributes to a better understanding of the dynamics within the ECM, paving the way for potential new tools in diagnosing and treating human and canine mammary tumors. [ABSTRACT FROM AUTHOR]

The gut microbiome is an epigenetic effector that influences host gene expression. Recent studies suggest that the host is also able to influence its gut microbiome through epigenetic changes, including histone and DNA modifications affecting host gene expression of immune genes, gut barrier function, and through noncoding RNAs. This outlines a bidirectional 'epigenome–microbiome axis', which embeds environmental variation and could mediate rapid adaptive evolution of host–microbe relationships. New tools for epigenetic engineering allow us to investigate the causality of the epigenome–microbiome axis and offer alternative approaches to engineering beneficial microbial traits. Targeting host epigenetic mechanisms of microbiome regulation could provide new treatments for gastrointestinal disease or dysbiosis due to epigenetic dysregulation. Recent studies of dynamic interactions between epigenetic modifications of a host organism and the composition or activity of its associated gut microbiota suggest an opportunity for the host to shape its microbiome through epigenetic alterations that lead to changes in gene expression and noncoding RNA activity. We use insights from microbiota-induced epigenetic changes to review the potential of the host to epigenetically regulate its gut microbiome, from which a bidirectional 'epigenome–microbiome axis' emerges. This axis embeds environmentally induced variation, which may influence the adaptive evolution of host–microbe interactions. We furthermore present our perspective on how the epigenome–microbiome axis can be understood and investigated within a holo-omic framework with potential applications in the applied health and food sciences. [ABSTRACT FROM AUTHOR]

Betaine is the major water-soluble component of Lycium chinensis. Although there are reports of a protective effect of betaine on fatty liver disease, the underlying mechanisms are unclear. We attempted to elucidate the molecular regulation of betaine on hyperglycemia-induced hepatic lipid accumulation via Forkhead box O (FoxO)6 activation. HepG2 cells and liver tissue isolated from db/db mice treated with betaine were used. The present study investigated whether betaine ameliorates hepatic steatosis by inhibiting FoxO6/peroxisome proliferator-activated receptor gamma (PPARγ) signaling in liver cells. Interestingly, betaine notably decreased lipid accumulation in tissues with FoxO6-induced mRNA expression of lipogenesis-related genes. Furthermore, betaine inhibited the FoxO6 interaction with PPARγ and cellular triglycerides in high-glucose- or FoxO6-overexpression-treated liver cells. In addition, we confirmed that betaine administration via oral gavage significantly ameliorated hepatic steatosis in db/db mice. We conclude that betaine ameliorates hepatic steatosis, at least in part, by inhibiting the interaction between FoxO6 and PPARγ, thereby suppressing lipogenic gene transcription. [ABSTRACT FROM AUTHOR]

Rationale. This article presents a study of the stress response to the effects of water deficiency in various genotypes of Scots pine, contrasting in seed yield, breeding genotypes of silver and downy birch, hybrids of black, white poplar and intersectional poplar hybrids. Results. A contrasting response of genotypes to the effects of drought was revealed, including activation of the pP5CS, pICDH, pNAC034, pDREB2, pCA genes in poplars 'E.s.-38', 'POK', and 'Veduga', while in black poplar the expression of all analyzed genes was reduced. In silver birch 'Ug.-1', all analyzed genes bPAL, bCA, bLEA8, bNAC19 и bDREB2 were upregulated, while in downy birch '15-1' bPAL and bNAC19 were downregulated and for the rest the changing in expression was not shown. An increase in the expression of the SAMS gene was detected in response to drought in pine genotypes of different seed production, medium-yielding No. 4 and low-yielding No. 23. For the low-yielding genotype No. 22, no changes in the expression of the analyzed genes were recorded under drought conditions. Conclusion. The results obtained indicate the development of a stress response and show differences in the adaptive response in different genotypes of pine, poplar and birch, which indicates the value of the tested genes in selection for drought conditions in different tree species.

In eukaryotic cells, gene expression begins with transcription in the nucleus, followed by the maturation of messenger RNAs (mRNAs). These mRNA molecules are then exported to the cytoplasm through the nuclear pore complex (NPC), a process that serves as a critical regulatory phase of gene expression. The export of mRNA is intricately linked to precursor mRNA (pre-mRNA) processing, ensuring that only properly processed mRNA reaches the cytoplasm. This coordination is essential, as recent studies have revealed that mRNA export factors not only assist in transport but also influence upstream processing steps, adding a layer of complexity to gene regulation. Furthermore, the export process competes with RNA processing and degradation pathways, maintaining a delicate balance vital for accurate gene expression. While these mechanisms are generally conserved across eukaryotes, significant differences exist between yeast and higher eukaryotic cells, particularly due to the more genome complexity of the latter. This review delves into the current research on mRNA export in higher eukaryotic cells, focusing on its role in the broader context of gene expression regulation and highlighting how it interacts with other gene expression processes to ensure precise and efficient gene functionality in complex organisms.

Objective: Ferroptosis has been described in association with acute kidney injury (AKI)-induced sepsis. Fibronectin type III domain containing protein 5 (FNDC5)/irisin plays a crucial role in renal protection. The objective of this study was to investigate whether FNDC5/irisin is involved in AKI-induced sepsis by modulating ferroptosis, and the molecular mechanisms that may be involved. Material and Methods: A sepsis-induced AKI model was built in vivo and in vitro through lipopolysaccharide (LPS) intervention. FNDC5, adenosine 5'-monophosphate-activated protein kinase (AMPK), phospho-AMPK (p-AMPK), nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), and acyl-CoA synthetase long-chain family member 4 (ACSL4) concentrations in cells and mouse kidney tissues were appraised by Western blot. Pro-inflammatory cytokines concentrations in cell supernatants and mouse kidney tissues were appraised by enzyme-linked immunosorbent assay. Fe2+ concentration in cells and mouse kidney tissue was appraised by kit. The apoptosis rate of cells and mouse kidney tissue was measured by flow cytometry. Automatic biochemical analyzer was to test serum creatinine (SCr) and blood urea nitrogen (BUN). The kidney tissue sections from each groups were observed by hematoxylin and eosin staining. Results: LPS abated FNDC5 concentration in human kidney-2 cells and mouse kidney tissue (P < 0.001). Overexpression of FNDC5 can abated proinflammatory cytokines concentrations in cells and mouse kidney tissue (P < 0.01). Meanwhile, overexpression of FNDC5 can boost GPX4 protein concentration, abate ACSL4 protein, and abate Fe2+ concentration in cells and mouse kidney tissues (P < 0.05). In addition, the overexpression of FNDC5 can reduce the rate of apoptosis (P < 0.01). In vivo experiments showed that FNDC5 overexpression reduced serum BUN and SCr concentrations and alleviated pathological damage in the mouse renal tissues (P < 0.05) and exhibited a certain renal protective effect. FNDC5 overexpression can boost p-AMPK/AMPK, Nrf2, and HO-1 protein concentrations (P < 0.01). Conclusion: FNDC5/irisin improves sepsis-induced acute renal injury by abating ferroptosis through the AMPK/Nrf2 signaling pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is the third most common type of childhood cancer but is responsible for over 15% of all pediatric cancer deaths. Children with advanced disease have less than a 50% survival rate and are in desperate need of new treatment options. Protein phosphatase 2A (PP2A) is a protein that inhibits tumors, but it is turned off in neuroblastoma. We have previously shown that we can activate PP2A in neuroblastoma with certain drugs and decrease tumor growth, but these drugs also affect the function of the immune system. We now have new small molecules that activate PP2A without upsetting the immune system. We observed a decrease in tumor growth, proliferation, and motility with our novel PP2A activators. We feel that a better understanding of how activating PP2A inhibits neuroblastoma will lead to better treatments for these children. Background: Neuroblastoma (NB) remains one of the deadliest pediatric solid tumors. Recent advancements aimed at improving outcomes have been insufficient, and patients with high-risk NB continue to have a poor prognosis. Protein phosphatase 2A (PP2A) is a tumor suppressor protein downregulated in many cancers, including NB. PP2A activation has been shown to affect the malignant phenotype in other solid tumors. The present studies aim to investigate the effects of two novel PP2A activators as a NB therapeutic. Methods: Four established NB cell lines and a patient-derived xenoline were utilized to study the effect on cell viability, proliferation, motility, and in vivo tumor growth using two novel tricyclic sulfonamide PP2A activators, ATUX-3364 and ATUX-8385. Results: ATUX-3364 and ATUX-8385 increased PP2A activity. These PP2A activators led to decreased viability, proliferation, and motility of NB cells. Treatment of animals bearing NB tumors with ATUX-3364 or ATUX-8385 resulted in decreased tumor growth in MYCN-amplified SK-N-BE(2) tumors. At the molecular level, PP2A-based reactivation led to dephosphorylation of MYCN-S62 and decreased MYCN protein expression. Conclusions: PP2A activators decreased NB cell viability, proliferation, and motility. In vivo experiments show that PP2A activators have more significant effects on tumorigenesis in MYCN-amplified tumors. Finally, phosphorylation of MYCN protein was decreased following treatment with novel sulfonamide PP2A activators. These data and mechanistic insights may be useful for developing new PP2A-based therapies that target MYCN for the treatment of NB. [ABSTRACT FROM AUTHOR]

Drought tolerance varies among different peanut (Arachis hypogaea L.) cultivars. Here, drought responses of two cultivars, Huayu 22 (HY22) with drought tolerance and Fuhua 18 (FH18) with drought sensitivity, were compared at the morphological, physiological, biochemical, photosynthetic, and transcriptional levels. Drought stress caused wilting and curling of leaves, bending of stems, and water loss in both cultivars. There was an increase in malondialdehyde (MDA) content under prolonged drought stress, more so in FH18. But the levels of reactive oxygen species (H2O2) and lipid peroxidation were low in HY22. The activities of superoxide dismutase (SOD), peroxidase (POD), and glutathione reductase (GR) were considerably elevated, corresponding with rapid increases in the accumulation of soluble proteins, soluble sugars, and proline. Transcriptional sequencing showed gene expression varied seriously in HY22, which was upregulated in both stems of two cultivars, though downregulation was less pronounced in HY22. KEGG pathway analysis revealed significant enrichment in four leaf and six stem pathways. Additionally, core genes relating to photosynthesis, carbon fixation, proline synthesis, and sucrose and starch synthesis pathways were identified by correlation analysis. Those gene expressions were variously upregulated in stems of two cultivars, especially in HY22, giving a novel view of the shoot as a whole participating in stress response. [ABSTRACT FROM AUTHOR]

In yeast, control of sulfur amino acid metabolism relies upon Met4 , a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCF Met30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30 's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCF Met30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids. [ABSTRACT FROM AUTHOR]

BACKGROUND: Environmental chemical exposures have been associated with metabolic outcomes, and typically, their binding to nuclear hormone receptors is considered the molecular initiating event (MIE) for a number of outcomes. However, more studies are needed to understand the influence of such exposures on cell membrane-bound adiponectin receptors (AdipoRs), which are critical metabolic regulators. OBJECTIVE: We aimed to clarify the potential interactions between AdipoRs and environmental chemicals, specifically organophosphorus flame retardants (OPFRs), and the resultant effects. METHODS: Employing in silico simulation, cell thermal shift, and noncompetitive binding assays, we screened eight OPFRs for interactions with AdipoR1 and AdipoR2. We tested two key events underlying AdipoR modulation upon OPFR exposure in a liver cell model. The Toxicological Prioritization Index (ToxPi)scoring scheme was used to rank OPFRs according to their potential to disrupt AdipoR-associated metabolism. We further examined the inhibitory effect of OPFRs on AdipoR signaling activation in mouse models. RESULTS: Analyses identified pi-pi stacking and pi-sulfur interactions between the aryl-OPFRs 2-ethylhexyl diphenyl phosphate (EHDPP), triphenyl phosphate (TPhP), and tricresyl phosphate (TCP) and the transmembrane cavities of AdipoR1 and AdipoR2. Cell thermal shift assays showed a >3°C rightward shift in the AdipoR proteins' melting curves upon exposure to these three compounds. Although the binding sites differed from adiponectin, results suggest that aryl-OPFRs noncompetitively inhibited the binding of the endogenous peptide ligand ADP355 to the receptors. Analyses of key events underlying AdipoR modulation revealed that glucose uptake was notably lower, whereas lipid content was higher in cells exposed to arylOPFRs. EHDPP, TCP, and TPhP were ranked as the top three disruptors according to the ToxPi scores. A noncompetitive binding between these aryl-OPFRs and AdipoRs was also observed in wild-type (WT) mice. In db/db mice, the finding of lower blood glucose levels after ADP355 injection was diminished in the presence of a typical aryl-OPFR (TCP). WT mice exposed to TCP demonstrated lower AdipoR1 signaling, which was marked by lower phosphorylated AMP-activated protein kinase (pAMPK) and a higher expression of gluconeogenesis-related genes. Moreover, WT mice exposed to ADP355 demonstrated higher levels of pAMPK protein and peroxisome proliferator-activated receptor-α messenger RNA. This was accompanied by higher glucose disposal and by lower levels of long-chain fatty acids and hepatic triglycerides; these metabolic improvements were negated upon TCP co-treatment. CONCLUSIONS: In silico, in vitro, and in vivo assays suggest that aryl-OPFRs act as noncompetitive inhibitors of AdipoRs, preventing their activation by adiponectin, and thus function as antagonists to these receptors. Our study describes a novel MIE for chemical-induced metabolic disturbances and highlights a new pathway for environmental impact on metabolic health. https://doi.org/10.1289/EHP14634 [ABSTRACT FROM AUTHOR]

Objective: Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes. Materials and Methods: The Pm–human oral keratinocyte (HOK) co‐culture model was established to detect monolayer permeability, zona occludens‐1 (ZO‐1) expression, and intracellular survival of Pm. We performed RNA‐seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML‐7 was utilized in Pm‐HOK co‐culture model to assess its effects on monolayer permeability and ZO‐1 expression. Results: HOK monolayer permeability was increased, and ZO‐1 expression was decreased after co‐culture (p < 0.05). Pm could survive in HOK cells. RNA‐seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm‐HOK co‐culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO‐1 expression was upregulated (p < 0.05). Conclusion: MLCK may be involved in disrupting epithelial barrier function by Pm. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNAs (lncRNAs) are reported that play an important role in regulating tumorigenesis. This study aims to develop a ferroptosis‐related lncRNA gene signature for predicting biochemical recurrence of prostate cancer (PCa) and prove a functional lncRNA BCRP3 as a promotor toward PCa progression. Methods: The ferroptosis‐related lncRNA were identified by interoperating the databases of The Cancer Genome Atlas (TCGA) and FerrDb. A predictive model for biochemical recurrence of PCa was established based on the LASSO Cox regression. Kaplan–Meier cures were applied in the measurement of the impact on patients' survival caused by key lncRNAs. Meanwhile, the molecular assays of CCK8, EdU, wound healing, and Transwell were conducted to evaluate the tumor‐suppressive effect of BCRP3 in vitro. Results: This study presented that 17 differentially expressed ferroptosis‐related lncRNAs were confirmed and further screened out 9 key lncRNAs for the establishment of risk model. The nomogram involved the risk model was also proved with an excellent predictive performance toward 1‐, 3‐, and 5‐year survival with the area of curves (AUC) as 0.77, 0.79, and 0.65 separately. Notably, the lncRNA BCRP3 was significantly correlated with the survival of PCa patients based on multivariate Cox regression. Additionally, downregulation of BCRP3 effectively inhibited the proliferation, migration, and invasion of PC3 cells which further proved its anti‐tumor function. Conclusion: We developed a ferroptosis‐related lncRNA risk model for predicting biochemical recurrence, which assisting in the clinical therapy of patients with PCa. [ABSTRACT FROM AUTHOR]

Introduction: The purpose of this study was to investigate the effects and potential mechanisms of ferroptosis‐related gene heat shock protein beta‐1 (HSPB1) on acute myeloid leukemia (AML). Methods: The RNA‐seq and clinical data of AML samples were obtained from the Genomic Data Commons database, and the FerrDb database was used to screen the marker, drive and suppressor of ferroptosis. Besides, DESeq2 was applied for differential expression analysis on AML samples and screening for differentially expressed genes (DEGs). The screened DEGs were subjected to the intersection analysis with ferroptosis‐related genes to identify the ferroptosis‐related DEGs. Next, the functional pathways of ferroptosis‐related DEGs were further be discussed by Gene Ontology as well as Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs. Additionally, lasso regression analysis was employed to determine the differential genes related to prognosis in patients with AML and the survival analysis was performed. Subsequently, quantitative real‐time polymerase chain reaction and western blot assay were applied to detect the mRNA and protein expression levels of HSPB1 in normal/AML bone marrow tissues and human normal (HS‐5)/AML (HL‐60) bone marrow cells, respectively. Furthermore, HSPB1 was knocked down to assess the expression changes of glutathione peroxidase 4 and acyl‐CoA synthetase long‐chain family member 4. Ultimately, the viability and oxidative stress levels of HL‐60 were analyzed by Cell Counting Kit‐8 and biochemical detection. Results: A total of 4986 DEGs were identified in AML samples, with 3324 up‐regulated and 1662 down‐regulated. The enrichment analysis illustrated that ferroptosis‐related DEGs were significantly enriched in response to metal irons, oxidative stress, and other pathways. After lasso regression analysis, 17 feature genes related to the prognosis of patients with AML were obtained, with HSPB1 exhibiting a significant correlation. The reliability of our models was verified by Cox regression analysis and survival analysis of the hazard model. Furthermore, the outcomes of quantitative real‐time polymerase chain reaction and western blot showed that mRNA and protein expression levels of HSPB1 were significantly increased in the AML Group and HL‐60 cells. The knockdown of HSPB1 in HL‐60 cells reduced the protein level of glutathione peroxidase 4, increased the protein level of acyl‐CoA synthetase long‐chain family member 4, decreased the cell viability, and aggravated oxidative stress. Conclusion: Ferroptosis‐related gene HSPB1 is highly expressed in patients with AML. In addition, HSPB1 may be involved in the occurrence and development of AML by regulating oxidative stress and ferroptosis‐related pathways. This study provides new clues for further understanding of AML molecular mechanisms. Also, HSPB1 is expected to be a potential therapeutic target for AML in the future. [ABSTRACT FROM AUTHOR]

Notch ligands and receptors, including JAG1/2, DLL1/4, and Notch1/3, are enriched on macrophages (MΦs), fibroblast‐like synoviocytes (FLS), and/or endothelial cells in rheumatoid arthritis (RA) compared with normal synovial tissues (ST). Power Doppler ultrasound‐guided ST studies reveal that the Notch family is highly involved in early active RA, especially during neovascularization. In contrast, the Notch family is not implicated during the erosive stage, evidenced by their lack of correlation with radiographic damage in RA ST. Toll‐like receptors and tumor necrosis factor (TNF) are the common inducers of Notch expression in RA MΦs, FLS, and endothelial cells. Among Notch ligands, JAG1 and/or DLL4 are most inducible by inflammatory responses in RA MΦs or endothelial cells and transactivate their receptors on RA FLS. TNF plays a central role on Notch ligands, as anti‐TNF good responders display JAG1/2 and DLL1/4 transcriptional downregulation in RA ST myeloid cells. In in vitro studies, TNF increases Notch3 expression in MΦs, which is further amplified by RA FLS addition. Specific disease‐modifying antirheumatic drugs reduced JAG1 and Notch3 expression in MΦ and RA FLS cocultures. Organoids containing FLS and endothelial cells have increased expression of JAG1 and Notch3. Nonetheless, Methotrexate, interleukin‐6 receptor (IL‐6R) antibodies, and B cell blockers are mostly ineffective at decreasing Notch family expression. NF‐κB, MAPK, and AKT pathways are involved in Notch signaling, whereas JAK/STATs are not. Although Notch blockade has been effective in RA preclinical studies, its small molecule inhibitors have failed in phase I and II studies, suggesting that alternative strategies may be required to intercept their function. [ABSTRACT FROM AUTHOR]