Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

The taxonomy and molecular phylogeny of two epibiotic colonial peritrich ciliates (Ciliophora, Peritrichia).

Two colonial sessilid peritrichs, Epistylis qingdaoensis n. sp. and Carchesium cyclopidarum Nenninger, 1948, were isolated from a marine crustacean and a freshwater mayfly, respectively. Morphological characters for each species were revealed by in vivo observations and silver staining methods. Epistylis qingdaoensis n. sp. is characterized by the apperance of the colony which is up to 250 µm high and usually contains fewer than eight zooids, the single-layered peristomial lip, the conspicuously conical peristomial disc, and the structure of infundibular polykinety 3 which comprises three isometric ciliary rows. Carchesium cyclopidarum is recognized by the colony height of about 600 µm, the small zooid size of 35-50 × 20-30 µm, and the low number of silverlines. Phylogenetic analyses based on small subunit ribosomal DNA sequences were performed to reveal their evolutionary relationships. Surprisingly, neither species clustered with its congeners. Epistylis qingdaoensis n. sp. nested within a clade of Zoothamnium species that was sister to a clade comprising core Epistylis species and several Zoothamnium species. Carchesium cyclopidarum nested within a clade comprising solitary peritrichs and was distantly related to the type species of Carchesium, C. polypinum. These findings imply for the first time that Carchesium is non-monophyletic.

Investigation of Parental Acceptance of General Anesthesia With Mask Induction in Children Undergoing Frenulectomy.

PURPOSE: To investigate parental acceptance of the use of general anesthesia with mask inhalation (GAMI) in the treatment of ankyloglossia. DESIGN: Parents of children with ankyloglossia received questionnaires to analyze the related factors of their acceptance of GAMI. METHODS: From July 2017 to November 2020, 131 parents of children with ankyloglossia in our hospital were enrolled and received investigation questionnaires. A total of 129 valid questionnaires were returned. The level of acceptance was evaluated using the visual analogue scale (VAS). We described the parental acceptance in a statistical method and performed univariant and multivariate analyses to identify related factors using SPSS 20.0. FINDINGS: A total of 129 (98.5%) parents completed the questionnaires. Only one patient (0.8%) experienced short-term (4 hours) abdominal bloating after surgery with GAMI. The average VAS regarding parental acceptance of the use of GAMI in the treatment was 43.80 mm (± 29.49), with only 17.8% of parents exhibiting a high level of acceptance of the anesthesia technique, while they had a relatively high level of satisfaction after surgery. CONCLUSIONS: Parents had a low level of acceptance of using GAMI in the treatment of ankyloglossia before surgery due to various factors.

Regulation of Semaphorin3A in the process of cutaneous wound healing.

Semaphorin 3A (Sema3A) has been recognized as a crucial regulator of morphogenesis and homeostasis over a wide range of organ systems. However, its function in cutaneous wound healing is poorly understood. In our study, we demonstrated that Sema3A adenovirus plasmids transfection limited keratinocyte proliferation and decreased migrative capacity as assessed by in vitro wound healing assay. Sema3A transduction inhibited TGF-ß1-mediated keratinocyte migration and EMT process. Besides, we applied mice with K14-Cre-mediated deletion of Sema3A and found that Sema3A depletion postponed wound closure with decreased re-epithelialization and matrix growth. Contrary to the results obtained with full-length Sema3A plasmids transfection, increased keratinocyte migration with recombinant Sema3A proteins resulted in quicker closure of the wounding area after a scratch. Further, exogenously applied recombinant Sema3A worked with EGF to maintain the activation of EGFR by interacting with NRP1 and thereby regulated the internalization of the EGFR-NRP1 complex. Taken together, these results indicated a paradoxical role of autonomous and non-autonomous Sema3A expression during wound healing. Combined administration of recombinant EGF and Sema3A proteins could accelerate the process of wound repair, thus providing promising treatment prospects in the future.

The hidden genomic diversity of ciliated protists revealed by single-cell genome sequencing.

BACKGROUND: Ciliated protists are a widely distributed, morphologically diverse, and genetically heterogeneous group of unicellular organisms, usually known for containing two types of nuclei: a transcribed polyploid macronucleus involved in gene expression and a silent diploid micronucleus responsible for transmission of genetic material during sexual reproduction and generation of the macronucleus. Although studies in a few species of culturable ciliated protists have revealed the highly dynamic nature of replicative and recombination events relating the micronucleus to the macronucleus, the broader understanding of the genomic diversity of ciliated protists, as well as their phylogenetic relationships and metabolic potential, has been hampered by the inability to culture numerous other species under laboratory conditions, as well as the presence of symbiotic bacteria and microalgae which provide a challenge for current sequencing technologies. Here, we optimized single-cell sequencing methods and associated data analyses, to effectively remove contamination by commensal bacteria, and generated high-quality genomes for a number of Euplotia species. RESULTS: We obtained eight high-quality Euplotia genomes by using single-cell genome sequencing techniques. The genomes have high genomic completeness, with sizes between 68 and 125 M and gene numbers between 14K and 25K. Through comparative genomic analysis, we found that there are a large number of gene expansion events in Euplotia genomes, and these expansions are closely related to the phenotypic evolution and specific environmental adaptations of individual species. We further found four distinct subgroups in the genus Euplotes, which exhibited considerable genetic distance and relative lack of conserved genomic syntenies. Comparative genomic analyses of Uronychia and its relatives revealed significant gene expansion associated with the ciliary movement machinery, which may be related to the unique and strong swimming ability. CONCLUSIONS: We employed single-cell genomics to obtain eight ciliate genomes, characterized the underestimated genomic diversity of Euplotia, and determined the divergence time of representative species in this subclass for the first time. We also further investigated the extensive duplication events associated with speciation and environmental adaptation. This study provides a unique and valuable resource for understanding the evolutionary history and genetic diversity of ciliates.

The Morphology, Taxonomy, and Phylogenetic Analyses of Five Freshwater Colonial Peritrich Ciliates (Alveolata, Ciliophora), Including the Descriptions of Two New Species.

The morphology and phylogeny of two new sessilid species, Zoothamnium weishanicum n. sp. and Epicarchesium sinense n. sp., two insufficiently known species, Zoothamnium arbuscula Ehrenberg, 1831 and Zoothamnium hentscheli Kahl, 1935, and a well-known species, Carchesium polypinum (Linnaeus, 1767) Ehrenberg, 1838, collected from freshwater habitats of China, were investigated. Zoothamnium weishanicum n. sp. is characterized by its inverted bell-shaped zooids, double-layered peristomial lip, alternately branched stalk, and two different-length rows in infundibular polykinety 3 (P3). Epicarchesium sinense n. sp. is recognized by its asymmetric-pyriform zooids, single-layered peristomial lip, conspicuous cortical blisters on the pellicle, dichotomously branched stalk, and P3 containing one short inner row and two long outer rows. Based on previous and newly obtained data of the three known species, improved diagnoses and redescriptions are provided including, for the first time, data on the infraciliature of Z. arbuscula and Z. hentscheli. In addition, we analyzed the phylogeny of each species based on SSU rDNA sequence data.

Isoform specific FBXW7 mediates NOTCH1 Abruptex mutation C1133Y deregulation in oral squamous cell carcinoma.

Our group previously identified that the NOTCH1 Abruptex domain contains the most mutations in Chinese OSCC patients, including a hotspot mutation (C1133Y). FBXW7 is an E3 ubiquitin ligase that regulates a network of proteins, including NOTCH1, via degradation. In this study, we first described the co-localization of isoform specific FBXW7-FBXW7ß and NOTCH1C1133Y mutation in the same cytoplasmic sites. Gain- and loss-of-function assays were performed to examine the tumor suppressor role of FBXW7ß in the proliferation and invasion of OSCC cells. The co-expression of NOTCH1C1133Y and FBXW7ß significantly attenuated tumor growth. Meanwhile, FBXW7ß reversed the oncogenic phenotype and the activation of the AKT/ERK/NFκB pathway induced by NOTCH1C1133Y mutation. FBXW7ß downregulated the stability of NOTCH1C1133Y protein and promoted protein ubiquitination. This was the first time that we selected a NOTCH1 hotspot mutation detected in clinical samples and identified the function of FBXW7ß that mediated NOTCH1 mutation degradation in OSCC. The newly identified interaction between FBXW7ß and NOTCH1C1133Y protein provides new insights into the progression of OSCC, especially regarding Abruptex domain mutations, and represents a valuable target for OSCC therapy.

CMTM6 promotes cell proliferation and invasion in oral squamous cell carcinoma by interacting with NRP1.

Previous studies have identified that both CKLF-like MARVEL transmembrane domain-containing member (CMTM6) and Neuropilin-1 (NRP1) played an essential part in regulating tumorigenesis and immune response. However, the potential connection between CMTM6 and NRP1 in oral squamous cell carcinoma (OSCC) remains unknown. In this study, we investigated the clinicopathologic significance of CMTM6 and NRP1 in OSCC. We examined the co-expression of CMTM6 and NRP1 in both OSCC tissues and cell lines. Co-overexpression of CMTM6 and NRP1 was generally highly expressed in cancer tissues and is associated with poor prognosis. Gain- and loss-of-function assays confirmed the oncogenic properties of CMTM6 in OSCC cells. Depletion of NRP1 abrogated tumorigenesis induced by CMTM6. By performing co-immunoprecipitation (co-IP), we discovered a potential interaction between CMTM6 and NRP1. Meanwhile, the stability of CMTM6 was significantly decreased in the NRP1-silencing cells, indicating the involvement of NRP1 in the degradation process of CMTM6. The crosstalk between CMTM6 and NRP1 provided a new insight into the progression of OSCC, which may indicate an alternative strategy for OSCC treatment.

Identification of extracellular vesicles-transported miRNAs in Erlotinib-resistant head and neck squamous cell carcinoma.

Erlotinib is an oral tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) pathway. Although our previous study has proved the efficacy of Erlotinib in head and neck squamous cell carcinoma (HNSCC), it has also demonstrated poor clinical response rates and disappointing results in clinical trials for HNSCC to date. In this study, we discovered elevated cell proliferation and invasion ability in erlotinib-resistant HNSCC cells. The contributions of miRNAs within extracellular vesicles (EVs) during the formation of chemoresistance were investigated in this study. Among up-regulated miRNAs in EVs derived from resistant cells, miR-7704, miR-21-5p and miR-3960 showed the most pro-tumorigenic alterations after transfection. Conversely, let-7i-5p, miR-619-5p and miR-30e-3p demonstrated tumor suppressive effects. By performing qRT-PCR and Western blot analysis, we found Vimentin played a pivotal role in modulating erlotinib resistance. Additionally, immune system was highlighted in the GO and KEGG analyses. Transfection of miR-7704, miR-21-5p significantly elevated CTLA-4 and LAG3 mRNA levels. Meanwhile, miR-3960 increased the relative mRNA expression of TIM3 in HNSCC cells. Transfection of let-7i-5p, miR-619-5p and miR-30e-3p decreased these checkpoint factors. To conclude, the present study described the roles of EVs-transmitted miRNAs on erlotinib resistance. Targeting the disregulated immune system could be the effective method to overcome erlotinib-resistance in HNSCC cells.

MiR-876-5p modulates head and neck squamous cell carcinoma metastasis and invasion by targeting vimentin.

BACKGROUND: Local or distant metastasis remains the main course of death in head and neck squamous cell carcinoma (HNSCC) patients. MicroRNAs (miRNAs) have been implicated in metastasis of HNSCC, but the mechanisms of their action are mainly undocumented. Through public head and neck cancer miRNA expression datasets, we found that miR-876-5p was a novel potential tumor suppressor targeting HNSCC metastasis. METHODS: Clinical significance and mechanism of miR-876-5P was systematically analyzed in HNSCC. Quantitative RT-PCR was used to evaluate miR-876-5p levels in HNSCC cell lines and in 20 pairs of HNSCC with associated regional nodal metastases and HNSCC without metastatic primary tumors. Scratch and invasion assays were evaluated to determine the role of miR-876-5p in the regulation of HNSCC cell migration and invasion, respectively. Western blotting was used to investigate the mechanism by which miR-876-5p suppresses HNSCC cell invasion and migration. Luciferase assays were performed to assess miR-876-5p binding to the vimentin gene. The animal model was used to support the in vitro experimental findings. RESULTS: MiR-876-5p mimics inhibited HNSCC cell migration and invasion. Vimentin protein and mRNA levels were decreased in the miR-876-5p mimics group but increased in the miR-876-5p inhibitors group, which demonstrated that miR-876-5p inhibits vimentin expression in HNSCC cells. By directly targeting the vimentin 3'-UTR, we used dual-luciferase reporter assays to verify that vimentin is a functional downstream target of miR-876-5p. Importantly, increased vimentin expression promoted cell migration and invasion, and co-transfection with miR-876-5p mimics and vimentin restored cell aggressiveness to the original level. Moreover, miR-876-5p overexpression significantly downregulated vimentin expression level and inhibited the distal metastasis of HNSCC cells in vivo. CONCLUSIONS: miR-876-5p, which functions as a tumor suppressor in HNSCC, inhibits metastasis by targeting vimentin and provides a novel therapeutic target for HNSCC treatment.

Construction of green fluorescence protein mutant to monitor STT3B-dependent N-glycosylation.

Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.

Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error.

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella, Blepharisma americanum and Strombidium stylifer We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

Stability of the recombinant anti­erbB2 scFv­Fc­interleukin­2 fusion protein and its inhibition of HER2­overexpressing tumor cells.

The anti­erbB2 scFv­Fc­IL­2 fusion protein (HFI) is the basis for development of a novel targeted anticancer drug, in particular for the treatment of HER2­positive cancer patients. HFI was fused with the anti­erbB2 antibody and human IL­2 by genetic engineering technology and by antibody targeting characteristics of HFI. IL­2 was recruited to target cells to block HER2 signaling, inhibit or kill tumor cells, improve the immune capacity, reduce the dose of antibody and IL­2 synergy. In order to analyse HFI drug ability, HFI plasmid stability was verified by HFI expression of the trend of volume changes. Additionally, HFI could easily precipitate and had progressive characteristics and thus, the buffer system of the additive phosphate­citric acid buffer, arginine, Triton X­100 or Tween­80, the establishment of a microfiltration, ion exchange, affinity chromatography and gel filtration chromatography­based purification process were explored. HFI samples were obtained according to the requirements of purity, activity and homogeneity. In vivo, HFI significantly delayed HER2 overexpression of non­small cell lung cancer (Calu­3) in human non­small cell lung cancer xenografts in nude mice, and the inhibition rate was more than 60% (P<0.05) in the group treated with 1 mg/kg the HFI dose; HFI significantly inhibited HER2 expression of breast cancer (FVB/neu) transgenic mouse tumor growth in 1 mg/kg of the HFI dose group, and in the following treatment the 400 mm3 tumors disappeared completely. Combined with other HFI test data analysis, HFI not only has good prospects, but also laid the foundation for the development of antibody­cytokine fusion protein­like drugs.