Hormone Transcription Research Articles

Limosilactobacillus reuteri promotes the expression and secretion of enteroendocrine- and enterocyte-derived hormones.

Observations that intestinal microbes can beneficially impact host physiology have prompted investigations into the therapeutic usage of such microbes in a range of diseases. For example, the human intestinal microbe Limosilactobacillus reuteri strains ATCC PTA 6475 and DSM 17938 are being considered for use for intestinal ailments including colic, infection, and inflammation as well as non-intestinal ailments including osteoporosis, wound healing, and autism spectrum disorder. While many of their beneficial properties are attributed to suppressing inflammatory responses in the gut, we postulated that L. reuteri may also regulate hormones of the gastrointestinal tract to affect physiology within and outside of the gut. To determine if L. reuteri secreted factors impact the secretion of enteric hormones, we treated an engineered jejunal organoid line, NGN3-HIO, which can be induced to be enriched in enteroendocrine cells, with L. reuteri 6475 or 17938 conditioned medium and performed transcriptomics. Our data suggest that these L. reuteri strains affect the transcription of many gut hormones, including vasopressin and luteinizing hormone subunit beta, which have not been previously recognized as being produced in the gut epithelium. Moreover, we find that these hormones appear to be produced in enterocytes, in contrast to canonical gut hormones which are produced in enteroendocrine cells. Finally, we show that L. reuteri conditioned media promotes the secretion of several enteric hormones including serotonin, GIP, PYY, vasopressin, and luteinizing hormone subunit beta. These results support L. reuteri affecting host physiology through intestinal hormone secretion, thereby expanding our understanding of the mechanistic actions of this microbe.

The SWI/SNF chromatin remodelling complex regulates pancreatic endocrine cell expansion and differentiation in mice in vivo.

Strategies to augment functional beta cell mass include directed differentiation of stem cells towards a beta cell fate, which requires extensive knowledge of transcriptional programs governing endocrine progenitor cell differentiation in vivo. We aimed to study the contributions of the Brahma-related gene-1 (BRG1) and Brahma (BRM) ATPase subunits of the SWI/SNF chromatin remodelling complex to endocrine cell development. We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1Δendo;Brm+/-) or homozygous (double knockout: DKOΔendo) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos. Brg1Δendo;Brm+/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKOΔendo mice were not recovered at weaning; however, postnatal day 6 DKOΔendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKOΔendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3)+ cells was reduced in Brg1Δendo;Brm+/- and DKOΔendo mutants. Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion. Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369.

Co-exposure to microplastic and plastic additives causes development impairment in zebrafish embryos

Since the run off of microplastic and plastic additives into the aquatic environment through the disposal of plastic products, we investigated the adverse effects of co-exposure to microplastics and plastic additives on zebrafish embryonic development. To elucidate the combined effects between microplastic mixtures composed of microplastics and plastic additives in zebrafish embryonic development, polystyrene (PS), bisphenol S (BPS), and mono-(2-ethylhexyl) phthalate (MEHP) were chosen as a target contaminant. Based on non-toxic concentration of each contaminant in zebrafish embryos, microplastic mixtures which is consisted of binary and ternary mixed forms were prepared. A strong phenotypic toxicity to zebrafish embryos was observed in the mixtures composed with non-toxic concentration of each contaminant. In particular, the mixture combination with ≤ EC10 values for BPS and MEHP showed a with a strong synergistic effect. Based on phenotypic toxicity to zebrafish embryos, change of transcription levels for target genes related to cell damage and thyroid hormone synthesis were analyzed in the ternary mixtures with low concentrations that were observed non-toxicity. Compared with the control group, cell damage genes linked to the oxidative stress response and thyroid hormone transcription factors were remarkably down-regulated in the ternary mixture-exposed groups, whereas the transcriptional levels of cyp1a1 and p53 were significantly up-regulated in the ternary mixture-exposed groups (P < 0.05). These results demonstrate that even at low concentrations, exposure to microplastic mixtures can cause embryonic damage and developmental malformations in zebrafish, depending on the mixed concentration-combination. Consequently, our findings will provide data to examine the action mode of zebrafish developmental toxicity caused by microplastic mixtures exposure composed with microplastics and plastic additives.

Introductory course-based research experience for university sophomores: Using testicular-feminized mice to identify genes underlying brain sexual differentiation

Like many educators, we are striving to incorporate more active learning and inquiry-driven exercises into our STEM curricula. Course-based undergraduate research experiences (CUREs) have become more common in undergraduate laboratory courses in many STEM fields. Here we detail a new CURE developed in a sophomore-level undergraduate molecular neuroendocrinology course at California State University Long Beach. The course centered on the mouse model carrying testicular feminization mutation ( Tfm). This mutation, resulting from a single base deletion, produces a nonfunctional androgen receptor (AR) protein. Thus, Tfm mice are insensitive to androgen through ARs, providing an excellent model to probe the physiological role of ARs in the development of the brain and behavior. Since AR is the main mediator of androgen activity by acting as a hormonal transcription regulator, gene expression profiling has been measured in the brains of Tfm mice and normal controls to identify the potential AR-regulated genes as downstream molecular effectors. Our CURE took a similar approach, aiming to determine (1) if the selected genes were expressed differentially between the sexes and (2) if their expression could be altered by loss of AR in specific brain regions. Three broad goals of having students think, communicate, and perform like scientists were organized in the learning objectives for the CURE. All participating students (n=21) were pursuing a biology or related major, and none had any experience conducting independent laboratory research. Here students first used polymerase chain reaction (PCR) genotyping to screen the mice carrying the Tfm mutation and their sexes. They next conducted a literature search and review, through which they learned how to select databases and how to combine key words to create a productive search string for identifying the potential AR-regulated genes. Last, students performed RT-qPCR in the wild-type and Tfm mouse hypothalamus and measured sex- and AR-dependent gene expression. These modules cover not only key concepts in genetics, molecular biology, and neuroendocrinology, but also the development of methods and techniques, including DNA/RNA extraction, electrophoresis, and RT-PCR/qPCR. We designed the CURE for a semester timeline with a single three-hour laboratory section meeting once a week for 15 weeks. All modules were taught and conducted in a research laboratory containing all materials and equipment necessary for research. At the end, students constructed and presented posters on their results. To assess how well students achieved the learning goals, we administered pre- and post-course surveys. Students reported striking gains related to the learning goals, with a high degree of satisfaction. Students also perceived the improved understanding of the scientific backgrounds and methods in the CURE. This CURE course illustrates that authentic research experiences, which use complex molecular techniques, can be offered to sophomore students. The course can serve as a template to ignite students’ intellectual curiosity and improve their understanding of the scientific process by engaging them in novel research. This work was supported by the Keck Undergraduate Research Experiences (KURE) Incubator funding. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeted Deletion of Pparβ/δ in Myeloid Cells Enhances Their Tumor-Promoting Function

Peroxisome proliferator activated receptors (PPARs) are transcription factors of the nuclear hormone transcription superfamily. Thee different PPAR subtypes have been described, PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ), with distinct tissue-specific allocations. All PPARs have been shown to be expressed in immune cells and, due to their ability to regulate lipid metabolism, these transcription factors can affect immune cell differentiation and function. While the effects of PPARα and PPARγ on M2 macrophage polarization are well established, the role of PPARβ/δ in myeloid cells is less studied. In the context of autoimmunity, it was shown that targeted deletion of PPARβ/δ in the myeloid compartment altered the Th1/Th17 polarization of CD4+ cells, and exacerbated experimental autoimmune encephalomyelitis in mice. In tumor mouse models, it was reported that M-CSF secreted by tumor cells could activate PPARβ/δ in tumor infiltrating myeloid cells, resulting in upregulation of genes responsible for their tumor-promoting properties such as IL-10, ARG-1 and VEGF. To investigate how PPARβ/δ can affect the myeloid cell responses in tumor immunity, we combined genetic and pharmacologic approaches. We generated mice with conditional targeting of Pparβ/δ gene and crossed them with LysMCre ( Pparβ/δ f/fLysMCre) to delete Pparβ/δ in myeloid cells. We used the MC38 colon adenocarcinoma syngeneic mouse tumor model and after implantation of tumor cells in Pparβ/δ f/fLysMCre and Pparβ/δ f/f control mice, we monitored tumor growth. Surprisingly, in contrast to what has been previously reported, we observed significantly larger and heavier tumors in PPAR β / δ f/fLysMCre mice compared to control mice, indicating that selective deletion of Pparβ/δ in the myeloid compartment was sufficient to impair anti-tumor immune responses in vivo. We assessed the intratumoral immune infiltrates including CD11b +F4/80 + tumor-associated macrophages (TAMs), CD11b +Ly6C hiLy6G - monocytic (M-MDSC), and CD11b +Ly6C loLy6G + polymorphonuclear (PMN-MDSC) MDSCs by flow cytometry. Although no numerical differences were detected in these populations between the two experimental groups, all myeloid cells isolated from tumor bearing Ppar β / δ f/fLysMCre mice demonstrated a less activated and pro-inflammatory phenotype, as indicated by lower levels of IFN-g expression. Notably, CD38 and CD88, two markers that have been associated with enhanced suppressive properties of MDSCs, were significantly upregulated in M-MDSCs and PMN-MDSC from Ppar β / δ f/fLysMCre tumor bearing mice compared to control Pparβ/δ f/f tumor-bearing mice. Hence, we sought to investigate how Pparβ/δ affects the suppressor properties of MDSCs. By using magnetic beads, we isolated GR1 + MDSC from the spleens of tumor-bearing Ppar β / δ f/fLysMCre and control mice and assessed suppression function in co-cultures with OTI TCR-transgenic T cells stimulated with cognate antigen, by DNA synthesis. Suppression capacity of MDSC from Ppar β / d f/fLysMCre tumor-bearing mice was significantly enhanced compared to MDSC from control tumor bearing mice. These results indicate that conditional deletion of Pparβ/δ in the myeloid cells prevented myeloid cell activation and augmented MDSCs suppressor properties by cell intrinsic mechanisms resulting in acceleration of tumor growth.

Effect of pregnancy on the expression of nutrient-sensors and satiety hormones in mice

Small intestinal satiation pathways involve nutrient-induced stimulation of chemoreceptors leading to release of satiety hormones from intestinal enteroendocrine cells (ECCs). Whether adaptations in these pathways contribute to increased maternal food intake during pregnancy is unknown.To determine the expression of intestinal nutrient-sensors and satiety hormone transcripts and proteins across pregnancy in mice.Female C57BL/6J mice (10–12 weeks old) were randomized to mating and then tissue collection at early- (6.5 d), mid- (12.5 d) or late-pregnancy (17.5 d), or to an unmated age matched control group. Relative transcript expression of intestinal fatty acid, peptide and amino acid and carbohydrate chemoreceptors, as well as gut hormones was determined across pregnancy. The density of G-protein coupled receptor 93 (GPR93), free fatty acid receptor (FFAR) 4, cholecystokinin (CCK) and glucagon-like peptide1 (GLP-1) immunopositive cells was then compared between non-pregnant and late-pregnant mice.Duodenal GPR93 expression was lower in late pregnant than non-pregnant mice (P < 0.05). Ileal FFAR1 expression was higher at mid- than at early- or late-pregnancy. Ileal FFAR2 expression was higher at mid-pregnancy than in early pregnancy. Although FFAR4 expression was consistently lower in late-pregnant than non-pregnant mice (P < 0.001), the density of FFAR4 immunopositive cells was higher in the jejunum of late-pregnant than non-pregnant mice.A subset of protein and fatty acid chemoreceptor transcripts undergo region-specific change during murine pregnancy, which could augment hormone release and contribute to increased food intake. Further investigations are needed to determine the functional relevance of these changes.

Palmitoyl acyltransferase ZDHHC7 inhibits androgen receptor and suppresses prostate cancer.

The hormonal transcription factor androgen receptor (AR) is a master regulator of prostate cancer (PCa). Protein palmitoylation, which attaches a palmitate fatty acid to a substrate protein, is mediated by a class of 23 ZDHHC (Zinc-Finger DHHC motif)-family palmitoyltransferases. Although palmitoylation has been shown to modify many proteins and regulate diverse cellular processes, little is known about ZDHHC genes in cancer. Here we examined ZDHHC family gene expression in human tissue panels and identified ZDHHC7 as a PCa-relevant member. RNA-seq analyses of PCa cells with ZDHHC7 de-regulation revealed global alterations in androgen response and cell cycle pathways. Mechanistically, ZDHHC7 inhibits AR gene transcription and therefore reduces AR protein levels and abolishes AR signaling in PCa cells. Accordingly, ZDHHC7 depletion increased the oncogenic properties of PCa cells, whereas restoring ZDHHC7 is sufficient to suppress PCa cell proliferation and invasion in vitro and mitigate xenograft tumor growth in vivo. Lastly, we demonstrated that ZDHHC7 is downregulated in human PCa compared to benign-adjacent tissues, and its loss is associated with worse clinical outcomes. In summary, our study reveals a global role of ZDHHC7 in inhibiting androgen response and suppressing PCa progression and identifies ZDHHC7 loss as a biomarker for aggressive PCa and a target for therapeutic intervention.

Stimulation of shrimp (Penaeus monodon) hemocytes by lipopolysaccharide-like molecules derived from Novacq™

Immune stimulation through feed additives is a promising strategy that can help to combat disease in shrimp farming and reduce the use of antibiotics and other chemotherapeutics. The present study investigated the in vitro immunostimulatory effects of lipopolysaccharide (LPS)-like molecules isolated from the microbial based feed additive Novacq™ (N-LPS). The presence of LPS-like molecules was confirmed and quantified Novacq™ using a HEK-TLR4 reporter cell line. Primary hemocytes isolated from adult Penaeus monodon were used to measure the immunostimulatory of N-LPS compared with the control group that were treated with E. coli derived LPS (E-LPS). The N-LPS stimulated a rapid and significant induction of the phenoloxidase (PO) response in the hemocytes. The PO response increased with exposure time and LPS concentration and was significantly higher compared with an E. coli LPS (E-LPS) control. In addition, using gene expression data, we quantified the transcriptome response of the hemocytes at 15, 30 and 60 mins post stimulation. Compared with the controls, the N-LPS treated hemocytes had a significant up-regulation of genes involved in the immune system modulation and control at all time-points. Most noteworthy was the significant induction of transcripts that function as serine protease inhibitors (namely SERPINs), that regulate the overexpression of the PO system. Transcription factors from the Notch family which directly regulate the expression of many immune genes were also induced within the hemocytes. Additionally, we also saw a strong up-regulation of crustacean hyperglycemic hormone (CHH) transcripts, an important neuropeptide involved in immune function. Overall, the transcriptome profile of the hemocytes suggests that the LPS component of Novacq™ is highly immunostimulatory and generates a strong PO response in vitro. The subsequent transcriptional response appears to be directed towards preventing further activation of the PO system most likely in an attempt to limit cytoxicity to the host. Our study highlights the immunostimulatory ability of Novacq™ and provides further evidence of the positive health benefits this microbial based feed additive can have in shrimp.

Crustacean Female Sex Hormone (CFSH) Transcript and Protein Profiles and Its Functions in Gradually Developing Adult-Specific-Features during the Prepuberty Molt Cycle of the Blue Crab, Callinectes sapidus

Crustacean female sex hormone (CFSH) plays a pivotal role in developing adult-specific female characteristics, specifically the mating and parental care system of the female blue crab, Callinectes sapidus, manifested after the puberty-terminal molt. Therefore, this study aimed to determine if there is a relationship between CFSH and the development of phenotypes specific to the female C. sapidus during its life cycle and if the continued presence of CFSH is required for developing adult-specific features during the prepuberty molt cycle. First, CasCFSH transcripts in females steadily increase, starting from crab stages 3-4 to prepuberty, except for a decrease at crab stages 15-16. The prepuberty females have the highest CasCFSH transcripts, with ∼5.6-fold higher than adults. CFSH protein levels at prepuberty are the highest but similar to adults. The first and visible secondary female characteristics include a pair of gonopores or a triangular abdomen and four pairs of pleopods seen as early as crab stages at ∼3 (carapace width, CW, 5-7 mm) - 5 (CW 8-10 mm), respectively. The adult-specific female morphological features that are developed gradually throughout the prepuberty molt cycle are manifested only after the puberty-terminal molt. A CasCFSH knockdown study by injecting CasCFSH-dsRNA into prepuberty females at specific molt stages provides further evidence: the continued presence of CasCFSH is required for gradually developing adult-specific morphological features during the prepuberty molt cycle. Overall, the abdomen size is first determined at the intermolt stage. Then, the abdominal shape, gonopores, sternal tubercles, spermathecae, and ovigerous setae and hairs are formed at the intermolt and early premolt stages. Finally, the plumose hairs are developed during intermolt, early, and late premolt stages.

CRHR1 mediates the transcriptional expression of pituitary hormones and their receptors under hypoxia.

Hypothalamus-pituitary-adrenal (HPA) axis plays critical roles in stress responses under challenging conditions such as hypoxia, via regulating gene expression and integrating activities of hypothalamus-pituitary-targets cells. However, the transcriptional regulatory mechanisms and signaling pathways of hypoxic stress in the pituitary remain to be defined. Here, we report that hypoxia induced dynamic changes in the transcription factors, hormones, and their receptors in the adult rat pituitary. Hypoxia-inducible factors (HIFs), oxidative phosphorylation, and cAMP signaling pathways were all differentially enriched in genes induced by hypoxic stress. In the pituitary gene network, hypoxia activated c-Fos and HIFs with specific pituitary transcription factors (Prop1), targeting the promoters of hormones and their receptors. HIF and its related signaling pathways can be a promising biomarker during acute or constant hypoxia. Hypoxia stimulated the transcription of marker genes for microglia, chemokines, and cytokine receptors of the inflammatory response. Corticotropin-releasing hormone receptor 1 (CRHR1) mediated the transcription of Pomc, Sstr2, and Hif2a, and regulated the function of HPA axis. Together with HIF, c-Fos initiated and modulated dynamic changes in the transcription of hormones and their receptors. The receptors were also implicated in the regulation of functions of target cells in the pituitary network under hypoxic stress. CRHR1 played an integrative role in the hypothalamus-pituitary-target axes. This study provides new evidence for CRHR1 involved changes of hormones, receptors, signaling molecules and pathways in the pituitary induced by hypoxia.

Investigation of the optimal culture time for warmed bovine ovarian tissues before transplantation†.

Ovarian tissue cryopreservation by vitrification is an effective technique, but there are still many unresolved issues related to the procedure. The aim of this study was to investigate the optimal culture time of postwarmed ovarian tissues and their viability before ovarian tissue transplantation. The bovine ovarian tissues were used to evaluate the effect of postwarming culture periods (0, 0.25, 0.5, 1, 2, 5, and 24h) in the levels of residual cryoprotectant, LDH release, ROS generation, gene and protein abundance, and follicle viability and its mitochondrial membrane potential. Residual cryoprotectant concentration decreased significantly after 1h of culture. The warmed ovarian tissues that underwent between 0 and 2h of culture time showed similar LDH and ROS levels compared with fresh nonfrozen tissues. The anti-Mullerian hormone transcript abundance did not differ in any of the groups. No increase in the relative transcript abundance and protein level of Caspase 3 and Cleaved-Caspase 3, respectively, in the first 2h of culture after warming. On the other hand, an increased protein level of double stranded DNA breaks (gamma-H2AX) was observed in postwarmed tissues disregarding the length of culture time, and a temporary reduction in pan-AKT was detected in postwarming tissues between 0 and 0.25h of culture time. Prolonged culture time lowered the percentage of viable follicles in warmed tissues, but it did not seem to affect the follicular mitochondrial membrane potential. In conclusion, 1-2h of culture time would be optimal for vitrified-warmed tissues before transplantation.

Molecular identification and putative role of insulin growth factor binding protein-related protein (IGFBP-rp) in the swimming crab Portunus trituberculatus

The insulin-like growth factor/insulin-like polypeptide (IGF/ILP) signaling is vital for growth, physiological metabolism, development, and reproduction. Insulin-like growth factor-binding protein (IGFBP) is involved in the insulin signaling pathway in both vertebrates and invertebrates and is critical for various physiology functions. Herein, we cloned and characterized the full-length cDNA of IGFBP-rp in the swimming crab, Portunus trituberculatus (PtIGFBP-rp). The deduced amino acid sequence of PtIGFBP-rp was found to contain three key domains (insulin-like binding (IB) domain, the kazale-type serine protease inhibitor (KAZAL) domain, and the immunoglobulin-like C2 (IGc2) domain). Results showed that PtIGFBP-rp shared the same expression pattern as P. trituberculatus insulin androgenic gland hormone (PtIAG) transcripts during the embryonic larval, juvenile crab stage and the androgenic gland (AG) developmental cycle. Moreover, PtIGFBP-rp transcripts were also present in high abundance in hepatopancreas, muscle, and androgenic glands. The regulatory relationship between PtIGFBP-rp and PtIAG was investigated by RNA interference and co-localization assays, which showed a co-localization relationship and feedback regulation between them. Bilateral eye stalk ablation (ESA) increased the expression of PtIGFBP-rp in the AG at 7 d after surgery. These results demonstrate the involvement of PtIGFBP-rp in the signaling regulatory network of IAG in P. trituberculatus.

Identification of Potential Pathways of Morella cerifera Seedlings in Response to Alkali Stress via Transcriptomic Analysis.

Alkali stress, a type of abiotic stress, severely inhibits plant growth. Only a few studies have investigated the mechanism underlying the transcriptional-level response of Morella cerifera to saline-alkali stress. Based on RNA-seq technology, gene expression differences in the fibrous roots of M. cerifera seedlings exposed to low- and high-concentration alkali stress (LAS and HAS, respectively) were investigated, and the corresponding 1312 and 1532 alkali stress-responsive genes were identified, respectively. According to gene set enrichment analysis, 65 gene sets were significantly enriched. Of these, 24 gene sets were shared by both treatment groups. LAS and HAS treatment groups exhibited 9 (all downregulated) and 32 (23 downregulated) unique gene sets, respectively. The differential gene sets mainly included those involved in trehalose biosynthesis and metabolism, phospholipid translocation, and lignin catabolism. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that M. cerifera seedlings were specifically enriched in stilbenoid, diarylheptanoid, and gingerol biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; and sesquiterpenoid and triterpenoid biosynthesis. Moreover, the related genes involved in hormone signaling pathways and transcription factors were determined through a localization analysis of core abiotic stress pathways. These genes and their molecular mechanisms will be the focus of future research.

Origin and evolutionary landscape of Nr2f transcription factors across Metazoa.

Nuclear Receptor Subfamily 2 Group F (Nr2f) orphan nuclear hormone transcription factors (TFs) are fundamental regulators of many developmental processes in invertebrates and vertebrates. Despite the importance of these TFs throughout metazoan development, previous work has not clearly outlined their evolutionary history. We integrated molecular phylogeny with comparisons of intron/exon structure, domain architecture, and syntenic conservation to define critical evolutionary events that distinguish the Nr2f gene family in Metazoa. Our data indicate that a single ancestral eumetazoan Nr2f gene predated six main Bilateria subfamilies, which include single Nr2f homologs, here referred to as Nr2f1/2/5/6, that are present in invertebrate protostomes and deuterostomes, Nr2f1/2 homologs in agnathans, and Nr2f1, Nr2f2, Nr2f5, and Nr2f6 orthologs that are found in gnathostomes. Four cnidarian Nr2f1/2/5/6 and three agnathan Nr2f1/2 members are each due to independent expansions, while the vertebrate Nr2f1/Nr2f2 and Nr2f5/Nr2f6 members each form paralogous groups that arose from the established series of whole-genome duplications (WGDs). Nr2f6 members are the most divergent Nr2f subfamily in gnathostomes. Interestingly, in contrast to the other gnathostome Nr2f subfamilies, Nr2f5 has been independently lost in numerous vertebrate lineages. Furthermore, our analysis shows there are differential expansions and losses of Nr2f genes in teleosts following their additional rounds of WGDs. Overall, our analysis of Nr2f gene evolution helps to reveal the origins and previously unrecognized relationships of this ancient TF family, which may allow for greater insights into the conservation of Nr2f functions that shape Metazoan body plans.

Use of Transcriptomic Analyses to Elucidate the Mechanism Governing Nodal Root Development in Eremochloa ophiuroides (Munro) Hack.

Centipedegrass [Eremochloa ophiuroides (Munro) Hack.] is a perennial warm-season grass that originated in China, and its speed of nodal rooting is important for lawn establishment. In our study, centipedegrass nodal rooting ability was limited by node aging. Transcriptome sequencing of nodal roots after 0, 2, 4, and 8 days of water culture was performed to investigate the molecular mechanisms of root development. GO enrichment and KEGG pathway analyses of DEGs indicated that plant hormone signal transduction and transcription factors might play important roles in centipedegrass nodal root growth. Among them, E3 ubiquitin-protein ligases participated in multiple hormone signal transduction pathways and interacted with transcription factors. Furthermore, an E3 ubiquitin protein ligase EoSINAT5 overexpressed in rice resulted in longer roots and more numerous root tips, while knockout of LOC_Os07g46560 (the homologous gene of EoSINAT5 in rice) resulted in shorter roots and fewer root tips. These results indicated that EoSINAT5 and its homologous gene are able to promote nodal root development. This research presents the transcriptomic analyses of centipedegrass nodal roots, and may contribute to elucidating the mechanism governing the development of nodal roots and facilitates the use of molecular breeding in improving rooting ability.

Comprehensive transcriptional analysis reveals salt stress-regulated key pathways, hub genes and time-specific responsive gene categories in common bermudagrass (Cynodon dactylon (L.) Pers.) roots

BackgroundDespite its good salt-tolerance level, key genes and pathways involved with temporal salt response of common bermudagrass (Cynodon dactylon (L.) Pers.) have not been explored. Therefore, in this study, to understand the underlying regulatory mechanism following the different period of salt exposure, a comprehensive transcriptome analysis of the bermudagrass roots was conducted.ResultsThe transcripts regulated after 1 h, 6 h, or 24 h of hydroponic exposure to 200 mM NaCl in the roots of bermudagrass were investigated. Dataset series analysis revealed 16 distinct temporal salt-responsive expression profiles. Enrichment analysis identified potentially important salt responsive genes belonging to specific categories, such as hormonal metabolism, secondary metabolism, misc., cell wall, transcription factors and genes encoded a series of transporters. Weighted gene co-expression network analysis (WGCNA) revealed that lavenderblush2 and brown4 modules were significantly positively correlated with the proline content and peroxidase activity and hub genes within these two modules were further determined. Besides, after 1 h of salt treatment, genes belonging to categories such as signalling receptor kinase, transcription factors, tetrapyrrole synthesis and lipid metabolism were immediately and exclusively up-enriched compared to the subsequent time points, which indicated fast-acting and immediate physiological responses. Genes involved in secondary metabolite biosynthesis such as simple phenols, glucosinolates, isoflavones and tocopherol biosynthesis were exclusively up-regulated after 24 h of salt treatment, suggesting a slightly slower reaction of metabolic adjustment.ConclusionHere, we revealed salt-responsive genes belonging to categories that were commonly or differentially expressed in short-term salt stress, suggesting possible adaptive salt response mechanisms in roots. Also, the distinctive salt-response pathways and potential salt-tolerant hub genes investigated can provide useful future references to explore the molecular mechanisms of bermudagrass.

Comparative transcriptome analysis reveals novel insights into transcriptional responses to phosphorus starvation in oil palm (Elaeis guineensis) root

BackgroundPhosphorus (P), in its orthophosphate form (Pi) is an essential macronutrient for oil palm early growth development in which Pi deficiency could later on be reflected in lower biomass production. Application of phosphate rock, a non-renewable resource has been the common practice to increase Pi accessibility and maintain crop productivity in Malaysia. However, high fixation rate of Pi in the native acidic tropical soils has led to excessive utilization of P fertilizers. This has caused serious environmental pollutions and cost increment. Even so, the Pi deficiency response mechanism in oil palm as one of the basic prerequisites for crop improvement remains largely unknown.ResultsUsing total RNA extracted from young roots as template, we performed a comparative transcriptome analysis on oil palm responding to 14d and 28d of Pi deprivation treatment and under adequate Pi supply. By using Illumina HiSeq4000 platform, RNA-Seq analysis was successfully conducted on 12 paired-end RNA-Seq libraries and generated more than 1.2 billion of clean reads in total. Transcript abundance estimated by fragments per kilobase per million fragments (FPKM) and differential expression analysis revealed 36 and 252 genes that are differentially regulated in Pi-starved roots at 14d and 28d, respectively. Genes possibly involved in regulating Pi homeostasis, nutrient uptake and transport, hormonal signaling and gene transcription were found among the differentially expressed genes.ConclusionsOur results showed that the molecular response mechanism underlying Pi starvation in oil palm is complexed and involved multilevel regulation of various sensing and signaling components. This contribution would generate valuable genomic resources in the effort to develop oil palm planting materials that possess Pi-use efficient trait through molecular manipulation and breeding programs.

Protein hormone fragmentation in intercellular signaling: hormones as nested information systems.

This study explores the hypothesis that protein hormones are nested information systems in which initial products of gene transcription, and their subsequent protein fragments, before and after secretion and initial target cell action, play additional physiological regulatory roles. The study produced four tools and key results: (1) a problem approach that proceeds, with examples and suggestions for in vivo organismal functional tests for peptide-protein interactions, from proteolytic breakdown prediction to models of hormone fragment modulation of protein-protein binding motifs in unrelated proteins; (2) a catalog of 461 known soluble human protein hormones and their predicted fragmentation patterns; (3) an analysis of the predicted proteolytic patterns of the canonical protein hormone transcripts demonstrating near-universal persistence of 9±7 peptides of 8±8 amino acids even after cleavage with 24 proteases from four protease classes; and (4) a coincidence analysis of the predicted proteolysis locations and the 1939 exon junctions within the transcripts that shows an excess (P<0.001) of predicted proteolysis within 10 residues, especially at the exonal junction (P<0.01). It appears all protein hormone transcripts generate multiple fragments the size of peptide hormones or protein-protein binding domains that may alter intracellular or extracellular functions by acting as modulators of metabolic enzymes, transduction factors, protein binding proteins, or hormone receptors. High proteolytic frequency at exonal junctions suggests proteolysis has evolved, as a complement to gene exon fusion, to extract structures or functions within single exons or protein segments to simplify the genome by discarding archaic one-exon genes.

Impact of high-fat diet on lifespan, metabolism, fecundity and behavioral senescence in Drosophila

Excess consumption of high-fat diet (HFD) is likely to result in obesity and increases the predisposition to associated health disorders. Drosophila melanogaster has emerged as an important model to study the effects of HFD on metabolism, gut function, behavior, and ageing. In this study, we investigated the effects of HFD on physiology and behavior of female flies at different time-points over several weeks. We found that HFD decreases lifespan, and also with age leads to accelerated decline of climbing ability in both virgins and mated flies. In virgins HFD also increased sleep fragmentation with age. Furthermore, long-term exposure to HFD results in elevated adipokinetic hormone (AKH) transcript levels and an enlarged crop with increased lipid stores. We detected no long-term effects of HFD on body mass, or levels of triacylglycerides (TAG), glycogen or glucose, although fecundity was diminished. However, one week of HFD resulted in decreased body mass and elevated TAG levels in mated flies. Finally, we investigated the role of AKH in regulating effects of HFD during aging. Both with normal diet (ND) and HFD, Akh mutant flies displayed increased longevity compared to control flies. However, both mutants and controls showed shortened lifespan on HFD compared to ND. In flies exposed to ND, fecundity is decreased in Akh mutants compared to controls after one week, but increased after three weeks. However, HFD leads to a similar decrease in fecundity in both genotypes after both exposure times. Thus, long-term exposure to HFD increases AKH signaling, impairs lifespan and fecundity and augments age-related behavioral senescence.

Piperine sensitizes radiation-resistant cancer cells towards radiation and promotes intrinsic pathway of apoptosis.

Piperine, a bioactive alkaloid, is known to have anticancer activities. Hence, in this study, the effectiveness of piperine pretreatment as a strategy for radio-sensitizing colorectal adenocarcinoma cell line (HT-29) was analyzed. For this, HT-29 cells were pretreated with piperine (12.5 and 25µg/mL) and exposed to γ-radiation (1.25Gy) and analyzed for various effector pathways to elucidate the possible mode of action in comparison to individual treatments. The proliferation efficiency of the cells was analyzed by trypan blue dye exclusion assay and MTT assay. The synergistic effects of the combination treatment were analyzed with compuSyn software. Downstream signaling pathways leading to apoptosis were studied using flowcytometry, immunofluorescence, and immunoblot assays. It was observed that combination treatment arrested HT-29 cells at G2/M phase nearly 2.8 folds higher than radiation treatment alone, inducing the radio-resistant cells to undergo apoptosis through mitochondria-dependent pathway. In addition, activation of caspase-3 and cleavage of poly(ADP-ribose) polymerases-1, the key molecular events in apoptotic signaling, were significantly enhanced. Activation of estrogen receptor beta (ERβ), a nuclear hormone transcription factor promoting tumor suppression represents a novel clinical advance towards management and prevention of cancers. Interestingly, the expression of ERβ was increased in the cells treated with piperine. In conclusion, piperine pretreatment enhances radio-sensitization in HT-29 cells by inducing the cells to undergo apoptosis hence, can be used as a classic candidate for colon cancer sensitization towards radiotherapy. PRACTICAL APPLICATION: Piperine induces enhanced radiosensitization of colon cancer cell line (HT-29) by interfering with the cancer cell line proliferation, DNA damage, and apoptosis.