Preferential Expression Research Articles

Aberrant BCAT1 expression augments MTOR activity and accelerates disease progression in chronic lymphocytic leukemia.

We performed gene expression profiling of mRNA/cDNA isolated from N = 117 flow sorted CLL. We detected aberrant expression of the metabolic enzyme branched chain amino acid transferase (BCAT1) in CLL with del17p/TP53mut. Through extensive validation, we confirmed the highly preferential expression of BCAT1 in CLL with del17p/TP53mut (66%) or trisomy 12 (77%). BCAT1 was not expressed in B cells isolated from normal human lymph nodes. The products of the bidirectional BCAT1 reaction, including leucine, acetyl-CoA, and alpha-ketoglutarate are known activators of MTOR. We measured an ~two-fold higher MTOR activity via normalized p-S6K levels in primary CLL with BCAT1 high versus absent expression before and after sIgM crosslinking. Through steady state metabolomics and heavy isotope metabolic tracing in primary CLL cells, we demonstrate that CLL cells are avid consumers of branched chain amino acids (BCAAs) and that BCAT1 in CLL engages in bidirectional substrate reactions. Of additional interest, CLL with aberrant BCAT1 expression were less sensitive to Venetoclax-induced apoptosis. Biologically, three CLL-derived cell lines with disruption of BCAT1 had substantially reduced growth ex vivo. Clinically, the expression of any detectable BCAT1 protein in CLL independently associated with shorter median survival (125 months versus 296 months; p < 0.0001), even after exclusion of del17p/TP53mut cases.

Aspartic Acid Binding on Hydroxyapatite Nanoparticles with Varying Morphologies Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulation.

Hydroxyapatite (HAP) exhibits a highly oriented hierarchical structure in biological hard tissues. The formation and selective crystalline orientation of HAP is a process that involves functional biomineralization proteins abundant in acidic residues. To obtain insights into the process of HAP mineralization and acidic residue binding, synthesized HAP with specific lattice planes including (001), (100), and (011) are structurally characterized following the adsorption of aspartic acid (Asp). The adsorption affinity of Asp on HAP surfaces is evaluated quantitatively and demonstrates a high dependency on the HAP morphological form. Among the synthesized HAP nanoparticles (NPs), Asp exhibits the strongest adsorption affinity to short HAP nanorods, which are composed of (100) and (011) lattice planes, followed by nanosheets with a preferential expression of the (001) facet, to which Asp displays a similar but slightly lower binding affinity. HAP nanowires, with the (100) lattice plane preferentially developed, show significantly lower affinity to Asp and evidence of multilayer adsorption compared to the previous two types of HAP NPs. A combination of solid-state NMR (SSNMR) techniques including 13C and 15N CP-MAS, relaxation measurements and 13C-31P Rotational Echo DOuble Resonance (REDOR) is utilized to characterize the molecular structure and dynamics of Asp-HAP bionano interfaces with 13C- and 15N-enriched Asp. REDOR is used to determine 13C-31P internuclear distances, providing insight into the Asp binding geometry where stronger 13C-31P dipolar couplings correlate with binding affinity determined from Langmuir isotherms. The carboxyl sites are identified as the primary binding groups, facilitated by their interaction with surface calcium sites. The Asp chelation conformations revealed by SSNMR are further refined with molecular dynamics (MD) simulation where specific models strongly agree between the SSNMR and MD models for the various surfaces.

Identification of LRP1+CD13+ human periosteal stem cells that require LRP1 for bone repair.

Human periosteal skeletal stem cells (P-SSCs) are critical for cortical bone maintenance and repair. However, their in vivo identity, molecular characteristics, and specific markers remain unknown. Here, single-cell sequencing revealed human periosteum contains SSC clusters expressing known SSC markers, PDPN and PDGFRA. Notably, human P-SSCs, but not bone marrow SSCs (BM-SSCs), selectively expressed newly identified markers, LRP1 and CD13. These LRP1+CD13+ human P-SSCs were perivascular cells with high osteochondrogenic but minimal adipogenic potential. Upon transplantation into bone injuries in mice, they preserved self-renewal capability in vivo. Single-cell analysis of mouse periosteum further supported the preferential expression of LRP1 and CD13 in Prx1+ P-SSCs. When Lrp1 was conditionally deleted in Prx1-lineage cells, it led to severe bone deformity, short statue, and periosteal defects. By contrast, local treatment with a LRP1 agonist at the injury sites induced early P-SSC proliferation and bone healing. Thus, human and mouse periosteum contains unique osteochondrogenic stem cell subsets, and these P-SSCs express specific markers, LRP1 and CD13, with regulatory mechanism through LRP1 that enhances P-SSC function and bone repair.

Deciphering Transcriptomic Variations in Hematopoietic Lineages: HSCs, EBs, and MKs.

In the realm of hematopoiesis, hematopoietic stem cells (HSCs) serve as pivotal entities responsible for generating various blood cell types, initiating both the myeloid and lymphoid branches within the hematopoietic lineage. This intricate process is marked by genetic variations that underscore the crucial role of genes in regulating cellular functions and interactions. Recognizing the significance of genetic factors in this context, this article delves into a genetic perspective, aiming to unravel the biological factors that govern the transition from one cell's fate to another within the hematopoietic system. To gain deeper insights into the genetic traits of three distinct blood cell types-HSCs, erythroblasts (EBs), and megakaryocytes (MKs)-we conducted a comprehensive transcriptomic analysis. Leveraging diverse hematopoietic cell datasets from healthy individuals, sourced from The BLUEPRINT consortium, our investigation targeted the identification of genetic variants responsible for changes in gene expression levels and epigenetic modifications across the entire human genome in each of these cell types. The total number of normalized expressed transcripts includes 14,233 novel trinity lncRNAs, 13,749 mRNAs, and 3092 lncRNAs. This scrutiny revealed a total of 31,074 transcripts, with a notable revelation that 14,233 of them were previously unidentified or novel lncRNAs, highlighting a substantial reservoir of genetic information yet to be explored. Examining their expression across distinct lineages further unveiled 2845 differentially expressed (DE) mRNAs and 354 DE long noncoding RNAs (lncRNAs) notably enriched among the three distinct blood cell types: HSCs, EBs, and MKs. Our investigation extended beyond mRNA to focus on the dynamic expression of lncRNAs, revealing a well-defined pattern that played a significant role in regulating differentiation and cell-fate specification. This coordination of lncRNA dynamics extended to aberrations in both mRNA and lncRNA transcriptomes within HSCs, EBs, and MKs. We specifically characterized lncRNAs with preferential expression in HSCs, as well as in various downstream differentiated lineage progenitors of EBs and MKs, providing a comprehensive perspective on lncRNAs in human hematopoietic cells. Notably, the expression of lncRNAs exhibited substantial cell-to-cell variation, a phenomenon discernible only through single-cell analysis. The comparative analysis undertaken in this study provides valuable insights into the distinctive genetic signatures guiding the differentiation of these crucial hematopoietic cell types.

Evolution and Functional Dynamics of TCP Transcription Factor Gene Family in Passion Fruit (Passiflora edulis).

Passion fruit is a valued tropical fruit crop that faces environment-related growth strains. TCP genes are important for both growth modulation and stress prevention in plants. Herein, we systematically analyzed the TCP gene family in passion fruit, recognizing 30 members. Genes exhibiting closer phylogenetic relationships exhibited similar protein and gene structures. Gene members of the TCP family showed developmental-stage- or tissue-specific expression profiles during the passion fruit life cycle. Transcriptome data also demonstrated that many PeTCPs showed induced expression in response to hormonal treatments and cold, heat, and salt stress. Based on transcriptomics data, eight candidate genes were chosen for preferential gene expression confirmation under cold stress conditions. The qRT-PCR assays suggested PeTCP15/16/17/19/23 upregulation, while PeTCP1/11/25 downregulation after cold stress. Additionally, TCP19/20/29/30 exhibited in silico binding with cold-stress-related miRNA319s. GFP subcellular localization assays exhibited PeTCP19/1 were localized at the nucleus. This study will aid in the establishment of novel germplasm, as well as the further investigation of the roles of PeTCPs and their cold stress resistance characteristics.

Regulatory networks of coresident subgenomes during rapid fiber cell elongation in upland cotton

Cotton, an intriguing plant species shaped by polyploidization, evolution, and domestication, holds particular interest due to the complex mechanisms governing fiber traits across its two subgenomes. However, the regulatory elements or transcriptional networks between subgenomes during fiber elongation remain to be fully clarified. Here, we analyzed 1462 cotton fiber samples to reconstruct the gene-expression regulatory networks that influence fiber cell elongation. Inter-subgenome expression quantitative trait loci (eQTLs) largely dictate gene transcription, with a notable tendency for the D subgenome to regulate A-subgenome eGenes. This regulation reveals synchronized homoeologous gene expression driven by co-localized eQTLs and divergent patterns that diminish genetic correlations, thus leading to preferential expression in the A and D subgenomes. Hotspot456 emerged as a key regulator of fiber initiation and elongation, and artificial selection of trans-eQTLs in hotspot456 that positively regulate KCS1 has facilitated cell elongation. Experiments designed to clarify the roles of trans-eQTLs in improved fiber breeding confirmed the inhibition of GhTOL9 by a specific trans-eQTL via GhWRKY28, which negatively affects fiber elongation. We propose a model in which the GhWRKY28–GhTOL9 module regulates this process through the ESCRT (endosomal sorting complex required for transport) pathway. This research significantly advances our understanding of cotton’s evolutionary and domestication processes and the intricate regulatory mechanisms that underlie significant plant traits.

Regulatory mechanisms orchestrating cellular diversity of Cd36+ olfactory sensory neurons revealed by scRNA-seq and scATAC-seq analysis

Recent discoveries have revealed remarkable complexity within olfactory sensory neurons (OSNs), including the existence of two OSN populations based on the expression of Cd36. However, the regulatory mechanisms governing this cellular diversity in the same cell type remain elusive. Here, we show the preferential expression of 79 olfactory receptors in Cd36+ OSNs and the anterior projection characteristics of Cd36+ OSNs, indicating the non-randomness of Cd36 expression. The integrated analysis of single-cell RNA sequencing (scRNA-seq) and scATAC-seq reveals that the differences in Cd36+/- OSNs occur at the immature OSN stage, with Mef2a and Hdac9 being important regulators of developmental divergence. We hypothesize that the absence of Hdac9 may affect the activation of Mef2a, leading to the up-regulation of Mef2a target genes, including teashirt zinc finger family member 1 (Tshz1), in the Cd36+ OSN lineage. We validate that Tshz1 directly promotes Cd36 expression through enhancer bindings. Our study unravels the intricate regulatory landscape and principles governing cellular diversity in the olfactory system.

Genome-wide identification of phytosulfokine (PSK) peptide family reveals TaPSK1 gene involved in grain development of wheat (Triticum aestivum L.)

BackgroundPhytosulfokine (PSK) functions as a plant peptide growth factor that plays important and diverse roles in plant development and stress responses. Nevertheless, PSKs have not been systematically analyzed in wheat.ResultsA genome-wide comparative analysis of PSK genes in wheat was conducted and 15 TaPSKs were identified and divided into four subgroups in the wheat genome based on sequence similarity. The examination of motif compositions of TaPSK genes revealed the presence of the YIYTQ signature motif in the C-terminus of all TaPSK polypeptide precursors, with a highly conserved feature across different species. Exogenous application of TaPSK peptide promoted root growth in wheat. Quantitative RT-PCR analysis revealed that the TaPSKs exhibited preferential or tissue-specific expression patterns in wheat. In particular, three homologs of the TaPSK1 genes were specifically expressed in grains, with the strongest expression observed in the developing grains at 15 days after anthesis. Compared with wild type, transgenic rice lines overexpressing TaPSK1-A exhibited larger grain size and higher thousand-grain weight. The promoter region and genomic sequence of the wheat TaPSK1-A gene were cloned. Sequence polymorphism showed that five single-nucleotide polymorphisms (SNPs) were identified in the promoter region of TaPSK1-A. A Kompetitive Allele-Specific PCR (KASP) marker was developed for TaPSK1-A based on –806 bp SNP (C/T transition), and two haplotypes, TaPSK1-A-HapI and TaPSK1-A-HapII were detected in 260 wheat accessions collected from different regions. The expression of TaPSK1-A, promoter activity, and thousand-grain weight (TGW) in the TaPSK1-A-HapII haplotype were higher than those in the TaPSK1-A-HapI haplotype. Furthermore, yeast one-hybrid assays revealed the binding of TaNF-YB1 and TaERF39 to the promoter regions of the TaPSK1-A gene, and TaMADS29 could bind to the promoters of TaPSK1-B and TaPSK1-D genes.ConclusionsComparative genome-wide analysis of TaPSK peptide family revealed that the TaPSK1 gene is involved in wheat grain development, and the developed TaPSK1-A-KASP marker could be utilized for marker-assisted selection breeding of wheat.Graphical

Bitter tastants relax the mouse gallbladder smooth muscle independent of signaling through tuft cells and bitter taste receptors

Disorders of gallbladder motility can lead to serious pathology. Bitter tastants acting upon bitter taste receptors (TAS2R family) have been proposed as a novel class of smooth muscle relaxants to combat excessive contraction in the airways and other organs. To explore whether this might also emerge as an option for gallbladder diseases, we here tested bitter tastants for relaxant properties and profiled Tas2r expression in the mouse gallbladder. In organ bath experiments, the bitter tastants denatonium, quinine, dextromethorphan, and noscapine, dose-dependently relaxed the pre-contracted gallbladder. Utilizing gene-deficient mouse strains, neither transient receptor potential family member 5 (TRPM5), nor the Tas2r143/Tas2r135/Tas2r126 gene cluster, nor tuft cells proved to be required for this relaxation, indicating direct action upon smooth muscle cells (SMC). Accordingly, denatonium, quinine and dextromethorphan increased intracellular calcium concentration preferentially in isolated gallbladder SMC and, again, this effect was independent of TRPM5. RT-PCR revealed transcripts of Tas2r108, Tas2r126, Tas2r135, Tas2r137, and Tas2r143, and analysis of gallbladders from mice lacking tuft cells revealed preferential expression of Tas2r108 and Tas2r137 in tuft cells. A TAS2R143-mCherry reporter mouse labeled tuft cells in the gallbladder epithelium. An in silico analysis of a scRNA sequencing data set revealed Tas2r expression in only few cells of different identity, and from in situ hybridization histochemistry, which did not label distinct cells. Our findings demonstrate profound tuft cell- and TRPM5-independent relaxing effects of bitter tastants on gallbladder smooth muscle, but do not support the concept that these effects are mediated by bitter receptors.

GRB7 Plays a Vital Role in Promoting the Progression and Mediating Immune Evasion of Ovarian Cancer.

Despite breakthroughs in treatment, ovarian cancer (OC) remains one of the most lethal gynecological malignancies, with an increasing age-standardized mortality rate. This underscores an urgent need for novel biomarkers and therapeutic targets. Although growth factor receptor-bound protein 7 (GRB7) is implicated in cell signaling and tumorigenesis, its expression pattern and clinical implications in OC remain poorly characterized. To systematically investigate GRB7's expression in OC, our study utilized extensive datasets from TCGA, GTEx, CCLE, and GEO. The prognostic significance of GRB7 was evaluated by means of Kaplan-Meier and Cox regression analyses. Using a correlation analysis and gene set enrichment analysis, relationships between GRB7's expression and gene networks, immune cell infiltration and immunotherapy response were investigated. In vitro experiments were conducted to confirm GRB7's function in the biology of OC. Compared to normal tissues, OC tissues exhibited a substantial upregulation of GRB7. Reduced overall survival, disease-specific survival, and disease-free interval were all connected with high GRB7 mRNA levels. The network study demonstrated that GRB7 is involved in pathways relevant to the course of OC and has a positive connection with several key driver genes. Notably, GRB7's expression was linked to the infiltration of M2 macrophage and altered response to immunotherapy. Data from single-cell RNA sequencing data across multiple cancer types indicated GRB7's predominant expression in malignant cells. Moreover, OC cells with GRB7 deletion showed decreased proliferation and migration, as well as increased susceptibility to T cell-mediated cytotoxicity. With respect to OC, our results validated GRB7 as a viable prognostic biomarker and a promising therapeutic target, providing information about its function in tumorigenesis and immune modulation. GRB7's preferential expression in malignant cells highlights its significance in the biology of cancer and bolsters the possibility that it could be useful in enhancing the effectiveness of immunotherapy.

Abstract We117: Preclinical Evidence of Long-term Efficacy and Safety of LX2020, an AAV based Plakophilin-2 Gene Therapy, for the Treatment of Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a fatal inherited cardiac disease caused by mutations in desmosomal anchoring genes, with plakophilin-2 (PKP2) being the most frequently mutated gene (70%). While AAV9-PKP2 gene therapy shows promise in mitigating ACM-related deficits in mice, high dose toxicity and organ failure of AAV9 in human clinical trials raises safety concerns. Prior studies showed AAVrh.10 exhibiting preferential cardiac gene expression at lower doses that could potentially be safer. We hypothesized that AAVrh.10.PKP2 (LX2020) could restore PKP2 to scaffold the desmosomal complex and ameliorate cardiac deficits with long-term efficacy at relatively lower doses and safety in non-human primates (NHP). We employed LX2020 to assess its efficacy in severe (PKP2 homozygous (Hom)) and mild (PKP2 heterozygous (Het)) adult ACM diseased mice, as well as safety in NHP at doses from 2E13 to 1E14 vg/kg and up to 12 weeks post-administration. A dose-dependent increase in hPKP2 expression was seen in all models, with rescue of desmosomal/gap junction protein deficits at 2E13 and 6E13 vg/kg doses in PKP2-Het and PKP2-Hom mouse hearts. Long term LX2020 efficacy was observed at the same doses in PKP2-Hom mice with dose-dependent improvements in cardiac function, arrhythmia reduction and histological improvements, such that LX2020 treated PKP2-Hom hearts resembled wild-type hearts, indicating a potential for reversal of late-stage pathology. The extended lifespan was striking with 100% of PKP2-Hom mice surviving using 2E13 vg/kg, completely averting premature death. Safety assessments in NHP also revealed no adverse events up to the highest dose of 1E14 vg/kg. These data support advancing LX2020 as a potentially viable treatment for PKP2 ACM patients, based on safety and long-term efficacy in rescuing desmosomal and cardiac deficits in ACM models at lower doses reported in the field.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

The invertase gene PWIN1 confers chilling tolerance of rice at the booting stage via mediating pollen development.

Pollen fertility is a primary regulator of grain yield and is highly susceptible to cold and other environmental stress. We revealed the roles of rice cell wall invertase gene PWIN1 in pollen development and chilling tolerance. We uncovered its preferential expression in microspores and bicellular pollen and identified its knock-down and knock-out mutants. pwin1 mutants produced a higher proportion of abnormal pollen than wild-type plants. The contents of sucrose, glucose, and fructose were increased, while ATP content and primary metabolism activity were reduced in the mutant pollen. Furthermore, the loss of function of PWIN1 coincided with an increase in SnRK1 activity and a decrease in TOR activity. Under chilling conditions, pwin1 mutants displayed significantly reduced pollen viability and seed-setting rate, while overexpressing PWIN1 notably increased pollen viability and seed-setting rate as compared with the wild-type, indicating that PWIN1 is essential for rice pollen development and grain yield under cold stress. This study provides insights into the molecular mechanisms underlying rice pollen fertility during chilling stress, and a new module to improve chilling tolerance of rice at the booting stage by molecular design.

ZFP36 family expression is suppressed by Th2 cells in asthma, leading to enhanced synthesis of inflammatory cytokines and cell surface molecules

Asthma is a chronic inflammatory airway disease, in which inflammatory cytokines play a pivotal role. The zinc finger binding protein 36 (ZFP36) family includes ZFP36, ZFP36L1, and ZFP36L2 and is among the RNA-binding proteins (RBPs) reported to cause inflammation. The present study aimed to clarify the roles of the ZFP36 family in asthma, particularly highlighting the relationship between the ZFP36 family and Th2 cells, which are key players in type 2 inflammation in asthma. Real-time PCR analysis revealed the preferential expression of ZFP36 family mRNAs in human white blood cells. Gene expression analysis using public datasets from the GEO database (https://www.ncbi.nlm.nih.gov/gds) showed significantly suppressed expression of ZFP36 family mRNAs in patients with asthma compared to that in healthy controls. Using multiple cytokine assays, Th2 cell transfection with ZFP36 family siRNAs enhanced the expression of inflammatory cytokines IL-8, IFN-γ, CCL3/MIP-1α, CCL4/MIP-1β, and TNF-α and cell surface molecules CCR4 (CD194) and PSGL-1 (CD162). Treatment with IL-2, 4, and 15 significantly suppressed, and corticosteroid significantly enhanced the expressions of ZFP36 family mRNAs by Th2 cells. In conclusion, the ZFP36 family expressed by Th2 cells was suppressed in patients with asthma, leading to the enhanced expression of cytokines and cell surface molecules. Suppressed ZFP36 expression in asthma may be involved in the enhancement of airway inflammation, and the ZFP36 family may be a therapeutic target for inflammatory diseases, including asthma.

Over-expression of a plant-type phosphoenolpyruvate carboxylase derails Arabidopsis stamen formation

We examined whether plant-type phosphoenolpyruvate carboxylase (PEPC) is involved in flower organ formation or not by over-expression in Arabidopsis. A wheat PEPC isogene Tappc3A, belonging to the ppc3 group, was targeted due to its preferential expression pattern in pistils and stamens. Transgenic Arabidopsis over-expressing Tappc3A exhibited irregular stamen formation, i.e., a lesser number of stamens per flower and shorter filaments in T2 and T3 generations. Irregular stamens were frequently observed in homozygous T4 lines, but no morphological change was observed in other floral organs. High-degree gene co-expression of Tappc3 isogenes with wheat SEEDSTICKs but not with other homeotic transcription factor genes for flower formation implicates that Tappc3 is under control by the class D genes of the ABCDE model to flower development. In addition, the conservation of CArG box sequences on the Tappc3 promoters supported the developmentally programmed gene expression of ppc3 in wheat flowering organs. Thus, this study provides the first experimental evidence for the critical regulation of plant-type PEPC for flower formation.

Investigation of Functional Cytochrome P450 4A Enzymes in Liver and Kidney of Pigs, Cats, Tree Shrews, and Dogs in Comparison with the Metabolic Capacity of Human P450 4A11.

Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli, preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω-position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω-hydroxylation of fatty acids.

Autophagy, apoptosis, and inflammation response of Sebastiscus marmoratus to different concentrations of hydroxychloroquine

Hydroxychloroquine (HCQ) is an important public health therapeutic agent widely used in the prevention and treatment of malaria and autoimmune diseases, with some antiviral effects, as well as a common autophagy inhibitor. Its autophagy-inhibiting effect attracts great research interest in mammals but is still little studied in fish. We even have no idea about the effects of HCQ on different tissues of fish and what concentrations should be used for safety studies. This study investigated the effect of different concentration of HCQ treatments on the survival, tissue structure, and expressions of genes related to autophagy, apoptosis, and inflammation in the gill, spleen, testis, and ovary of Sebastiscus marmoratus. The results showed that the higher HCQ concentration (77.40 and 154.80 mg/mL) led to mass mortality within an hour. The half-lethal concentration (LC50, 24 h) of HCQ for S. marmoratus was approximately 48.95 mg/mL. When exposed to 30.96 mg/mL HCQ for 24 hours, autophagy was blocked as revealed by electron microscopy from gill, spleen and testis of fish. The expressions of autophagy-related genes (LC3/Cx43), apoptosis-related genes (Cas3/p53), and inflammation-related genes (TNF-α/IL8) exhibited tissue-specific and dose-dependent responses. The gonads showed preferential expression of all these genes and were found to be sensitive and regular after HCQ treatment. For example, at a concentration of 30.96 mg/mL, the testis demonstrated a regularity that suggests it is an ideal candidate tissue for studying the role of HCQ or autophagy. This study systematically revealed the response of S. marmoratus to different concentrations of HCQ and provided optional assay concentrations for key tissues, serving as an important reference for the future studies on HCQ and autophagy in S. marmoratus. Furthermore, the potential crosstalk between autophagy, apoptosis, and inflammatory pathways initially identified in this study could be helpful for the future research on autophagy regulation in marine fish.

The Human Developing Cerebral Cortex Is Characterized by an Elevated De Novo Expression of Long Noncoding RNAs in Excitatory Neurons.

The outstanding human cognitive capacities are computed in the cerebral cortex, a mammalian-specific brain region and the place of massive biological innovation. Long noncoding RNAs have emerged as gene regulatory elements with higher evolutionary turnover than mRNAs. The many long noncoding RNAs identified in neural tissues make them candidates for molecular sources of cerebral cortex evolution and disease. Here, we characterized the genomic and cellular shifts that occurred during the evolution of the long noncoding RNA repertoire expressed in the developing cerebral cortex and explored putative roles for these long noncoding RNAs in the evolution of the human brain. Using transcriptomics and comparative genomics, we comprehensively annotated the cortical transcriptomes of humans, rhesus macaques, mice, and chickens and classified human cortical long noncoding RNAs into evolutionary groups as a function of their predicted minimal ages. Long noncoding RNA evolutionary groups showed differences in expression levels, splicing efficiencies, transposable element contents, genomic distributions, and transcription factor binding to their promoters. Furthermore, older long noncoding RNAs showed preferential expression in germinative zones, outer radial glial cells, and cortical inhibitory (GABAergic) neurons. In comparison, younger long noncoding RNAs showed preferential expression in cortical excitatory (glutamatergic) neurons, were enriched in primate and human-specific gene co-expression modules, and were dysregulated in neurodevelopmental disorders. These results suggest different evolutionary routes for older and younger cortical long noncoding RNAs, highlighting old long noncoding RNAs as a possible source of molecular evolution of conserved developmental programs; conversely, we propose that the de novo expression of primate- and human-specific young long noncoding RNAs is a putative source of molecular evolution and dysfunction of cortical excitatory neurons, warranting further investigation.

DsRNA formation leads to preferential nuclear export and gene expression

When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67–Mtr2 in the yeast Saccharomyces cerevisiae (TAP–p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.

Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.