Serotonin or 5-hydroxytryptamine (5-HT) is an important neurotransmitter that is found in mammalian taste buds and can regulate the output of intragemmal signaling networks onto afferent nerve fibers. However, it is unclear how 5-HT is produced, synthesized locally inside taste buds or absorbed from outside sources. In this study, we attempt to address this question by delineating the process of possible 5-HT biosynthesis within taste buds. First, we verified that the rate-limiting enzyme tryptophan hydroxylase (TPH2) responsible for converting L-tryptophan into the intermediate 5-hydroxy-L-tryptophan (5-HTP) is expressed in a subset of type II taste bud cells (TBCs) whereas the enzyme aromatic L-aromatic amino acid decarboxylase (AADC) capable of converting 5-HTP into 5-HT is found in type III TBCs. And abolishment of TPH2 did not affect the production of intragemmal 5-HT or alter TBCs; the mutant mice did not show any changes in behavioral responses to all five primary taste qualities: sweet, umami, bitter, salty, and sour. Then we identified that 5-HTP as well as AADC are abundant in type III TBCs; and application of an AADC inhibitor significantly blocked the production of 5-HT in taste buds. In contrast, administration of an inhibitor on serotonin-reuptake transporters had minimal impact on the 5-HT amount in taste buds, indicating that exogenous 5-HT is not a major source for the intragemmal transmitter. Taken together, our data indicate that intragemmal serotonin is not biosynthesized de novo from tryptophan; instead, it is produced by AADC-mediated conversion of 5-HTP absorbed from the plasma and/or nerve fibers into 5-HT. Thus, our results suggest that the overall bodily 5-HTP level in the plasma and nervous system can regulate taste buds' physiological function, and provide an important molecular mechanism connecting these peripheral taste organs with the circulatory and nervous systems.

MicroRNAs (miRNAs) are important regulators involved in the antiviral response to influenza virus infection, however, an analytical comparison of miRNA and mRNA expression changes induced by several H7N9 host-adapting PB2 mutants remains undone. Here, miRNA microarray and transcriptome sequencing of BALB/c mouse lungs infected with A/Anhui/1/2013 (H7N9) [hereafter referred to as H7N9/AH1-PB2-627K(WT)] and mutant variants with PB2 amino acid substitutions (avian-like H7N9/AH1-PB2-627E and mammalian-adapted H7N9/AH1-PB2-627E/701N) were directly compared. The results showed that influenza virus infection induced dysregulation of numerous host cell processes. In a miRNA-mRNA network associated with immunity, changes in the expression of 38 miRNAs and 58 mRNAs were detected following influenza virus infection. Notably, the miRNAs of mmu-miR-188-5p, mmu-miR-511-5p, mmu-miR-483-5p, and mmu-miR-690 were specifically associated with the replication of the avian-like virus H7N9/AH1-PB2-627E. Likewise, the miRNAs of mmu-miR-691, mmu-miR-329-3p, and mmu-miR-144-3p were specifically associated with the mammalian-adapted virus H7N9/AH1-PB2-627E/701N. Finally, the miRNAs of mmu-miR-98-5p, mmu-miR-103-3p, mmu-miR-199a-5p, and mmu-miR-378a-3p were specifically associated with H7N9/AH1-PB2-627K(WT) virus replication. This is the first report of comparative integration analysis of miRNA-mRNA expression of these three H7N9 influenza viruses with different host-adapting PB2 mutations. Our results highlight potential miRNAs of importance in influenza virus pathogenesis.

To preserve genome integrity, eukaryotic cells use small RNA-directed mechanisms to repress transposable elements (TEs). Paradoxically, in order to silence TEs, precursors of the small RNAs must be transcribed from TEs. However, it is still poorly understood how these precursors are transcribed from TEs under silenced conditions. In the otherwise transcriptionally silent germline micronucleus (MIC) of Tetrahymena, a burst of non-coding RNA (ncRNA) transcription occurs during meiosis. The transcripts are processed into small RNAs that serve to identify TE-related sequences for elimination. The Mediator complex (Med) has an evolutionarily conserved role for transcription by bridging gene-specific transcription factors and RNA polymerase II. Here, we report that three Med-associated factors, Emit1, Emit2, and Rib1, are required for the biogenesis of small ncRNAs. Med localizes to the MIC only during meiosis, and both Med localization and MIC ncRNA transcription require Emit1 and Emit2. In the MIC, Med occupies TE-rich pericentromeric and telomeric regions in a Rib1-dependent manner. Rib1 is dispensable for ncRNA transcription but is required for the accumulation of double-stranded ncRNAs. Nuclear and sub-nuclear localization of the three Med-associated proteins is interdependent. Hence, Emit1 and Emit2 act coordinately to import Med into the MIC, and Rib1 recruits Med to specific chromosomal locations to quantitatively or qualitatively promote the biogenesis of functional ncRNA. Our results underscore that the transcription machinery can be regulated by a set of specialized Med-associated proteins to temporally transcribe TE-related sequences from a silent genome for small RNA biogenesis and genome defense.

The application of fire-retardant fabrics is essential for providing thermal protective function of the garments. Appropriate clothing design are beneficial for preventing the wearers from skin burn injuries and heat strains simultaneously. The intention of this work was to investigate the effects of clothing ventilation designs on its thermal protective performance by bench-scale tests. Four boundary conditions were designed to simulate the garment aperture structures on fabric level. Tests of thermal shrinkage, mass loss and time-to-second-degree-burns were performed with and without air gap under three heat-flux levels for two kinds of inherently fire-retardant fabrics. The impacts of fabric type, heat-flux level, air gap and boundary condition were analyzed. The presence of a 6.4-mm air gap could improve thermal protective performance of the fabrics, however, the garment openings would decrease this positive effects. More severe thermal aging found for spaced test configuration indicated the importance of balancing the service life and thermal protective performance of the clothing. The findings of this study implied that the characteristics of fabric type, air gap, boundary condition, and their effects on fabric thermal aging should be considered during clothing ventilation designs, to balance the thermal protection and comfort of the protective gear.

In most eukaryotes, the meiotic chromosomal bouquet (comprising clustered chromosome ends) provides an ordered chromosome arrangement that facilitates pairing and recombination between homologous chromosomes. In the protist Tetrahymena thermophila, the meiotic prophase nucleus stretches enormously, and chromosomes assume a bouquet-like arrangement in which telomeres and centromeres are attached to opposite poles of the nucleus. We have identified and characterized three meiosis-specific genes [meiotic nuclear elongation 1-3 (MELG1-3)] that control nuclear elongation, and centromere and telomere clustering. The Melg proteins interact with cytoskeletal and telomere-associated proteins, and probably repurpose them for reorganizing the meiotic prophase nucleus. A lack of sequence similarity between the Tetrahymena proteins responsible for telomere clustering and bouquet proteins of other organisms suggests that the Tetrahymena bouquet is analogous, rather than homologous, to the conserved eukaryotic bouquet. We also report that centromere clustering is more important than telomere clustering for homologous pairing. Therefore, we speculate that centromere clustering may have been the primordial mechanism for chromosome pairing in early eukaryotes.

Influenza A virus is a single-stranded RNA virus that can cause great mortality and economic loss worldwide. Circular RNAs (circRNAs) are non-coding RNAs that have been shown to have important functions in the regulation of biological processes. However, their functions during the influenza A virus infection process remain unclear. Herein, RNA sequencing technology was used to identify circRNAs expressed in mouse lungs during infection with H7N9/PB2-627 K/701D (H7N9/Wild-type) virus and PB2 mutant viruses (H7N9/PB2-627E/701D and H7N9/PB2-627E/701 N). We identified 7126 circRNAs at different genomic locations during H7N9 influenza virus and its mutant virus infections, of which 186 were differentially expressed. Enrichment analysis revealed that the differentially expressed circRNAs were associated with the viral infection process. Our study shows that circRNA expression profiles were altered following H7N9 influenza A virus infection and the differentially expressed circRNAs may have an important immune-regulating function during viral infection.

Untargeted metabolomics based on liquid chromatography coupled with mass spectrometry (LC-MS) can detect thousands of features in samples and produce highly complex datasets. The accurate extraction of meaningful features and the building of discriminant models are two crucial steps in the data analysis pipeline of untargeted metabolomics. In this study, pure ion chromatograms were extracted from a liquor dataset and left-sided colon cancer (LCC) dataset by K-means-clustering-based Pure Ion Chromatogram extraction method version 2.0 (KPIC2). Then, the nonlinear low-dimensional embedding by uniform manifold approximation and projection (UMAP) showed the separation of samples from different groups in reduced dimensions. The discriminant models were established by extreme gradient boosting (XGBoost) based on the features extracted by KPIC2. Results showed that features extracted by KPIC2 achieved 100% classification accuracy on the test sets of the liquor dataset and the LCC dataset, which demonstrated the rationality of the XGBoost model based on KPIC2 compared with the results of XCMS (92% and 96% for liquor and LCC datasets respectively). Finally, XGBoost can achieve better performance than the linear method and traditional nonlinear modeling methods on these datasets. UMAP and XGBoost are integrated into KPIC2 package to extend its performance in complex situations, which are not only able to effectively process nonlinear dataset but also can greatly improve the accuracy of data analysis in non-target metabolomics.

Microplastics (MPs) are of environmental concern due to their bioavailability and potential impacts on a wide range of marine biota. In this study, we investigated the ingestion, bioaccumulation and egestion of fluorescent polystyrene (PS) micospheres (10 μm) in both larvae and adults of marine medaka (Oryzias melastigma), with or without food supply. The post-exposure effects of non-fluorescent PS (10 μm) on the survival, growth and reproduction of medaka larvae were also explored. Results showed that the PS microspheres could be ingested by both larvae and adults during the 48 h-exposure. Notably, feeding status was found to significantly affect the ingestion in medaka adults, which was not observed in the larvae. The egestion process of PS was rapid during the first recovery day but there was still certain percent of particles retained in digestive tracts at the end of 7 d recovery for either larvae or adults. After a 14 d pre-exposure with the non-fluorescent PS microspheres, the subsequent survival, growth and reproduction of medaka larvae were all significantly affected at the end of 120 d of experiment without PS. Overall, these results indicate that fishes might ingest or retain more MPs if the environmental abundance of MPs continues to increase while the available food decreases. Medaka fishes in larval stage have no capacity to select natural food sources like the adults. The chronic and "legacy effect" of MPs might also be a problem worthy paid more attention in future research instead of acute and immediate effect studies.

Condensins are highly conserved proteins that are important for chromosome maintenance in nearly all forms of life. Although many organisms employ two forms of the condensin complex, the condensin genes in Tetrahymena have expanded even further. Here we report a form of condensin that is specifically active during sexual reproduction. This complex, condensin D, is composed of the core condensin proteins, Smc2 and Smc4, and two unique subunits, the kleisin Cph5 and Cpd2. Cpd2 is also found in somatic nuclei in vegetative cells, but is dispensable for growth and nuclear division. Immunoprecipitation experiments show that condensin D interacts with a putative member of a chromatin-remodeling complex during development. Condensin D is required for sexual reproduction and for endoreplication and genome reduction of the progeny's somatic nuclei. Altogether, Tetrahymena possesses at least four forms of condensin to fulfill the needs of maintaining chromosomes in two different nuclei containing the somatic and germline genomes.

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Nonsense-mediated mRNA decay (NMD) is essential for removing premature termination codon-containing transcripts from cells. Studying the NMD pathway in model organisms can help to elucidate the NMD mechanism in humans and improve our understanding of how this biologically important process has evolved. Ciliates are among the earliest branching eukaryotes; their NMD mechanism is poorly understood and may be primordial. We demonstrate that highly conserved Upf proteins (Upf1a, Upf2 and Upf3) are involved in the NMD pathway of the ciliate, Tetrahymena thermophila. We further show that a novel protozoa-specific nuclease, Smg6L, is responsible for destroying many NMD-targeted transcripts. Transcriptome-wide identification and characterization of NMD-targeted transcripts in vegetative Tetrahymena cells showed that many have exon-exon junctions downstream of the termination codon. However, Tetrahymena may lack a functional exon junction complex (EJC), and the Tetrahymena ortholog of an EJC core component, Mago nashi (Mag1), is dispensable for NMD. Therefore, NMD is EJC independent in this early branching eukaryote.

Fibromyalgia (FM) and obesity are closely related. However, little is known about how obesity contributes to FM. Importantly, adequate evidence has shown that tumor necrosis factor alpha (TNF-α) plays a critical role in obesity. Thus, we hypothesized that obesity-induced TNF-α release may potentiate FM-associated pain. To test this hypothesis, we investigated the role of TNF-α in the development of FM-like pain in a mouse model of acid saline injection-induced FM. Consistent with previous reports, we showed that repeated acid saline injections induced bilateral mechanical hyperalgesia, and this effect lasted for at least 4 days after acid saline injections. This phenomenon was associated with increased levels of TNF-α in plasma, muscles, and spinal cord. Furthermore, we found that 24 weeks of high fat diet treatment significantly potentiated acid saline-induced bilateral mechanical hyperalgesia. High fat diet-treated mice exhibited robustly increased levels of TNF-α in plasma, muscles, and spinal cord after acid saline injections compared with low fat diet-treated mice. Additionally, using immunofluorescence staining, we found that the number of TNF-α positive cells in dorsal root ganglion (DRG) was increased after acid saline injections, and high fat diet treatment further sensitized this increase. Finally, we reported that acid saline-induced FM-like pain behaviors were abolished in TNFRp55-/- mice, confirming the critical role of TNF-α in the development of FM-like pain. Taken together, our results suggested that high fat diet treatment may sensitize acid saline-induced FM-like pain via increasing TNF-α levels in plasma, muscles, and DRG.

Several types of retinal interneurons exhibit spikes but lack axons. One such neuron is the AII amacrine cell, in which spikes recorded at the soma exhibit small amplitudes (<10 mV) and broad time courses (>5 ms). Here, we used electrophysiological recordings and computational analysis to examine the mechanisms underlying this atypical spiking. We found that somatic spikes likely represent large, brief action potential-like events initiated in a single, electrotonically distal dendritic compartment. In this same compartment, spiking undergoes slow modulation, likely by an M-type K conductance. The structural correlate of this compartment is a thin neurite that extends from the primary dendritic tree: local application of TTX to this neurite, or excision of it, eliminates spiking. Thus, the physiology of the axonless AII is much more complex than would be anticipated from morphological descriptions and somatic recordings; in particular, the AII possesses a single dendritic structure that controls its firing pattern.

The Chinese soft-shelled turtle (Pelodiscus sinesis) is a widely cultured commercial species in East and Southeast Asian countries. The turtles frequently suffer from acute cold stress during farming in China. Stress-induced factor such as Interleukin-6 (IL6) is a multifunctional molecule that plays important roles in innate and adaptive immune response. In the present study, we found that the turtle possessed two IL6 transcripts, where one IL6 transcript contained a signal peptide sequence (psIL6), while the other IL6 transcript (psIL6ns) possessed no such signal peptide gene. To test any differential expression of the two isoforms during temperature and microbial stress, turtles were adapted to optimal environmental water temperature (25 °C), stressed by acute cooling for 24 h, followed with the challenge of Aeromonas hydrophila (1.8 × 108 CFU) or Staphylococcus aureus (5.8 × 108 CFU). Gene characterization revealed that psIL6ns, a splicer without codons encoding a signal peptide and identical to the one predicted from genomic sequence, and psIL6, a splicer with codons encoding a signal peptide, were both present. Inducible expression was documented in primary spleen cells stimulated with ConA and poly I: C. The splenic and intestinal expression of psIL6ns and psIL6 was increased in response to temperature stress and bacterial infection.

The purpose of this study was to construct a deep learning system for rapidly and accurately screening retinal detachment (RD), vitreous detachment (VD), and vitreous hemorrhage (VH) in ophthalmic ultrasound in real time.

Cubosomes, a product of nanobioengineering, are self-structured lipid nanoparticles that act like drug-loaded theranostic probes. Here, we describe a simple method for the preparation of combinatorial drug-loaded cubosomes with, proof-of-principle, therapeutic effect against cancer cells, along with diagnostic capabilities. Anticancer drugs cisplatin and paclitaxel were loaded in the cubosomes in combination. The cubosomes were coated with a layer of poly-Ɛ-lysine, which helped avoid the initial burst release of drug and allowed for a slow and sustained release for better efficacy. Cubosomes were imaged by transmission electron microscope, and their dispersion analyzed in vitro by differential scanning calorimetric and X-ray diffractogram studies. The microscopic images depicted spherical polyangular structures, which are easily distinguishable. The analyses revealed that the drug is uniformly dispersed all through the cubosomes. Further characterization was carried out by zeta-potential measurement, in vitro release, and entrapment efficiency studies. The in vitro studies established that the coating of cubosomes successfully reduced the burst release of drugs initially and confirmed a slow, sustained release over increased time. Comparative cytotoxicity of coated, uncoated, and blank cubosomes was evaluated, using human hepatoma HepG2 cell line, and the formulations were found to be entirely nontoxic, similar to the blank ones. The therapeutic efficiency of the cubosomes against HeLa cells was confirmed by the impedance measurement and fluorescent imaging. Furthermore, the reduction in impedance in cells treated with coated combinatorial cubosomes proved the impairment of HeLa cells, as confirmed by fluorescence microscopy.

Choanephora rot of Chenopodium plants (CRC) was observed at the flowering stages in seven plantations of Shanxi Province, China. CRC had caused leaf, stem, and panicle neck rot of C. quinoa, panicle neck and stem rot of C. formosanum, and stem rot of C. album. Typical symptoms included water-soaked, rapid soft rotting, and abundant sporulation on the whole panicle necks, stems, and leaves. Based on morphological characteristics, phylogenetic analyses, and pathogenicity tests, the pathogens were identified as Choanephoraceae cucurbitarum. Sporangiola and sporangiospore of C. cucurbitarum germinated at 30 °C and were able to germinate by two h post-inoculation (hpi). The germination rates of sporangiola and sporangiospore significantly increased at 3 to 4 hpi, and the germination rates ranged from 91.53 to 97.67%. The temperature had a significant effect on the pathogenicity of C. cucurbitarum the optimum pathogenic temperatures for stems of C. quinoa, C. formosanum and C. album were 30 °C after one day post-inoculation. Choanephoraceae cucurbitarum could infect white and red quinoa panicle necks between 20 and 30 °C, and the average lesion lengths were 0.21 to 3.62 cm. Among the five tested fungicides (boscalid, dimethomorph, isopyrazam, propiconazole, and tebuconazole), isopyrazam showed higher sensitivity to sporangiola germination of C. cucurbitarum, with an EC50 value of 0.6550 μg/mL. Isopyrazam and tebuconazole strongly inhibited the sporangiospore germination of C. cucurbitarum, which showed EC50 values of 0.4406 and 0.3857 μg/mL. To our knowledge, the present study found for the first time that C. cucurbitarum is a pathogen causing panicle neck of C. formosanum and stem rot of C. formosanum and C. album, while CRC first appeared in the quinoa panicle necks, and gradually expanded to stems and leaves.

Members of the E2F family of transcription factors have been reported to regulate the expression of genes involved in cell cycle control, DNA replication, and DNA repair in multicellular eukaryotes. Here, E2FL1, a meiosis-specific E2F transcription factor gene, was identified in the model ciliate Tetrahymena thermophila. Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I. Finally, by searching for E2F DNA-binding motifs in the entire T. thermophila genome, we identified 714 genes containing at least one E2F DNA-binding motif; of these, 235 downregulated represent putative E2FL1 target genes.

Certain ciliates of the subclass Scuticociliatia (scuticociliates) are facultative parasites of fishes in which they cause a suite of diseases collectively termed scuticociliatosis. Hitherto, comparatively little was known about genetics and genomics of scuticociliates or the mechanism of scuticociliatosis. In this study, a laboratory culture of the facultatively pathogenic scuticociliate Pseudocohnilembus persalinus was established and its genome sequenced, giving the first genome of a marine ciliate. Genome-wide horizontal gene transfer (HGT) analysis showed P. persalinus has acquired many unique prokaryote-derived genes that potentially contribute to the virulence of this organism, including cell adhesion, hemolysis and heme utilization genes. These findings give new insights into our understanding of the pathology of scuticociliates.

The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation.