mRNA Transcripts Research Articles

Molecular cloning and functional characterization of eight Galectin genes from Takifugu obscurus

Galectins are a family of animal lectins involved in the immune response against pathogens. However, the roles of fish galectins during pathogen infection require comprehensive studies. In the present research, eight different galectin genes from Takifugu obscurus (named ToGalec1-8) were identified and characterized. ToGalec1-8 encoded proteins of 240, 182, 373, 145, 452, 135, 359 and 346 amino acids, respectively. All predicted ToGalec1-8 proteins possessed one or more conserved carbohydrate recognition domains (CRDs). Phylogenetic analysis revealed that ToGalec1-8 were evolutionarily closely related to their counterparts in other selected vertebrates, hinting their genetic relationship. Tissue distribution analysis showed that most ToGalec genes were distributed ubiquitously in all detected tissues, with relatively high expression in immune tissues. After stimulation by Vibrio harveyi and Staphylococcus aureus, the mRNA transcripts of ToGalec1-8 in liver and kidney were significantly upregulated. In addition, RNA interference experiments showed that knockdown of ToGalec1 and ToGalec7 inhibited the clearance of bacteria in vivo. Taken together, these obtained results suggested that ToGalec1-8 play an important role in innate immunity and defense against bacterial infection in T. obscurus.

The hidden architects of the genome: a comprehensive review of R-loops.

Three-stranded DNA: RNA hybrids known as R-loops form when the non-template DNA strand is displaced and the mRNA transcript anneals to its template strand. Although R-loop formation controls DNA damage response, mitochondrial and genomic transcription, and physiological R-loop formation, imbalanced formation of R-loop can jeopardize a cell's genomic integrity. Transcription regulation and immunoglobulin class switch recombination are two further specialized functions of genomic R-loops. R-loop formation has a dual role in the development of cancer and disturbed R-loop homeostasis as observed in several malignancies. R-loops transcribe at the telomeric and pericentromeric regions, develop in the space between long non-coding RNAs and telomeric repeats, and shield telomeres. In bacteria and archaea, R-loop development is a natural defence mechanism against viruses which also causes DNA degradation. Their emergence in the mammalian genome is controlled, suggesting that they were formed as an inevitable byproduct of RNA transcription but also co-opted for regulatory functions. R-loops may be engaged in cell physiology by regulating gene expression. R-loop biology is probably going to remain a fascinating field of study for a very long time as it offers many avenues for R-loop research.

A multidimensional recommendation framework for identifying biological targets to aid the diagnosis and treatment of liver metastasis in patients with colorectal cancer

The quest to understand the molecular mechanisms of tumour metastasis and identify pivotal biomarkers for cancer therapy is increasing in importance. Single-omics analyses, constrained by their focus on a single biological layer, cannot fully elucidate the complexities of tumour molecular profiles and can thus overlook crucial molecular targets. In response to this limitation, we developed a multiobjective recommendation system (RJH-Metastasis 1.0) anchored in a multiomics knowledge graph to integrate genome, transcriptome, and proteome data and corroborative literature evidence and then conducted comprehensive analyses of colorectal cancer with liver metastasis (CRCLM). A total of 25 key genes significantly associated with CRCLM were recommended by our system, and GNB1, GATAD2A, GBP2, MACROD1, and EIF5B were further highlighted. Specifically, GNB1 presented fewer mutations but elevated RNA transcription and protein expression in CRCLM patients. The role of GNB1 in promoting the malignant behaviours of colon cancer cells was demonstrated via in vitro and in vivo studies. Aberrant expression of GNB1 could be regulated by METTL1-driven m7G modification. METTL1 knockdown decreased m7G modification in the 3’ UTR of GNB1, increasing its mRNA transcription and translation during liver metastasis. Furthermore, GNB1 induced the formation of an immunosuppressive microenvironment by promoting the CLEC2C-KLRB1 interaction between memory B cells and KLRB1+PD-1+CD8+ cells. GNB1 expression and the efficacy of PD-1 antibody-based treatment in CRCLM patients were significantly correlated. In summary, our recommendation system can be used for effective exploration of key molecules in colorectal cancer, among which GNB1 was identified as a critical CRCLM promoter and immunotherapy biomarker in colorectal cancer patients.

Codon optimality influences homeostatic gene expression in zebrafish.

The ribosome plays a crucial role in translating mRNA into protein; however, the genetic code extends beyond merely specifying amino acids. Upon translation, codons, the three-nucleotide sequences interpreted by ribosomes, have regulatory properties affecting mRNA stability, a phenomenon known as codon optimality. Codon optimality has been previously observed in vertebrates during embryogenesis, where specific codons can influence the stability and degradation rates of mRNA transcripts. In our previous work, we demonstrated that codon optimality impacts mRNA stability in human cell lines. However, the extent to which codon content influences vertebrate gene expression in vivo remained unclear. In this study, we expand on our previous findings by demonstrating that codon optimality has a robust effect on homeostatic mRNA and protein levels in whole zebrafish during normal physiological conditions. Using reporters with nearly identical nucleotide sequences but different codon compositions, all expressed from the same genomic locus, we show that codon composition can significantly influence gene expression. This study provides new insights into the regulatory roles of codon usage in vertebrate gene expression and underscores the importance of considering codon optimality in genetic and translational research. These findings have broad implications for understanding the complexities of gene regulation and could inform the design of synthetic genes and therapeutic strategies targeting mRNA stability.

Tissue oxidative stress and expression of chicken UCP and ANT mRNA in laying hens exposed to acute cold stress

ABSTRACT 1. Exposure to stress alters normal homoeostasis and, hence, the antioxidant defence system. The aim of this study was to examine the effect of acute cold temperature on the antioxidant defence system in hens. 2. Hy-line grey commercial layers (80 40-week-old) were randomly assigned to one of eight groups. In groups 1 to 5, hens were exposed to low temperature at −8.68°C (cool stressed) for 2, 4, 6, 8 and 10 h, respectively. In groups 6 and 7, post 10 h cool stressed, hens were quickly transferred to room at 21°C to recovery for 2 h and 4h, respectively. In treatment groups 6 and 7, post 10 h cool stressed, hens were quickly transferred to room at 21°C for 2 h and 4 h, respectively. Group 8 was the control, where hens were housed under regular condition at 21°C as controls. 3. Antioxidant enzymes (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA), in skeletal muscle, the kidney, liver and pancreas were measured. The transcription of avUCP and ANT mRNA was tested by RT-PCR. 4. The T-AOC activity was increased in the skeletal muscle of hens cold stressed for 2, 4, 6, 8 and 10 h and the 2 h recovery groups compared with control hens (p < 0.05). The GPx activity was increased in the liver and skeletal muscle after cold stress 4 h and in the pancreas of cold stress 2 h compared with the control group (p < 0.05). Antioxidant SOD activity was increased in the kidney after cold stress 6 h and in the liver after cold stress 10 h compared to the control group (p < 0.05). Measured MDA activity was increased in the pancreas after 2 h cold stress (p < 0.05). 5. UCP mRNA expression level was increased in the pectoral muscle for 2 h and 4 h recovery groups compared with the control hens (p < 0.05) and avian uncoupling protein (UPC), adenine nucleotide translocator (ANT) expression level was increased in the leg muscle of hens cold stress for 2, 6, 8 h and recovery 2 and 4 h. 6. The observed changes in the antioxidant defence system were tissue specific. Increments in levels of ANT (leg muscle) and UCP (pectoral and leg muscle) mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.

Differential Expression of Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) and Interleukin12B (IL12B) in the Bursa of Fabricius of chicken infected with Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) caused by Infectious Bursal Disease virus (IBDV), poses a significant threat to the poultry industry, causing immunosuppression and economic losses since its discovery in 1957. To understand the host response to IBDV infection, this study examined the modulation of MAP2K3 and IL12B in chickens infected with IBDV. A local IBDV isolate propagated in embryonated chicken eggs was inoculated into 18-day-old chicken. The mRNA transcription levels of MAP2K3 and IL12B in bursal tissue were quantified by real-time quantitative polymerase chain reaction on day two post-infection and compared with those of uninfected, age-matched control birds (sham inoculated). The study revealed increased expression of the MAP2K3 and IL12B genes, indicating their role in inflammatory response during IBDV infection. Thus, the present investigation sheds light on the involvement of MAP2K3 and IL12B in the inflammatory response to IBDV infection in chickens. Keywords: Infectious bursal disease virus, immunosuppression, MAP2K3, IL12B, pro-inflammatory cytokine gene expression

The dynamics and functional impact of tRNA repertoires during early embryogenesis in zebrafish.

Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis.

β2-integrins control HIF1α activation in human neutrophils.

During inflammation, human neutrophils engage β2-integrins to migrate from the blood circulation to inflammatory sites with high cytokine but low oxygen concentrations. We tested the hypothesis that the inhibition of prolyl hydroxylase domain-containing enzymes (PHDs), cytokines, and β2-integrins cooperates in HIF pathway activation in neutrophils. Using either the PHD inhibitor roxadustat (ROX) (pseudohypoxia) or normobaric hypoxia to stabilize HIF, we observed HIF1α protein accumulation in adherent neutrophils. Several inflammatory mediators did not induce HIF1α protein but provided additive or even synergistic signals (e.g., GM-CSF) under pseudohypoxic and hypoxic conditions. Importantly, and in contrast to adherent neutrophils, HIF1α protein expression was not detected in strictly suspended neutrophils despite PHD enzyme inhibition and the presence of inflammatory mediators. Blocking β2-integrins in adherent and activating β2-integrins in suspension neutrophils established the indispensability of β2-integrins for increasing HIF1α protein. Using GM-CSF as an example, increased HIF1α mRNA transcription via JAK2-STAT3 was necessary but not sufficient for HIF1α protein upregulation. Importantly, we found that β2-integrins led to HIF1α mRNA translation through the phosphorylation of the essential translation initiation factors eIF4E and 4EBP1. Finally, pseudohypoxic and hypoxic conditions inducing HIF1α consistently delayed apoptosis in adherent neutrophils on fibronectin under low serum concentrations. Pharmacological HIF1α inhibition reversed delayed apoptosis, supporting the importance of this pathway for neutrophil survival under conditions mimicking extravascular sites. We describe a novel β2-integrin-controlled mechanism of HIF1α stabilization in human neutrophils. Conceivably, this mechanism restricts HIF1α activation in response to hypoxia and pharmacological PHD enzyme inhibitors to neutrophils migrating toward inflammatory sites.

The Drosophila EcR-Hippo component Taiman promotes epithelial cell fitness by control of the Dally-like glypican and Wg gradient.

Rapidly dividing cells can eliminate slow growing neighbors through the apoptotic process of cell competition. This process ensures that only high fitness cells populate embryonic tissues and is proposed to underlie the ability of oncogene-transformed cells to progressively replace normal cells within a tissue. Patches of cells in the Drosophila wing disc overexpressing the oncogenic Taiman (Tai) transcriptional coactivator kill normal neighbors by secreting Spz ligands that trigger pro-apoptotic Toll signaling in receiving cells. However, extracellular signaling mechanisms responsible for elimination of slow growing cells by normal neighbors remain poorly defined. Here we show that slow growing cells with reduced Tai (Tai low ) are killed by normal neighbors through a mechanism involving competition for the Wingless (Wg/Wnt) ligand. Elevated Wg signaling significantly rescues elimination of Tai low cells in multiple organs, suggesting that Tai may normally promote Wg activity. Examining distribution of Wg components reveals that Tai promotes extracellular spread of the Wg ligand from source cells across the wing disc, thus ensuring patterned expression of multiple Wg-regulated target genes. Tai controls Wg spread indirectly through the extracellular glypican Dally-like protein (Dlp), which binds Wg and promotes its extracellular diffusion and capture by receptors. Data indicate that Tai likely controls Dlp at two levels: transcription of dlp mRNA and Dlp intracellular trafficking. Overall, these data indicate that the Tai acts through Dlp to enable Wg transport and signaling, and that cell competition in the Tai low model arises due to inequity in the ability of epithelial cells to sequester limiting amounts of the Wg growth factor.

HEXIM1 homodimer binds two sites on 7SK RNA to release autoinhibition for P-TEFb inactivation.

Hexim proteins are RNA-dependent regulators whose main target is 7SK long non-coding RNA, a major regulator of eukaryotic mRNA transcription. 7SK RNPs control available intracellular concentrations of the kinase P-TEFb (Cdk9-CyclinT1/2) by sequestering it in an inactive form. Active P-TEFb phosphorylates NELF, DSIF, and the RNA polymerase II CTD to transition it from promoter-proximal pausing to productive elongation. P-TEFb associates with 7SK RNP via Hexim, which directly binds 7SK RNA. However, free Hexim is in an autoinhibited state that cannot inactivate P-TEFb, and how Hexim autoinhibition is released by 7SK remains unknown. Here, we show that one Hexim1 homodimer binds two sites on linear 7SK RNA in a manner that exposes the Cdk9 binding sites, which are otherwise masked within the autoinhibited dimer. These results provide mechanistic insights into Hexim-RNA specificity and explain how P-TEFb can be effectively regulated to respond to changing levels of transcriptional signaling.

HPV16 E6-induced M2 macrophage polarization in the cervical microenvironment via exosomal miR-204-5p

The persistent infection of high-risk human papillomavirus (HPV) and the progression of cervical cancer necessitate the involvement of microenvironmental immunity. As cervical lesions advance, there is an observed increase in the infiltration of type 2 (M2) macrophages. However, the precise mechanism driving this increased infiltration of M2 macrophages remains unclear. In this study, we investigated the role of exosomes in polarising M2 macrophages in cervical lesions associated with HPV E6. Through the analysis of bioinformatics data and clinical specimens, we discovered a positive correlation between HPV E6/E7 mRNA copy number and the level of M2 macrophage infiltration. Exosomes derived from HPV E6 overexpressed (HPV E6+) cervical squamous cell carcinoma (CESC) cells were found to induce the polarisation of macrophages towards M2 type. Specifically, miR-204-5p, enriched in HPV E6 + CESC exosomes, was transported into macrophages and triggered M2 macrophage polarisation by inhibiting JAK2. The clinical relevance of exosomal miR-204-5p in the progression of cervical lesions was validated through serum samples from 35 cases. Exosomal miR-204-5p emerges as a critical factor influencing M2 macrophage polarisation and is correlated with the severity of cervical lesions. Consequently, miR-204-5p could be used as a potential treatment and a candidate biomarker for cervical lesions.

Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced.

The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3' loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant.

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

8602 Classical progesterone signaling in kisspeptin neurons is not required for negative feedback regulation of the GnRH pulse generator in mice

Abstract Disclosure: K.M. Dillon: None. D.B. Lohr: None. A.G. Novak: None. A.V. Petriv: None. N.T. Neifert: None. A.M. Moore: None. Mammalian reproductive function depends on the ability of progesterone to suppress pulsatile gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Impairments in progesterone negative feedback are associated with the development of polycystic ovary syndrome (PCOS), the leading cause of infertility for people with ovaries. Although previous research indicates cells upstream from GnRH neurons expressing the classical progesterone receptor (PR) are required for negative feedback, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion remain unclear. In this study, we aimed to address the hypothesis that PR expressed by a neuronal population in the arcuate nucleus expressing Kisspeptin, Neurokinin B, and Dynorphin, recently defined as the GnRH pulse generator, is required for negative feedback regulation of LH pulses. To achieve this, we utilized Cre-lox technology in mice to conditionally delete PR from kisspeptin cells (KPRKO mice) and assessed fecundity, LH pulsatility, and the mRNA expression of KNDy neuropeptides. Female KPRKO mice paired with B6 stud males for 3 months produced significantly fewer litters (p&amp;lt;0.05, n=5) and pups per litter (p&amp;lt;0.05) compared to WT controls (n=5) (students t-tests), recapitulating a previously reported subfertile phenotype in this model. In a separate cohort of animals, serial blood samples were collected every 6 minutes for 2 hours from the tail-tip of habituated WT and KPRKO mice in diestrus (n=6/group) and estrus (n=6/group), and an ultra-sensitive ELISA was used to detect LH pulses. However, compared to WT controls, KPRKO mice did not exhibit any significant changes in total and baseline LH release, pulse frequency or pulse amplitude in either cycle stages (p&amp;gt;0.05, students t-tests). At the completion of serial blood sampling, brains were collected from WT and KPRKO mice and RNAscope in situ hybridization was used to quantify mRNA for kisspeptin (KISS1) and dynorphin (PDYN), which are excitatory and inhibitory to GnRH/LH secretion, respectively. No differences in the number of KISS1- and PDYN-expressing cells or the number of KISS1 and PDYN mRNA transcripts were observed between WT and KPRKO mice (n=4/group, p&amp;gt;0.05, students t-tests). These data suggest that progesterone signaling in kisspeptin cells through PR is not essential for negative feedback regulation of LH pulses and is unlikely to contribute to the infertile phenotype of KPRKO mice. These results suggest that negative feedback may be maintained via non-classical mechanisms or an unidentified PR-positive neuronal population. Presentation: 6/1/2024

TMT-Based Quantitative Proteomics Revealed the Antibacterial Mechanism of Cinnamaldehyde against MRSA.

Natural plant extracts have demonstrated significant potential in alternative antibiotic therapies. Cinnamaldehyde (CA) has garnered considerable attention as a natural antibacterial agent. In this study, Tandem mass tag (TMT) quantitative proteomics combined with Western blot and RT-qPCR methods were employed to explore the antibacterial mechanism of CA against Methicillin-Resistant Staphylococcus aureus (MRSA) at the protein level. The results showed that a total of 254 differentially expressed proteins (DEPs) were identified in the control group and CA treatment group, of which 161 were significantly upregulated and 93 were significantly downregulated. DEPs related to nucleotide synthesis, homeostasis of the internal environment, and protein biosynthesis were significantly upregulated, while DEPs involved in the cell wall, cell membrane, and virulence factors were significantly downregulated. The results of GO and KEGG enrichment analyses demonstrated that CA could exert its antibacterial effects by influencing pyruvate metabolism, the tricarboxylic acid (TCA) cycle, teichoic acid biosynthesis, and the Staphylococcus aureus (S. aureus) infection pathway in MRSA. CA significantly inhibited the expression of recombinant protein MgrA (p < 0.05), significantly reduced the mRNA transcription levels of mgrA, hla, and sdrD genes (p < 0.05), and thermostability migration assays demonstrated that CA can directly interact with MgrA protein, thereby inhibiting its activity. These findings suggest that CA exerts its antibacterial mechanism by regulating the expression of related proteins, providing a theoretical basis for further development of clinical applications of antimicrobial agents derived from natural plant essential oils in the treatment of dairy cow mastitis.

UBTF haploinsufficiency associated with UBTF-related global developmental delay and distinctive facial features without neuroregression

BackgroundThe Upstream Binding Transcription Factor (UBTF) gene encodes two nucleolar proteins, UBTF1 and UBTF2. UBTF1 regulates rRNA transcription by RNA polymerase I, while UBTF2 regulates mRNA transcription by RNA polymerase...

Glutamine Attenuates Inflammation and Stimulates Amniotic Cell Proliferation in Premature Rupture of Membranes-related in vitro Models.

Premature rupture of membranes (PROM), with a prevalence of 15.3% in China, frequently results in adverse pregnancy outcomes. In this study, we aimed to identify amino acid metabolites that were differentially expressed in PROM versus healthy controls (HC) using targeted metabolomics and further explored their mechanisms of action with in vitro models.Inclusion and exclusion criteria were established to recruit 50 PROM and 50 HC cases for targeted metabolomics analysis. Twenty-three amino acid metabolites were quantified in the secretions of the posterior vaginal fornix of pregnant women between 31 and 36 weeks of gestation. Glutamine (0.0216 vs. 0.037μg/mg, P = 0.003, AUC = 72.1%) was identified as the most differentially expressed amino acid metabolite between PROM and HC groups, and had a negative correlation with the abundance of Gardnerella (r=-0.3868, P = 0.0055), Megasphaera (r=-0.3130, P = 0.0269), and Prevotella (r=-0.2944, P = 0.0380), respectively.In amniotic epithelial cell and macrophage co-culture model, Glutamine reduced inflammatory cytokines and chemokines expression and suppressed macrophage chemotaxis. In LPS stimulated RAW 264.7 inflammation model, Glutamine inhibited the expression of inflammatory proteins iNOS and COX-2, down-regulated mRNA transcription of TNF, IL-6, and IL-1β, and reduced the production of reactive oxygen species through inhibiting NF-κB signaling pathway, and therefore demonstrated its anti-inflammatory effect. Furthermore, Glutamine protected amniotic epithelial cell from autophagy and stimulated its proliferation, therefore may intensify fetal membrane and prevent PROM in vivo.Our results suggested that low Glutamine level in vaginal secretion can be used as an indicator for PROM, and local Glutamine supplementation is a potential intervention and prevention strategy for PROM.

Combined Dietary Spirulina platensis and Citrus limon Essential Oil Enhances the Growth, Immunity, Antioxidant Capacity and Intestinal Health of Nile Tilapia.

The dietary presence of feed additives is crucial for boosting fish growth and immunity. Accordingly, this feeding trial aimed to investigate the effects of the separate and concurrent dietary supplementation of Spirulina platensis (SP) and bitter lemon (Citrus limon) peel essential oil (LEO) on the growth, immunity, antioxidant capacity, and intestinal health of Nile tilapia (Oreochromis niloticus). Four groups of male Nile tilapia were employed. The first group (control) was given the basal diet, while the second and third groups received the basal diet supplemented with LEO extract (1%) and SP (1 g/kg diet), respectively. The fourth group received the basal diet supplemented with a mix of LEO (1%) and SP at 1 g/kg. After two months of feeding, using LEO or/and SP improved the overall growth and immunological parameters, with their combination yielding the best outcomes. The supplementation of LEO or/and SP improved the Nile tilapia's growth metrics and transcriptomic levels of growth-regulating genes such as (oligo-peptide transporter 1 (Pep1), growth hormone receptors 1 (GHR1), and insulin-like growth factor (IGF1). The improved growth performance was linked to significant increases in the expression levels of mucin and fat metabolism-related genes. Moreover, fish supplemented with LEO, SP, or their combination showed enhanced non-specific immunological measures, including phagocytic and lysozyme activities and the mRNA copies of its regulating genes. Additionally, remarkable increases in the antioxidant enzyme activities and the mRNA levels of their related genes were detected. The complement (C3) gene's transcriptomic level was also significantly increased. Furthermore, the dietary supplementation of LEO, SP, or their combination improved the histological structures of the spleen, hepatopancreas, and intestine. The enhanced effects of LEO, SP, or their combination on fish immunity and growth are suggested to be due to their contents of bioactive compounds with anti-inflammatory, antioxidant, and antimicrobial properties. Thus, using the LOE and SP blends as feed additives is recommended for better growth and immunity of Nile tilapia.

The TOP-2/condensin II axis silences transcription during germline specification in C. elegans.

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 protein's ability to globally silence mRNA transcription in germline precursor cells, also known as the P lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of TOP-2/condensin II activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P lineage. We also present evidence that while both PIE-1 and TOP-2/condensin II are required for genome silencing in the P lineage, PIE-1 can silence transcription independently of TOP-2/condensin II when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active. Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

Stocking density affects growth, feed utilisation, metabolism, welfare and associated mRNA transcripts in liver and muscle of rainbow trout more pronouncedly than dietary fish meal inclusion level

In aquaculture, stocking density and diet composition are critical factors that influence the performance and welfare of the farmed animal. However, there is limited information on their interactive effects. In this context, applying a 2 × 2 factorial design, we conducted a 12-week feeding trial to evaluate the effect of stocking density (low, LSD: 3.3–15 kg m−3 and high, HSD: 5.8–25 kg m−3; initial-final values) and dietary fish meal content (high, HFM: 30 % and low, LFM: 15 %) on growth, feed utilisation, metabolic oxygen consumption, tissue indices, carcass composition, plasma metabolites, and transcriptional regulation of growth, metabolism and welfare-related biomarker mRNAs in liver and muscle of the juvenile rainbow trout. At the whole-animal level, the HSD group showed significantly higher routine metabolic rate and feed intake indicating higher energy demand; but growth and feed efficiency was lower in HSD than the LSD group. Morpho-anatomically, HSD fish had lower relative gut length and higher intra-peritoneal fat index than LSD fish. Lower plasma free amino acids and glucose levels reiterated high energy expenditure in HSD, and elevated albumin levels indicated potential oxidative stress in the HSD group. Correspondingly, at the transcriptional level, hepatic mRNA levels of antioxidative enzymes (sod, cat, gst, g6pd) and stress-related molecular chaperone hsp90 were up-regulated in HSD; while the transcript abundance of growth-axis markers (ghr, igf1 igf2) and nutrient-sensor mtor were decreased. In muscle, proteolytic pathway components fbx32 and murf1 were upregulated in HSD, and concurrently mtor was also upregulated suggesting higher protein turnover in this group. With respect to the effect of dietary fish meal inclusion levels, LFM showed decrease in growth, hepatic igf2 expression, and lower plasma triglycerides and free amino acid levels than HFM group. However, the sensory appearance of flesh was found to be slightly better in LFM. Significant diet-husbandry interactions were not observed phenotypically, but the mRNA abundance of hepatic markers (ampk1, hsp70, mhc1 and tnfa) linked to energy-sensing, stress and immune response were differentially regulated. Overall, the study shows that high stocking density has a greater degree of impact on fish growth and welfare than reduction in dietary fishmeal inclusion, with limited interactional effects.