Quantitative Analysis of Isopimpinellin from Ammi majus L. Fruits and Evaluation of Its Biological Effect on Selected Human Tumor Cells.

Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma.

CRISPR/Cas9 editing reveals IRF8 regulated gene signatures restraining plasmablast differentiation.

The transcription factor Interferon regulatory factor 8 (IRF8) is involved in maintaining B cell identity. However, how IRF8 regulates T cell independent B cell responses are not fully characterized. Here, an in vivo CRISPR/Cas9 system was optimized to generate Irf8-deficient murine B cells and used to determine the role of IRF8 in B cells responding to LPS stimulation. Irf8-deficient B cells more readily formed CD138+ plasmablasts in response to LPS with the principal dysregulation occurring at the activated B cell stage. Transcriptional profiling revealed an upregulation of plasma cell associated genes prematurely in activated B cells and a failure to repress the gene expression programs of IRF1 and IRF7 in Irf8-deficient cells. These data expand on the known roles of IRF8 in regulating B cell identity by preventing premature plasma cell formation and highlight how IRF8 helps evolve TLR responses away from the initial activation towards those driving humoral immunity.

IL-6/STAT3 Signaling Axis Enhances and Prolongs Pdcd1 Expression in Murine CD8 T Cells.

CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.

H3K27me3 Demethylase UTX Restrains Plasma Cell Formation.

B cell differentiation is associated with substantial transcriptional, metabolic, and epigenetic remodeling, including redistribution of histone 3 lysine 27 trimethylation (H3K27me3), which is associated with a repressive chromatin state and gene silencing. Although the role of the methyltransferase EZH2 (Enhancer of zeste homolog 2) in B cell fate decisions has been well established, it is not known whether H3K27me3 demethylation is equally important. In this study, we showed that simultaneous genetic deletion of the two H3K27 demethylases UTX and JMJD3 (double-knockout [Utx fl/fl Jmjd3 fl/fl Cd19 cre/+] [dKO]) led to a significant increase in plasma cell (PC) formation after stimulation with the T cell-independent Ags LPS and NP-Ficoll. This phenotype occurred in a UTX-dependent manner as UTX single-knockout mice, but not JMJD3 single-knockout mice, mimicked the dKO. Although UTX- and JMJD3-deficient marginal zone B cells showed increased proliferation, dKO follicular B cells also showed increased PC formation. PCs from dKO mice upregulated genes associated with oxidative phosphorylation and exhibited increased spare respiratory capacity. Mechanistically, deletion of Utx and Jmjd3 resulted in higher levels of H3K27me3 at proapoptotic genes and resulted in reduced apoptosis of dKO PCs in vivo. Furthermore, UTX regulated chromatin accessibility at regions containing ETS and IFN regulatory factor (IRF) transcription factor family motifs, including motifs of known repressors of PC fate. Taken together, these data demonstrate that the H3K27me3 demethylases restrain B cell differentiation.

Inhibition of H3K27me3 Demethylases Promotes Plasmablast Formation.

B cell differentiation into Ab-secreting plasma cells requires transcriptional, metabolic, and epigenetic remodeling. Histone H3 lysine 27 trimethylation (H3K27me3), a histone modification associated with gene silencing, is dynamically regulated during B cell differentiation. Although several studies have focused on mechanisms involving the gain of this modification in plasmablasts (PB), the role of active demethylation of H3K27me3 by ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji domain-containing protein 3 (JMDJ3) during B cell differentiation has not been examined. In this study, this process was assessed using a pharmacological inhibitor of UTX and JMJD3, GSK-J4. Treatment of ex vivo stimulated mouse B cells with GSK-J4 led to an increase in PB frequency without affecting the ability of the newly formed PB to secrete Abs. Consistent with the role of UTX and JMJD3 in promoting gene expression, the majority of differentially expressed were downregulated upon GSK-J4 treatment. GSK-J4-treated cells downregulated genes associated with signaling and P53 pathways. Inhibitor treated cells upregulated genes associated with cell cycle and proliferation, which correlated with an increase in actively proliferating cells. Unexpectedly, a majority of the downregulated transcripts corresponded to genes that in the wild-type setting were genes that gain H3K27me3 and downregulated in PB. Together, our results show that UTX and JMDJ3 are required to restrain B cell differentiation and suggest that they function as a rheostat for H3K27me3 to control this process.

An IRF4-MYC-mTORC1 Integrated Pathway Controls Cell Growth and the Proliferative Capacity of Activated B Cells during B Cell Differentiation In Vivo.

Cell division is an essential component of B cell differentiation to Ab-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell-specific deletion of IRF4. IRF4-deficient B cells responding to influenza, 4-hydroxy-3-nitrophenylacetyl-Ficoll, and LPS divided but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient cells was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4-MYC-mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.

Epigenetic gene regulation in plasma cells.

Humoral immunity provides protection from pathogenic infection and is mediated by antibodies following the differentiation of naive B cells (nBs) to antibody-secreting cells (ASCs). This process requires substantial epigenetic and transcriptional rewiring to ultimately repress the nB program and replace it with one conducive to ASC physiology and function. Notably, these reprogramming events occur within the framework of cell division. Efforts to understand the relationship of cell division with reprogramming and ASC differentiation in vivo have uncovered the timing and scope of reprogramming, as well as key factors that influence these events. Herein, we discuss the unique physiology of ASC and how nBs undergo epigenetic and genome architectural reorganization to acquire the necessary functions to support antibody production. We also discuss the stage-wise manner in which reprogramming occurs across cell divisions and how key molecular determinants can influence B cell fate outcomes.

Conserved Epigenetic Programming and Enhanced Heme Metabolism Drive Memory B Cell Reactivation.

Memory B cells (MBCs) have enhanced capabilities to differentiate to plasma cells and generate a rapid burst of Abs upon secondary stimulation. To determine if MBCs harbor an epigenetic landscape that contributes to increased differentiation potential, we derived the chromatin accessibility and transcriptomes of influenza-specific IgM and IgG MBCs compared with naive cells. MBCs possessed an accessible chromatin architecture surrounding plasma cell-specific genes, as well as altered expression of transcription factors and genes encoding cell cycle, chemotaxis, and signal transduction processes. Intriguingly, this MBC signature was conserved between humans and mice. MBCs of both species possessed a heightened heme signature compared with naive cells. Differentiation in the presence of hemin enhanced oxidative phosphorylation metabolism and MBC differentiation into Ab-secreting plasma cells. Thus, these data define conserved MBC transcriptional and epigenetic signatures that include a central role for heme and multiple other pathways in augmenting MBC reactivation potential.

EZH2 Represses the B Cell Transcriptional Program and Regulates Antibody-Secreting Cell Metabolism and Antibody Production.

Epigenetic remodeling is required during B cell differentiation. However, little is known about the direct functions of epigenetic enzymes in Ab-secreting cells (ASC) in vivo. In this study, we examined ASC differentiation independent of T cell help and germinal center reactions using mice with inducible or B cell-specific deletions of Ezh2 Following stimulation with influenza virus or LPS, Ezh2-deficient ASC poorly proliferated and inappropriately maintained expression of inflammatory pathways, B cell-lineage transcription factors, and Blimp-1-repressed genes, leading to fewer and less functional ASC. In the absence of EZH2, genes that normally gained histone H3 lysine 27 trimethylation were dysregulated and exhibited increased chromatin accessibility. Furthermore, EZH2 was also required for maximal Ab secretion by ASC, in part due to reduced mitochondrial respiration, impaired glucose metabolism, and poor expression of the unfolded-protein response pathway. Together, these data demonstrate that EZH2 is essential in facilitating epigenetic changes that regulate ASC fate, function, and metabolism.

Cortical Auditory Evoked Potentials in (Un)aided Normal-Hearing and Hearing-Impaired Adults.

Cortical auditory evoked potentials (CAEPs) are influenced by the characteristics of the stimulus, including level and hearing aid gain. Previous studies have measured CAEPs aided and unaided in individuals with normal hearing. There is a significant difference between providing amplification to a person with normal hearing and a person with hearing loss. This study investigated this difference and the effects of stimulus signal-to-noise ratio (SNR) and audibility on the CAEP amplitude in a population with hearing loss. Twelve normal-hearing participants and 12 participants with a hearing loss participated in this study. Three speech sounds-/m/, /g/, and /t/-were presented in the free field. Unaided stimuli were presented at 55, 65, and 75 dB sound pressure level (SPL) and aided stimuli at 55 dB SPL with three different gains in steps of 10 dB. CAEPs were recorded and their amplitudes analyzed. Stimulus SNRs and audibility were determined. No significant effect of stimulus level or hearing aid gain was found in normal hearers. Conversely, a significant effect was found in hearing-impaired individuals. Audibility of the signal, which in some cases is determined by the signal level relative to threshold and in other cases by the SNR, is the dominant factor explaining changes in CAEP amplitude. CAEPs can potentially be used to assess the effects of hearing aid gain in hearing-impaired users.

Prevalence and remediation of spatial processing disorder (SPD) in Indigenous children in regional Australia.

OBJECTIVE: This study aimed to determine the prevalence of spatial processing disorder (SPD) in the Indigenous Australian population and the benefit of and logistical issues arising from remediation of the disorder. DESIGN: Participants were assessed for SPD with the Listening in Spatialized Noise - Sentences test (LiSN-S). Participants diagnosed with SPD were instructed to use the LiSN & Learn auditory training software until 100 games had been completed. STUDY SAMPLE: Participants were 144 Indigenous Australian children (aged between 6;0 [years;months] and 12;2). RESULTS: Ten participants (6.9%) presented with SPD. Nine took part in the auditory training study. Post-training LiSN-S performance improved on average by 0.9 population standard deviations (1.4 dB). There was a significant correlation (r = 0.71, p = 0.031, η(2) = 0.51) between total number of LiSN & Learn games played (mean = 65, SD = 27) and improvement in LiSN-S performance. Teachers rated all participants as improving in their listening abilities post-intervention. CONCLUSIONS: There is a high prevalence of SPD in the Indigenous Australian population. LiSN & Learn training is effective in remediating SPD in this population and is considered a beneficial intervention by teachers, however improvement in spatial processing is dependent on training program uptake.

Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.

In fungi, transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-mannosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.

Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes. Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases. Moreover, disruption of the pmt1 gene also led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain was defective in septa formation, producing only half the number of septa per unit length of hypha compared with the wild type. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.

Assessing the role of evidence in patients' evaluation of complementary therapies: a quality study.

BACKGROUND: Making the decision to use complementary and alternative medicine (CAM) for cancer treatment is difficult in light of the limited available evidence for these treatments. It is unclear how patients use evidence to make these decisions. OBJECTIVES: (1) Describe the type of information about CAM that cancer patients use in their decision making; (2) understand why certain types of information about CAM are accepted as evidence by cancer patients; and (3) explore the role of scientific evidence in treatment decision making. METHODS: A qualitative study design using in-depth semistructured interviews with cancer patients attending 4 conventional and integrative health care institutions in Alberta and British Columbia, Canada, was used. RESULTS: Twenty-seven patients were interviewed. Patients sought CAM information from a range of sources, including the Internet, health care providers, friends, relatives, and newspapers. Many expressed frustration about the overwhelming amount of available information and found it difficult to identify reliable information. Information was described as reliable if it supported them in arriving at a decision about CAM. Types of information participants identified included anecdotes, expert opinion, gut feeling, popular literature, scientific evidence, testimonials, advertising and trial and error. Profound differences were found between new CAM users, experienced CAM users, and users with late-stage cancer in type of information sought, the role of scientific evidence in decision making, and overall information needs. CONCLUSION: Although this was a relatively small qualitative study, the results suggest that (1) many patients do not value scientific evidence as highly as conventional providers and (2) it is important for clinicians and other information providers to be aware of the different types of information that patients seek out and access when making choices and decisions regarding CAM treatments and why they seek out these sources.

Stroke, myocardial infarction, acute and chronic inflammatory diseases: caspases and other apoptotic molecules as targets for drug development.

Mapping of the human and other eukaryotic genomes has provided the pharmacological industry with excellent models for drug discovery. Control of cell proliferation, differentiation, activation and cell removal is crucial for the development and existence of multicellular organisms. Each cell cycle progression, with sequences of DNA replication, mitosis, and cell division, is a tightly controlled and complicated process that, when deregulated, may become dangerous not only to a single cell, but also to the whole organism. Regulation and the proper control of the cell cycle and of programmed cell death (apoptosis) is therefore essential for mammalian development and the homeostasis of the immune system. The molecular networks that regulate these processes are critical targets for drug development, gene therapy, and metabolic engineering. In addition to the primary, intracellular apoptotic suicide machinery, components of the immune system can detect and remove cells and tissue fragments that no longer serve their defined functions. In this review we will focus on apoptotic pathways converging on caspase family proteases, summarizing pharmacological attempts that target genes, proteins, and intermolecular interactions capable of modulating apoptosis and the inflammatory response. The upcoming pharmacological development for treatment of acute pathologies, such as sepsis, SIRS, stroke, traumatic brain injury, myocardial infarction, spinal cord injury, acute liver failure, as well as chronic disorders such as Huntington's disease, Parkinson's disease, ALS, and rheumatoid arthritis, will be discussed in details. We also suggest new potential molecular targets that may prove to be effective in controlling apoptosis and the immune response in vivo.